Retinal Origins of Vigabatrin Toxicity In Infantile Spasms · ERG growth curves, for each component, recorded from children with IS were generated using data recorded pre-VGB treatment
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Retinal Origins of Vigabatrin Toxicity In Infantile Spasms
by
Julianna Sienna
A thesis submitted in conformity with the requirements for the degree of Master of Medical Science
Institute of Medical Science University of Toronto
Retinal Origins Of Vigabatrin Toxicity In Infantile Spasms
Julianna Sienna
Master of Science
Institute of Medical Science University of Toronto
2011
Vigabatrin (VGB) is an anti-epileptic drug used to treat children with Infantile
Spasms (IS). The 3.0 flicker amplitude of the electroretinogram (ERG) is
currently used to monitor visual function changes in infants on VGB. To find
a more specific marker of permanent changes due to VGB, sedated ERGs
were performed on 31 IS patients and 13 retinally normal controls to isolate
components of the cone pathway. ERG growth curves, for each component,
recorded from children with IS were generated using data recorded pre-VGB
treatment and for controls. Only the cone off response (from Off bipolar
cells) and cone photoreceptor sensitivity were associated with decreased
flicker amplitude. Twenty nine percent of patients had an abnormal cone off
response. No patient had an abnormal cone off response at baseline. No
patient with an abnormal cone off response recovered normal function. The
cone off response could serve as a marker VGB retinal toxicity.
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Acknowledgments
This thesis would not have been possible without the help and support of many people. First and foremost, I would like to thank Dr. Carol Westall for her unending patience, commitment to my learning and for sharing so much of her knowledge and expertise with me. Dr. Westall has given me so many opportunities to grow both as a scientist and as a person over the last two years and I will always be grateful for that. Thank you for standing in my corner as I pursued my dreams, believing in my project and truly serving as a role model for me.
I would also like to thank Tom Wright for his support in the development and execution of this project and Carole Panton and Melissa Cotesta for performing ERGs. I have appreciated your guidance, your technical knowledge. Tom, Carole & Melissa’s participation in this project and my life made coming to the hospital everyday a treat. Thank you for always believing I could pull it out in the end and always helping me to get there.
Thanks must also be given to Dr. Raymond Buncic, and the sedation nurses Beverly Griffiths and Yasmin Sherrif for their help in testing. Thank you for being teachers as well as team members. I have appreciated the support from all the members of the Ophthalmology department at Sick Kids.
The supervision and advice from the members of my program advisory committee must be acknowledged. Drs. Agnes Wong, Carter Snead & Gideon Koren were instrumental in ensuring the quality of this research project.
Lastly, thank you to my family and friends whose constant support and encouragement helped me to stay focused.
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Table of Contents
Acknowledgments .............................................................................. iii
Table of Contents ................................................................................ iv
List of Tables ...................................................................................... vi
List of Figures .................................................................................... vii
List of Appendices .............................................................................. ix
List of Abbreviations………………………………………………………………………………………xii
1. Symptomatic causes of Infantile Spasms by category.......................2 2. Initial case reports of vigabatrin-associated visual field loss.............11 3. Case reports of two IS patients with visual field loss.......................14 4. Summary of studies investigating visual field defects
in children on VGB using perimetry...............................................16 5. Summary of ERG findings in adults taking VGB…............................26 6. Summary of ERG results in children and infants taking
vigabatrin.................................................................................29 7. Additional stimulus condition testing parameters............................35 8. Group demographics of patients with IS at baseline........................42 9. Drug history and visual acuity in Infantile Spasms patients at
baseline....................................................................................44 10. Group demographics of controls...................................................46 11. Longitudinal drug information and visual acuity in IS patients..........56 12. Diagnostic characteristics of abnormal tests using flicker, cone off,
and sensitivity…………………………………………………………………………………....68 13. Individual patient data for all those with at least one
abnormal test. ........................................................................76 14. Distribution of sex and mean daily VGB dose for normal vs.
abnormal test using different definitions of abnormality.................78
vii
List of Figures
1. Structure of vigabatrin.............................................................5 2. Schematic diagram of the retina..............................................18 3. Light adapted (photopic) 3.0 ERG............................................23 4. 3.0 Flicker response from IS patient........................................36 5. Photopic negative response from patient..................................37 6. Hood & Birch equation to describe the leading edge of
the a-wave………………………………………………………………………………………38 7. Cone off response.................................................................38 8.
a. Control flicker amplitude plotted by age in months............48 b. Baseline IS flicker amplitude plotted by age in months.......49
9. a. Control PhNR amplitude plotted by age in months.............50 b. Baseline IS PhNR amplitude plotted by age in months........50
10. a. Control cone sensitivity plotted by age in months..............51 b. Baseline IS cone sensitivity plotted by age in months.........51
11. a. Control cone maximum response plotted by age in
months. ………………………………………………………………………………..52 b. Baseline IS cone maximum response plotted by age in
months. …………………………………………………………………………………52 12. Control (solid line) and subject (dashed line) developmental curves
for: a. Flicker amplitude……………………………………………………………………53 b. PhNR amplitude……………………………………………………………………..54 c. Cone sensitivity……………………………………………………………………..54 d. Cone maximum response……………………………………………………..55
13. Boxplot comparing cone off response amplitude in controls with IS subjects at baseline.................................................55
14. a. Box plot comparing adjusted flicker amplitude over time on
vigabatrin.. …………………………………………………………………………..60 b. Box plot comparing adjusted PhNR amplitude over time on
vigabatrin.……………………………………………………………………………..61
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c. Box plot comparing adjusted maxiumum response (Rmax) over time on vigabatrin.. …………………………………………………….62
d. Box plot comparing adjusted cone sentivity over time on vigabatrin………………………………………………………..63
15. Cone off response in normal and abnormal test................65 16.
a. Adjusted Flicker amplitude for patients with normal vs. abnormal cone off response...........................................66
b. Adjusted Flicker amplitude for patients with normal vs. abnormal photopic negative response .............................66
c. Adjusted Flicker amplitude for patients with normal vs. abnormal Cone Rmax ..................................................67
d. Adjusted Flicker amplitude for patients with normal vs. abnormal cone Sensitivity .............................................67
17. Survival plots for cone off response, flicker amplitude and cone sensitivity ..................................................................70
18. Venn Diagram of the number test points where patients had overlap between each test. ..................................................71
19. Mosaic plots of agreement in classifying tests between: a. flicker and cone off.......................................................72 b. flicker and sensitivity ...................................................73 c. cone off and sensitivity ................................................73 d. flicker, cone off, and sensitivity .....................................74
20. Structure of D- alpha amino adipic acid (left) and vigabatrin (right)................................................................93
21. Structure of 4-methryl glutamic acid (left) and GYKI 52466 (right).……………………………………………………………………..93
ix
List of Appendices
1. Current lab protocol …………………………………………………………………………….115
2. Vigabatrin and infantile epilepsy subject consent form………………………116
3. Vigabatrin and infantile epilepsy control consent form……………………….122
vs 9/31). There was acceptable agreement between both tests in 9 VGB treated
patients and in 10 control subjects (ages 4-7 years). Caution was advised in
interpreting the results as those VGB treated patients who could not complete
Goldmann perimetry tended to have smaller fields than those who did cooperate
(Agrawal et al., 2009). The number of children that could not be included because
of difficulty with perimetry is listed for each study in table 4. In one study, patients
less than 6 years old or with mental handicap were excluded from visual field
analysis (Ascaso, Lopez, Mauri & Cristobal, 2003).
In order to monitor vision in this population, electroretinograms are routinely
used. When VGB was re-approved for use in the USA, it was done under the
SHARE (Support, Help and Resources for Epilepsy) program, where physicians
must take part in the REMS (Risk Evaluation and mitigation Strategy). On top of
requiring routine assessments of effectiveness, this strategy also incorporates
baseline and regular vision monitoring using ERGs when and where possible.
16
Several additional studies have shown results of both visual field and ERG
monitoring in this population (Agrawal et al., 2009; Gaily, Johnson, & Lappi, 2009;
Camposano, Major, Halpern & Thiele, 2008). Monitoring in infants less than 2
years old who cannot complete perimetry was also recommended in a recent
review by Sergott (2010).
Table 4. Summary of studies investigating visual field defects in children on VGB using perimetry
Author (year) Design Control grp
Test Method
Mean VGB duration
% VFD (exp’d)
Cant do VFs
Wohlrab (1999)
CS Y G -- 42% 92%
Gross-Tsur (2000)
CS N G & HF 3.0 years 65% 29%
Ianetti (2000) CS N G & HF -- 19% 30%
Pelosse(2001) CS N G 3.4 years 55% --
Roccella (2001)
L N HF -- 33% --
Vanhatalo (2002)
L N G 2.2 years 24% Only inc if could do G
Spencer (2003)*
CS N G & HF 3 mos-9 years
36% 72%
Ascaso(2003) L N HF 3.5 20% --
Pojda-Wilczek (2005)
L N HF -- 53% --
Werth (2006) CS Y G -- 33% 46%
You (2006) L N HF 4.0 years 22% Only inc if
17
could do HF
Wild (2009) 8-12 y only
L Y G + HF 29.3 mos 20% 91-92.7%
Camposano (2008)*
CS N G 17.7 mos resp. 6.3 mos Non-resp
4% 58%
Gaily (2009)* CS N G 21.0 mos 7% 0
Agrawal (2009)*
CS Y WSK + G
Not reported
WSK – (29%)
71% (G) 10% (WSK)
Legend: grp – group, exp’d – exposed, VFD – Visual Field Defect, VFs – Visual Field Testing, CS – Control Study, L – Longitudinal, Y – Yes, N – No, G -Goldmann Perimetry, HF – Humphrey Field Analyzer, mos – months, inc - included, resp – responder, WSK – White Sphere Kinetic Perimetry
18/18 Latency of the 3.0 flicker b-wave and a–b amp, <52 uV flicker amp – VFL (18/18)
Miller (1999) 32/32 Reduced OP amps (32/32), reduced photoreceptor sensitivity (9/32), reduced rod and cone b waves (32/32), reduction in cone flicker responses correlated strongly with the degree of VFL as measured by kinetic perimetry, 7/20 VFL
Hardus (2001) 15/30 b-wave abnormalities in 15 pts with VFL
Coupland (2001)
18/76 eyes
Reduced 3.0 flicker amp (18/76), reduced photopic (30/76) and scotopic b wave (30/76), reduced Ops (22/76), VFL not measured
Legend: Tx – treatment, VFD – visual field defect, ND – Not done, Ab – Abnormal, N – Normal, amp – amplitude, IT – Implicit time, assoc. – associated, R – maximum response, pts – patients, VF - visual field testing
The current research was designed to reduce the pre-test variability and increase
sensitivity and specificity for an electrophysiological test for VA- function loss by
using tests that isolates targeted areas of the retina. The 30 Hz flicker response is
a measure of integrity of the entire cone pathway. We have identified three ERG
tests used to isolate different parts of the cone pathway: cone a wave modeling,
the photopic negative response and the photopic off response. The first, cone a
wave modeling, isolates the response of the cone photoreceptors. The photopic
1/3 delayed mixed rod cone
Pojda-Wilczek (2005) Abstract only
8-20 years old
- More than half VFD
Decreased or borderline b-wave amp “after flicker 3.0”
Eklund (2006) Abstract only
Med 22 months (6-69 months)
- ND R rod and R cone reduced in majority of pts, photopic b wave IT prolonged
Camposano (2008)
- - 1/25 VFD 1/20 Ab ERG, Ab photopic and scotopic responses, prolonged 3.0 latency (no VF testing)
Legend: * - not reported or unknown; Wks- weeks; sx – seizure; mos – months; M – male; F – female; init – initiated; MCA- middle cerebral artery; ACA- Anterior cerebral artery; 2*- secondary; L – left; CP-Cerebral Palsy; HIE – Hypoxic ischemic event
Subjects were tested within one month of starting Vigabatrin using sedated ERGs.
This data was used to create developmental curves. All fundus exams were
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normal. Drug and Visual acuity information at baseline testing is provided in table
9.
Table 9. Drug history and visual acuity in Infantile Spasms patients at baseline.
STUDY ID
Mos on VGB
Age at test
Other AED meds
Dose of VGB VA (Binoc) logMAR
VA test
VIE01 0.23 7.61 600 mg BID No co-op
VIE02 0.23 9.80 800 mg BID No co-op
VIE03 0.13 4.00 CO, PH 600 mg BID NT
VIE04 0.07 12.49 600 mg BID >1.6 T
VIE05 0.23 5.70 625 mg bid LP only
VIE06 0.23 7.51 600 mg bid NT
VIE07 0.16 9.02 1.1 T
VIE08 0.00 16.03 625 mg bid 1.3 T
VIE09 0.26 6.23 750 mg bid No co-op
VIE10 0.82 9.61
500 mg am, 750 mg pm
NR
VIE11 0.23 18.79 CO 1000 mg bid 0.3 C
VIE12 0.26 13.93 650 mg bid 0.1 C
VIE13 CO 750 mg bid 1.1 T
VIE14 0.26 23.64 650 mg bid 0.8 T
VIE15 0.33 5.67 450 mg bid NR
VIE16 0.26 8.75 600 mg bid Fixes and
45
follows
VIE17 1.11 12.92 1000 mg bid 1.1 T
VIE18 0.23 8.30 ACTH 625 mg bid 1.1 T
VIE19 0.13 7.02 750 mg bid 1.3 T
VIE20 0.59 9.44 600 mg bid NT
VIE21 0.16 3.18
PH, ACTH 500 mg bid Fixes and follows
VIE22 0.20 7.57 720 mg bid 1.0 T
VIE23 0.23 6.23 650 mg bid 1.0 T
VIE25 0.43 4.62 750 mg bid NR
VIE26 0.39 10.03 LE 500 mg bid >1.1 T
VIE27 0.20
9.87
375 mg bid Slow to pick up fixation
VIE28 0.46 6.23 750 mg bid 0.2 C
VIE29 0.66 6.89 300 mg bid NT
VIE30 0.46 9.61
650 mg bid No attention
VIE31 1.05 17.54 PH, LE 725 mg bid >1.1 T
Legend: mos – months; AED – anti-epileptic drug; VA – visual acuity; binoc – binocular; T – Teller, C – Cardiff, co-op – cooperation, NT – Not tested; cpd – cycles per degree; LP – Light Perception; NR – No response; CO – Clobazam; LE – Levetiracetam; ACTH –Adrenocorticotrophic hormone; PH – Phenobarbital
46
9.2 Controls:
Group demographics for controls are presented in table 10. Seventeen children
were tested in the VEU to rule out retinal reasons for early onset horizontal
nystagmus. Six were tested prospectively and data from eleven patients were
gathered retrospectively. Three patients were excluded because ERGs showed
retinal abnormalities that were responsible for their nystagmus. Thus, 9 male and
4 female patients, ages ranging from 5.6 – 47.02 months, with a (mean age of
18.7 months) were included.
At baseline, visual acuity was assessed when possible and subjects underwent
cycloplegic exams and ophthalmoscopy exams.
Ophthalmoscopy results included an examination of the fundus, macula and disc.
Table 10. Group demographics of controls
ID Referred for Age (mos)
Normal ERG
Comorbidities VA (binoc) logMAR
VA test
C01 EOHN 41.54 No 0.1 C
C02 EOHN with vertical component
13.84 No Left head tilt 0.4 C
C03 EOHN 9.21 Yes 0.2 C
C04 EOHN, photophobia
13.28 Yes 0.0 C
47
Legend: #=retrospective; mos – months; VA – visual acuity; binoc – binocular; EOHN – early onset horizontal nystagmus; C – Cardiff; T – Teller; LE – left eye; RE – right eye; LX(T)- left
C05 R jerk nystagmus
31.80 No Trisomy 21, roving eye mvts, poor visual attention
1.4 T
C06 EOHN, photophobia
23.18 Yes Left head turn 0.1 C
#C07 EOHN, poor fixation, no following
5.93 Yes Plagiocephaly, proximal hypotonia
1.4 T
#C08 EOHN, LX(T) c LH(T)
5.61 Yes 1.7 T
#C09 RE nystagmus
15.51 Yes R strab, limited adduction LE, R face turn, intranuclear ophthalmoplegia
0.2 C
#C10 EOHN 18.36 Yes Trisomy 21 0.2 C
#C11 EOHN 16.26 Yes Twin A, 38 wks gestation, ON hypoplasia, hypopigmented fundi
0.2 C
#C12 EOHN 15.51 Yes Delayed milestones 0.1 C
#C13 EOHN 14.85 Yes R face turn 0.0 C
#C14 EOHN 10.39 No 1.3 T
#C15 Nystagmus LE
14.03 Yes L cataract extraction, anterior vitrectomy, wears L contact lens
1.0 T
#C16 EOHN, photophobia
22.07 Yes LE only
LET, decreased stereopsis 1.6 T
#C17 EOHN 47.02 Yes AHP c chin elevation, high hyperopia
0.1 C
48
intermittent exotropia ; LH(T) left intermittent hypertropia- ; mvts – movements; strab – strabismus; ON – optic nerve; LET – left esotropia; AHP-abnormal head posture; c –with
Analysis 10
10.1 Developmental curves
In both controls and subjects, flicker amplitude increased with age. Flicker
amplitude increase from baseline approximately 25% over 24 months in patients
(baseline IS) and 15% over the first 24 months in controls (Figure 8a and b).
Three patients have abnormal flicker (falling greater than 45 uV below the
developmental curve). The two curves follow a similar pattern. The development
curve for subjects is only 2-3 uV lower than controls over the entire line.
Controls
Figure 8 a. Control flicker amplitude plotted by age in months. Solid line is the line of best fit and represents developmental curve.
49
In both patients and controls, PhNR amplitude increases with age, this is reflected
in the curve becoming more negative over time (Figure 9a & b). The curves follow
a similar trend though subjects increase approximately 400% over 24 months
while controls increase approximately 240% over the first 24 months. The
absolute value of PhNR is on average between 15 - 18 uV less in patients than
controls at any point along the curve.
y = 8.82ln(x) + 67.629 R² = 0.01761
0 20 40 60 80
100 120 140 160 180
0.00 10.00 20.00 30.00 40.00 50.00
Flicker a
mplitu
de (u
V)
Age (months)
Subjects
Figure 8 b. Baseline IS flicker amplitude plotted by age in months. Dashed line is the line of best fit and represents developmental curve.
Figure 11b. Baseline IS cone maximum response plotted by age in months. Dashed line is the line of best fit and represents developmental curve.
53
y = 8.82ln(x) + 67.629 R² = 0.01761
y = 7.3404ln(x) + 74.923 R² = 0.09322
0
20
40
60
80
100
120
140
0.00 10.00 20.00 30.00 40.00 50.00
Flicker a
mplitu
de (u
V)
Age (months)
Subjects and controls have similar developmental curves for maximum response
(figure 11 a and b) with maximum response amplitude increasing with age; this is
reflected in the line becoming more negative over time. The absolute value of the
maximum response in subjects increases from baseline approximately 22% over
24 months. Controls maximum response increase approximately 13% from
baseline to 24 months.
Developmental curves for subjects and controls for each marker, overlayed for
comparison are shown in Figure 12 a-d. In each figure, the dotted line represents
subjects with IS and the solid line represents control subjects.
Figure 12. Control (solid line) and subject (dashed line) developmental
curves for, a) flicker amplitude, and b) PhNR Amplitude.
Figure 12 a.
54
y = -‐15.46ln(x) + 24.628 R² = 0.12962
y = -‐16.19ln(x) + 17.821 R² = 0.42643
-‐70
-‐60
-‐50
-‐40
-‐30
-‐20
-‐10
0
10
20
30
0.00 10.00 20.00 30.00 40.00 50.00
PhNR Am
plitu
de (u
V)
Age (months)
y = 15.564ln(x) + 9.8971 R² = 0.06027
y = 2.8424ln(x) + 36.672 R² = 0.02539
0
20
40
60
80
100
120
0.00 10.00 20.00 30.00 40.00 50.00
SensiQvity (p
hot td-‐
1 s-‐3)
Age (months)
Figure 12 b.
Figure 12. Control (solid line) and subject (dashed line) developmental curves for c) cone sensitivity, and d) cone maximum response. Figure 12 c
55
4060
8010
012
0
Con
e O
ff R
espo
nse
Ampl
itude
(uV)
Controls Subjects Baseline
Cone off response amplitude did not display any effect of age in either subjects or
controls. In figure 11, box plots display the range (whisker to whisker), median,
(solid line through square), 25th and 75th percentile (bottom and top of square) of
cone off response amplitudes. Controls range from 39 uV – 87 uV (median67 uV).
Subjects range from 49 uV – 118 uV (median 83 uV).
Figure 13. Boxplot comparing cone off response amplitude in controls with IS subjects at baseline. Dots represent outliers (greater than two standard deviations away from the mean).
Data regarding visual acuity, drug dosage and other AED use in longitudinal tests is provided in table 11. Table 11. Longitudinal drug information and visual acuity in IS patients
STUDY ID
Test #
Age (mos)
Other AED meds
Dose of VGB VA (binoc) (logMAR)
VA test
VIE01 2 11.4 400 mg BID NT T
VIE02 2 15.0 800 mg BID No attention
VIE02 3 20.9
VIE04 2 15.7 TO, CO, LE 1875 mg per day > 1.6 T
VIE04 3 21.7 TO, CO, LE 1875 mg per day NR, fixes and follows
T
VIE04 4 25.3 TO, CO, LE 1875 mg per day >1.4 T
VIE05 2 9.7 CO, LE D/C 1.4 T
VIE05 3 14.1 CO, LE D/C 0.1 C
VIE07 2 12.8 750 mg bid 0.1 C
VIE07 3 18.9 750 mg bid 0.2 C
VIE08 2 19.8 635 mg bid 0.8 T
VIE08 3 24.1 NT
VIE08 4 29.3 D/C 0.1 C
VIE09 2 10.4 625 mg bid NT
VIE09 3 15.4 D/C 0.1 C
VIE10 2 13.2 1250 mg per day 0.4 C
VIE10 3 18.6 D/C 0.2 C
57
VIE11 2 22.4 1000 mg bid 0.2 C
VIE11 3 28.9 D/C 0.2 C
VIE12 2 17.9 TO 750 mg bid 0.8 T
VIE12 3 21.3 TO, CO 750 mg bid 0.9 T
VIE12 4 25.2 CO, VA 750 mg bid 0.8 T
VIE13 1 16.1 CO 750 mg bid 1.1 T
VIE13 2 21.1 250 mg bid NR
VIE13 3 27.3 D/C 1.6 T
VIE14 2 27.5 575 mg bid 0.8 T
VIE14 3 31.2 575 mg bid 0.2 C
VIE14 4 38.1 D/C 0.0 C
VIE15 2 9.9 450 mg bid NLP
VIE15 3 13.5 450 mg bid LP only
VIE15 4 17.2 CO 300 mg bid fixes, no following
VIE16 2 13.1 ACTH (d/c) 200 mg bid 1.0 T
VIE16 3 16.8 D/C 0.3 C
VIE16 4 19.7 D/C 0.2 C
VIE17 2 16.7 1000 mg bid 1.1 T
VIE17 3 20.1 NT
VIE17 4 24.4 1000 mg bid 0.0 C
VIE17 5 28.1 1000 mg bid 0.0 C
VIE17 6 35.9 1000 mg bid 0.2 C
58
VIE17 7 40.3 D/C 0.0 C
VIE19 2 10.2 750 mg bid 0.8 T
VIE19 3 13.6 750 mg bid 0.7 T
VIE19 4 18.0 250 mg bid 0.2 C
VIE19 5 21.9 D/C 0.0 C
VIE20 2 13.1 750 mg bid 1.4 T
VIE20 3 17.2 750 mg bid NR T
VIE20 4 22.1 VA 750 mg bid NR T
VIE21 2 9.0 PH 375 mg bid NT
VIE22 2 11.9 1320 mg per day 0.8 T
VIE22 3 15.9 CL, CZ 1320 mg per day 0.8 T
VIE22 4 20.4 CL, CZ 450 mg bid 0.1 C
VIE22 5 26.1 TO, CO 200 mg bid 0.1 C
VIE22 6 30.3 TO, CO, LE, VA
D/C 0.0 C
VIE23 2 9.9 650 mg bid 0.0 C
VIE23 3 15.6 D/C 0.0 C
VIE25 2 8.5 TO 400 mg bid NT
VIE27 2 17.5 TO, LE 375 mg bid NT
VIE27 3 22.0 TO, LE 200 mg bid no eye movement
VIE28 2 10.1 750 mg bid 0.2 C
Legend: TO – Topiramate; CO – Clobazam; LE – Levetiracetam; VA – Valproic Acid; ACTH –Adrenocorticotrophic hormone; PH – Phenobarbital; CL –
59
Clonazepam; CZ – Carbamazepine; bid – twice daily; NT – Not tested; cpd – cycles per degree; NLP – No light perception; LP – Light Perception; NR – No response; C – Cardiff; T- Teller
Age-adjusted values for flicker, cone S, cone Rmax and PhNR were calculated
using the method described in Methods section 3.0. Time was divided into
months on VGB), 12 months (11.51-13.50 months on VGB) and 15+ months
(13.51 +). The longest duration of VGB treatment included in this study was 28.5
months. Off VGB was also included as a category for those children who had ERGs
after stopping VGB treatment. Data are presented as box plots and change over
the course of drug treatment (not including ‘off drug’ time band) was compared
using ANOVA (figure 12 a-d). If there was no effect of the drug, one would expect
that the box plots would centre around zero.
60
Figure 14a. Box plot comparing adjusted flicker amplitude over time on vigabatrin. Dots represent outliers.
Adjusted flicker amplitude decreases over time (P=0.020). In those patients
treated with vigabatrin for 15 months or more, the median adjusted flicker
amploitude is 62uV less than expected for age (figure 12a). There appears to be
recovery in the median after the drug is discontinued, however this difference is
not significant in patients who were on the drug for at least 9 months before
discontinuing vigabatrin (P=0.2).
-100
-50
050
100
Adju
sted
Flic
ker A
mpl
itude
(uV)
Time on Vgb
Baseline 3 mos 6-9 mos12 mos 15 + mos Off vgb
Adj
uste
d Fl
icke
r Am
plitu
de (
uV)
61
Figure 14b. Box plot comparing adjusted PhNR amplitude over time on vigabatrin. Dots represent outliers.
Adjusted photopic negative response amplitude decreases over time (p=0.013)
(figure 12 b). There appears to be recovery when the drug is stopped, as the
median PHNR amplitude in those tested once they have stopped the drug only is 1
uV smaller than expected for age. The difference between the last test on the drug
and off the drug results is not significant for patients treated at least 9 months on
vigabatrin (P=0.6)
-100
-50
050
Adju
sted
PhNR
Am
plitu
de (u
V)
Time on Vgb
Baseline 3 mos 6-9 mos12 mos 15+ mos Off vgb
Adj
uste
d Ph
NR A
mpl
itude
(uV
)
62
-40
-20
020
40
Adjus
ted
Rmax
(uV)
Time on Vgb
Baseline 3 mos 6-9 mos12 mos 15+ mos Off vgb
Figure 14 c. Box plot comparing adjusted maximum response (Rmax) over time on vigabatrin. Dots represent outliers.
Adjusted maximum response increases over time (p = 0.004) (Figure 12 c). This
means that the amplitude is becoming larger over time. When the drug is
discontinued, the median value returns to close to what is expected for age,
however this difference is not significant (P=0.5). This may be due to a drug
effect, which is why it returns to value expected for age when the drug is stopped.
Adj
uste
d Rm
ax (
uV)
63
-50
050
100
150
Adjus
ted
Sens
itivity
(pho
t td-
1 s-
3)
Time on Vgb
Baseline 3 mos 6-9 mos12 mos 15+ mos Off vgb
Figure 14 d. Box plot comparing adjusted cone sentivity over time on vigabatrin. Dots represent outliers.
Adjusted sensitivity decreases over time on vigabatrin (p=0.027) (figure 12 d). In
those patients who are treated with vigabatrin for 15 months or more, the median
sensitivity is 62 phot td-1 s-3 less than expected for age. There appears to be some
degree of recovery when the drug is stopped but the median remains 30 phot td-1
s-3 less than expected for age at that time. The difference between adjusted
sensitivity at last test on the drug and off the drug for patients treated with
vigabatrin for at least 9 months is not significant (P=0.7).
Adj
uste
d Sen
sitiv
ity (
phot
td-1
s-3
)
64
For each response, linear models were used to investigate whether adjusted flicker
amplitude was associated with a normal or abnormal test at any point. An ideal
marker would be assosciated with decreased age adjusted flicker response.
Abnormal versus normal was delineated by establishing the 95th percentile of
distance from the developmental curve at baseline and rounding to the nearest
whole number for all markers except cone off response. As cone off response did
not demonstrate a developmental curve, the 95%ile was calculated based on raw
values. Values for abnormal cutoff points are listed below.
Flicker response was considered abnormal if amplitude was >42 uV less than
expected for age. Photopic negative response was considered abnormal if
amplitude was > 25 uV than expected for age. Cone maximum response was
considered abnormal if amplitude was >24uV than expected for age. Cone
Sensitivity was considered abnormal if value was >40 scot td-1 s-3 less than
expected for age. Cone Off response was considered abnormal if amplitude was
<40 uV. In all cases, where cone off response was abnormal, the peak was
unmeasureable (Figure 15).
65
Figure 15. Cone off response in normal and abnormal test. Left panel shows normal cone off response ( 65 uV). Right panel shows abnormal cone off response in an Infantile Spasms patient.
Abnormal cone off respones were significantly assosciated with reduced age
expected flicker amplitude (p<0.001) (figure 14a). The median adjusted flicker
amplitude in those with an normal cone off response is 0uV (expected for age),
whereas in those with an abnormal cone off response the median adjusted flicker
is -50uV. Furthermore, the maxiumum adjusted flciker response in those patients
with an abnormal cone off response is – 20 uV.
0 100 200 300
-100
-50
0
50
100
Time (ms)
Am
plitu
de (
uV)
0 100 200 300
-100
-50
0
50
100
Time (ms) Am
plitu
de (
uV)
66
Figure 16a.
Adjusted Flicker amplitude for patients with normal vs abnormal cone off response
Adjusted flicker amplitude is not associated with a normal or abnormal photopic
negative response amplitude (P=.49) (figure 16b), or cone maximum response (P
=0.25) (figure 16c).
Figure 16b.
Adjusted Flicker amplitude for patients with normal vs abnormal photopic negative response
Adj
uste
d Fl
icke
r Am
plitu
de (
uV)
Cone Off Response
Adj
uste
d Fl
icke
r Am
plitu
de (
uV)
Photopic Negative Response
67
Figure 16c.
Adjusted Flicker amplitude for patients with normal vs abnormal Cone Rmax
Decreased adjusted flicker amplitude is significantly associated with abnormal
cone sensitivity (P<0.001) (figure 16 d). The median adjusted flicker amplitude in
patients with abnormal cone sensitivity is -45uV, where as in patients with normal
cone sensitivity, median adjusted flicker is 0uV (expected for age).
Figure 16d. Adjusted Flicker amplitude for patients with normal vs abnormal cone Sensitivity.
Adj
uste
d Fl
icke
r Am
plitu
de (
uV)
Cone Rmax
Adj
uste
d Fl
icke
r Am
plitu
de (
uV)
Cone Sensitivity
68
Table 12 is a comparison of each marker’s ability to detect abnormal tests, using
the guidelines set forth above. Patients were only considered abnormal if they had
abnormalities beyond baseline. Therefore, patients were only included if they had
at least two tests (Eight patients excluded).
Table 12. Diagnostic characteristics of abnormal tests using flicker, cone off, and sensitivity Legend: pts – patients
Flicker response identified, 46% of patients (26 % of 81 tests) as abnormal, cone
off identified 29% of patients (14% of 49 tests) and sensitivity identified 46% of
patients (24% of 76 tests) as abnormal.
In the one patient who recovered cone sensitivity once the drug had been stopped,
sensitivity was still reduced (-34 phot td-1 s-3) compared with that expected for
Time of peak abnormality 9 months 9 months 6-9 months
# Abnormal at baseline 3 0 1
Pts with 2 consecutive Abnormal tests
5 2 4
# with abnormal test followed by normal test
3 0 4
Maintain abnormality once drug stopped?
Yes (4) Yes (5) Yes (4), No (1)
69
age. The patient who had an abnormal sensitivity test at baseline was not one of
the three patients with an abnormal baseline flicker.
Figure 17. shows survival curves for each marker up to 15 months.
Cone off response has no abnormal tests at baseline and reaches a 69% survival
rate at 15 months. Flicker is 5% abnormal at baseline and reaches 64% survival
rate at 15 months. Cone sensitivity is 5% abnormal at baseline and reaches 48%
survival at 15 months.
70
Figure 17. Survival plots for cone off response (top), flicker amplitude (middle) and cone sensitivity (bottom). Solid lines indicate survival curves. Dashed lines indicate 95th confidence intervals. Y-axis represents proportion ‘survived’, in this case with normal test. X-axis is months on vigabatrin.
0 2 4 6 8 10 12 14
1.0 0.8 0.6 0.4 0.2 0.0
1.0 0.8 0.6 0.4 0.2 0.0
1.0 0.8 0.6 0.4 0.2 0.0
71
Mosaic plots were created to represent the overlap between the three markers in
classifying a particular patient’s test as normal or abnormal.
2 1 Normal 1 Abnormal
11 (a) 38 (d) 38 (b)
0 (c) 0
Figure 18. Venn Diagram of the number test points where patients had overlap between each test. Flicker and cone off (a), flicker and sensitivity (b), sensitivity and cone off (c), or all three tests (d) conducted.
In each mosaic plot, the bottom line represents one marker, in the first plot (figure
19a), flicker. The square is divided vertically into two sections. The left hand
section represents the proportion of tests that had a normal flicker response and
the right hand section represents the proportion of tests that had an abnormal
flicker response. On the right hand of the square cone off response is represented.
The two boxes that begin at the top side of the square had an abnormal cone off
response and the two boxes that meet the bottom side of the square had a normal
cone off response. In this way, four categories are created.
1) Normal Flicker – Normal Cone Off (Blue): 73%
2) Normal Flicker – Abnormal Cone Off (Green, top left): 11%
3) Abnormal Flicker – Normal Cone Off (Green, bottom right): 3%
4) Abnormal Flicker – Abnormal Cone Off (Yellow): 13%
Flicker
Sensitivity
Cone off
72
From this plot, it is also clear that of all patients who had both flicker and cone off
response tests performed:
a) Flicker was normal in 84% of cases and abnormal in 16%.
b) Cone off response was normal in 76% and abnormal in 24%.
Figure 19. Mosaic plots of agreement in classifying tests between (a) flicker and cone off, (b) flicker and sensitivity, (c) cone off and sensitivity, (d) flicker, cone off and sensitivity.
Abnormal
24%
Cone Off
76%
Normal 84% 16% Normal Flicker Abnormal
Figure19a.
73%
13%
3%
11%
73
Abnormal
20%
Cone Sens
80%
Normal
80% 20% Normal Flicker Abnormal
Abnormal
14%
Cone Sens
86%
Normal
75% 25%
Normal Cone Off Abnormal
Figure 19b.
9%
16%
5%
70%
Figure 19c.
10%
10%
10%
70%
74
Figure 19d. shows the agreement between all three tests. This plot is the same as the others except that there are two columns of normal and abnormal cone sensitivity, creating four columns. In the case where flicker response was normal and cone off response was abnormal, each test had a normal sensitivity; this explains why there is only one column in that section.
Sensitivity
Normal Abnormal Normal Abnormal
71% 19%
Abnormal
16%
Cone Off
84%
Normal
76% 24% Normal Flicker Response | Abnormal
Figure 19d
61% 13%
5% 5%
11% 2.5%
2.5%
75
Between all three markers, there is perfect agreement in 72% of tests. For cone
off and flicker, sensitivity and flicker, and sensitivity and cone off, there is 86%,
80% and 79% agreement between markers respectively.
Case Reports
Table 13 illustrates results of flicker, cone off and cone sensitivity for all patients
who had at least one abnormal test. All patients, with the exception of one
(patient 8), were initiated on vigabatrin within 3 weeks of seizure onset. In patient
8, there was a 3 month delay because they were being seen at another centre
where the type of seizure was not identified. Of the twelve patients who showed
any abnormal tests, only four had been treated with other AED’s (patient 13 -
compared to 12 of twenty patients without abnormal test who were taking other
AEDs (see table 9 & 11).
76
Table 13. Individual patient data for all those with at least one abnormal test
0 3 6 9 12 15 18 21 24 27
F
1 O
S
F
2 O
S
F x
8 O x
S x
F x
9 O x
S x
F x
10 O x
S x
F
12 O
S
F x
13 O x
S x
77
F
15 O
S
F x x
16 O x x
S x x
F
17 O
S
F x
19 O x
S x
F
27 O
S
F
28 O
S
All patients with at least one abnormal test on at least one of the three markers were included except for two patients (number 14 & 29) who were excluded because their only abnormal test was abnormal flicker at baseline.
Legend: F – flicker; O - cone off; S - cone sensitivity; Green colouring identifies a normal test result; red identifies an abnormal test result; White spaces identify that a test was not done; X’s indicate the patient was off vigabatrin at that time.
78
Table 14 below shows the distribution of sex for normal and abnormal groups for
different criteria (no values are significantly different by a t test between normal
and abnormal for each definition).
Table 14. Distribution of sex and mean daily VGB dose for normal vs abnormal test using different definitions of abnormality
% Male Mean Daily VGB dose (mg/d)
Abnormal (Ab) Definition
Abnormal Normal Abnormal Normal
Any Ab tests 9/13 = 61% 5/10 = 50% 1240 1305
Ab by flicker (excluding Baseline
only)
5/8 = 62% 9/15 = 60% 1200 1300
Ab for Cone off 5/7 = 71% 9/16 = 56% 1210 1290
Ab for Sens 7/ 11 = 64% 7/12 = 58% 1200 1330
79
Discussion 11The present study investigated four potential new electrophysiological markers of
changes due to vigabatrin: photopic negative response, cone sensitivity, cone
maximum response and cone off response. These measures were compared to the
3.0 flicker amplitude, which is currently the most sensitive measure of Vigabatrin
retinal toxicity. The major findings were that both cone sensitivity and cone off
response were negatively affected by Vigabatrin use over time and were correlated
with results from 3.0 flicker amplitudes. Cone maximum response was not altered
significantly with drug treatment and changes in the photopic negative response
were not related to changes in the 3.0 flicker amplitude. It is still unclear which
response may be the best marker of change but the cone off response represents
a promising marker because it is not abnormal at baseline and does not return to
normal after having an abnormal test. The cone off response identifies 30% of
patients as abnormal, a value similar to the estimated 34% of children who
experience visual field loss, (Maguire et al., 2010).
It has been widely established that in adult patients taking vigabatrin, visual field
loss and some degree of visual function loss occurs. While correlations between
visual field loss and retinal dysfunction have been problematic even in an adult
population, it is clear that there are major changes that happen to the
electroretinogram in some patients taking vigabatrin in both adults and children.
Vigabatrin continues to be used as a first-line treatment for Infantile Spasms and
80
its use has become even more widespread since it’s reintroduction into the
American market in 2009. The use of the electroretinogram to monitor visual
function in pediatric vigabatrin users has been agreed upon by many, including in
the official REMS strategy, however the most appropriate and reliable marker has
not been agreed upon.
It has become clear in research done by our lab that infants with IS may have
some degree of altered visual function, even before initiating vigabatrin (Mirabella
et al., 2007; McCoy et al., 2011; McFarlane et al., 2011). Thus, the ability of our
lab to take baseline measures in these children is key to the delineation of the
effects of seizures versus that of the drug.
This study also described the development of photopic negative response and cone
off response in retinally normal control patients and Infantile Spasms patients,
which has not previously been done. As well, while cone sensitivity and cone
maximum response have been studied in normally developing infants up to 10
weeks (Hansen & Fulton, 2005), they have not been studied previously in IS
patients as demonstrated in this study or normal controls from 10 weeks to 4
years old.
Cone off response and cone sensitivity have not been studied in adults taking
vigabatrin or children old enough to reliably conduct visual field testing, so it is
difficult to know if these changes are related to loss of visual fields. However,
81
though we cannot currently know whether these changes are directly responsible
for visual field loss, it is clear that there are significant, lasting defects in these
parameters in some patients taking vigabatrin.
The loss of cone off response could be explained by damage to the cone OFF
bipolar pathways. Off bipolar cells respond to glutamate by depolarizing. The loss
in function could either represent a blockage or alteration in synaptic transmission
from photoreceptors to off bipolar cells, or by direct damage to bipolar cells
themselves.
Reduction in PhNR amplitude, over time might also be contributing to dysfunction
at the level of the bipolar cells. Changes in adjusted PhNR amplitude may not be
related to changes in flicker because a lag exists between initial bipolar cell
damage and the downstream effect on ganglion cells.
Decreases in cone sensitivity, but not cone maximum response, suggests that
vigabatrin affects primarily the process of phototransduction and may not, at least
in the early stages, be the cause of degenerating cones. Cone degeneration has
been seen in animal models, however these studies employ more acute doses and
sensitivity of cones has not previously been reported in these vigabatrin treated
animals. In normal controls aged 8-40 years, cone sensitivity values were found
to range from 55 – 120 phot td-1 s-3, with a mean (+ SE) of 81 + 5.5 phot td-1 s-3
by Hansen and Fulton (2005). Although in our study, toxicity was defined as a
82
difference from age expected value, it is notable that the absolute values are also
well below these values (mean = 6.03 phot td-1 s-3). There are no normal values
for children aged 4 months to 48 months. However, in infants 10 weeks old,
Hansen and Fulton demonstrated average cone sensitivity to be 59 + 3.9 phot td-1
s-3 and the mean sensitivity in our control population was 52.37 phot td-1 s-3. It is
puzzling that the control patients did not experience a significant effect of
development, while the IS patients did. This is probably related to the small
sample size of controls (n=10). As well, the cone sensitivity was greatly reduced in
the youngest of infants with IS and the developmental curve will reflect a catch up
in early infancy.
That fact that alterations in cone off response and cone sensitivity are related to
changes in the 3.0 flicker amplitude is consistent with work by Bush and Sieving
(1996). Bush and Sieving conclude that along with the contributions of cone
photoreceptor potentials, post receptoral cells that normally produce the b and d
waves, i.e. On and Off Bipolar cells, are strong contributors to the photopic fast
flicker response. It was also noted that the flicker response is independent of inner
retinal responses.
There is not complete agreement between the flicker amplitude, cone off response
and cone sensitivity in identifying abnormal tests; it is still unclear which is the
optimum marker for diagnosing true vigabatrin-induced retinal toxicity. While
83
flicker is the only test that has been correlated to visual field loss in adults with
100% sensitivity, it has only been shown to be 75% specific (Harding et al.,
2000b). This suggests that flicker amplitude may over-diagnose visual field loss.
This might also explain why some patients have an abnormal flicker test followed
by a normal test. If this is true, it is promising that the cone off response identifies
fewer patients as abnormal (29% vs. 46% for flicker and cone sensitivity). While
there is overlap in the patients in which both flicker and cone off response are
deemed abnormal (13% of cases), in the cases where only cone off response is
abnormal (11%), it may be that cone off response is identifying damage earlier
(i.e. VIE 9 and 28). It may also be that the cone off response is under-diagnosing
the problem. It is difficult to confirm this for two reasons: the lack of visual field
testing and the lack of a complete data set for all patients (cone off response
protocol only performed from June 2010 – June 2011). Further research is needed
to confirm this.
The question of the true mechanism of vigabatrin-induced retinal toxicity still
remains. It would be ideal to have a marker of the first or direct source of toxicity
in these infants, however, any marker that correlates to visual field loss will be
useful in screening IS patients on Vigabatrin. As visual field testing is not currently
feasible in this population, it is interesting to consider what these results indicate
about a possible mechanism of toxicity.
84
Animal studies have identified two key phenomena in the development of retinal
damage with vigabatrin use. First, it is clear that light exposure is key to the
development of retinal dysfunction in this population. This has been shown in both
rats (Butler et al., 1987; Izumi et al., 2004; Jammoul et al., 2009) and mice
(Jammoul et al., 2009). As well, it seems that taurine levels may play some role in
the toxicological mechanism.
In adult (Jammoul et al., 2009), and neonatal rats (Jammoul et al., 2010) treated
with vigabatrin who develop retinal dysfunction, taurine levels have been shown to
be depleted. Jammoul also demonstrated that six Vigabatrin–treated IS patients
ranging from 8.5 months – 3 years of age had reduced taurine levels. Visual fields
and ERG findings were not presented for these patients, therefore it is unclear
whether reduced taurine is related to decreases in retinal function (Jammoul et al.,
2009). It has been suggested that taurine levels are normal pre-treatment and are
reduced by vigabatrin treatment, however this has only been shown in one patient
with Infantile Spasms (Jammoul et al., 2009). It is unclear what role the
reduction of taurine levels plays in the toxicological mechanism. It may be that
either:
a) Vigabatrin à Decreased taurine à Retinal damage
b) Vigabatrin à Retinal damage à Decreased taurine
85
In mechanism (a), it is hypothesized that an increase in GABA could decrease
taurine levels as GABA is a competitive inhibitor of the taurine transporter (Lee
and Kang, 2004; Jammoul et al., 2009) or VGB may directly affect taurine uptake
and release. Alternatively, taurine levels might be reduced as a result of decreased
taurine synthesis via a decreased cysteine pool. This would involve increased
enzymatic conversion, or by use of taurine as a free radical scavenger. For
example, if antioxidant glutathione levels were reduced as a result of oxidative
stress, this would lead to a reduction in cysteine, resulting in reduced taurine
synthesis (Hayes & Sturman, 1981). In Sprague-Dawley rats, dietary taurine
supplementation decreases malondialdehyde levels in the retina, and increase
retinal taurine levels, superoxide dismutase and glutathione peroxidase. This
supplementation prevented photochemical damage caused by fluorescent light.
Supplementation with taurine in VGB treated adult and neonatal rats partially
prevented retinal lesions (Jammoul et al., 2009, 2010).
It is clear that in general, there are no damaging effects of natural light exposure
to the eye / retina, as light serves as a key component in the process of vision and
is not known to cause retinal toxicity in healthy normal eyes (Roberts, 2001). In
this review, Roberts identifies seven factors that may affect whether light is
damaging:
a) Intensity
In general, the greater the intensity, the more likelihood a light will damage the
86
eye. Cumulative light damage has been generally recognized to occur as a result
of lower long term exposure when there is a loss of protective mechanisms in the
eye (often because of age).
b) Wavelength
Shorter wavelengths have greater potential to cause damage. The young human
retina may be exposed to light as short as 320nm-400nm. If light exposure plays
a role, it may be expected that higher levels of phototoxicity occur in areas that
and increased vitreal taurine levels in only albino Wistar rats and not Long – Evans
pigmented rats.
Bulley & Shen (2010) noted that off bipolar cells in salamander retina may release
taurine as well as glutamate. Taurine was found primarily in the Off bipolar
terminals in the IPL, but not amacrine or ganglion cells. It is believed that taurine
suppresses glutamate-elicited Ca2+ in third order neurons by ionotropic glutamate
receptors. Decreased taurine levels may account for bipolar cell abnormalities. If
so, taurine supplementation may help to recover bipolar cell function even after
the drug has been stopped.
Not all patients who receive vigabatrin develop retinal toxicity. Several
mechanisms could be responsible:
a) Dietary taurine levels
b) Light exposure levels
89
c) Metabolizing enzymes
d) Vigabatrin dose
e) Genetic background factors
f) Any combination of these factors
Dietary taurine levels and light exposure levels may affect the susceptibility of
patients to vigabatrin retinal toxicity. It may be that a combination of low dietary
taurine intake, a retinal antioxidant, and increased light exposure, an oxidizing
agent, lead to an increased chance of vigabatrin damage. Retinal cells would need
to be sensitized to the light damage by something other than low taurine levels. It
is unlikely that low taurine levels themselves would sensitize retinal cells to
phototoxicity; if this were the case we would expect to see these visual field defect
in malnourished but non vigabatrin treated children and adults.
A difference in metabolizing enzymes could explain the effects in targeted retinal
cells and would fit with the premise of oxidative damage. Clinical pharmacological
studies have identified that between 60-80% (Schechter, 1986; Haegele &
Schechter, 1986; Rey et al., 1990) of the active S enantiomer of vigabatrin is
excreted unchanged in urine. If vigabatrin were undergoing retinal metabolism,
the product of this reaction may account for the 20-50% of vigabatrin that was not
detected in previous pharmacokinetic studies. The structure of vigabatrin does not
appear to be a prime suspect for ocular phototoxicity, however metabolic
90
conversion could change that. It is plausible that vigabatrin undergoes local retinal
metabolism to convert it to a toxic metabolite, which sensitizes retinal cells to light
damage. A similar mechanism happens in methanol toxicity.
Methanol is converted to formaldehyde by alcohol dehydrogenase, which is further
converted to formic acid by aldehyde dehydrogenase (AHD-2), an enzyme that
metabolizes retinaldehyde to retinoic acid in the normal human retina. When
formate (formic acid salt) accumulates, retinal toxicity may occur. After
biotransformation, a combination of direct oxidative stress at the Muller cell,
combined with decreased anti-oxidants enzyme activity, leads to methanol-
induced retinal toxicity. The Muller cells are believed to be the site of
biotransformation and initial insult (Garner, Lee, & Louis Ferdinand, 2002). This
may be because a unique aldehyde dehydrogenase isoform, AlDH-2, has been
shown to be present almost exclusively in the Muller cells in the adult retina of
mice. Levels of this enzyme were highest in dorsal retina, with still many in the
temporal peripheral retina and very few in the central retina (McCaffery, Tempst,
Lara, & Drager, 1991).
A decrease in ATP is seen after methanol administration in Folate-reduced rats,
which has been shown to be a good model of human methanol toxicity (Eells,
Henry, Lewandowski, Seme, & Murray, 2000), and this corresponds to changes in
the ERG (Garner & Lee, 1994). Decreases in the b-wave and loss of potassium
91
induced Muller cell depolarization were similar between methanol treated folate
reduced rates and alpha AAD (a Muller cell toxin) in folate reduced rats. Seme,
Summerfelt, Henry, Neitz & Eells (1999) showed the ERG flicker amplitude of M-
cones, cone that respond to medium wavelengths of light (450-630nm), decreased
in a formate concentration and time dependent manner. In FR methanol treated
rats, reductions of glutathione have also been reported Rajamani, Muthuvel,
Senthilvelan, & Sheeladevi (2006). Formic acid also inhibits cytochrome oxidase, a
mitochondrial enzyme, in cultured Muller cells (Eells et al., 2003). This arrests
electron transport chain activity (Nicholls, 1975, 1976), which in turn stops
regeneration of ATP and thus leads to cell death (Treichel, Henry, Skumatz, Eells &
Burke, 2004). Photoreceptors and the RPE also accumulate formate and cytotoxic
effects are seen in both types of cells (Treichel et al. 2004b). It has been
postulated that photoreceptors undergo more methanol toxicity than RPE because
they have higher level of antioxidant enzymes including catalase.
A moderating effect of light has not been seen in methanol toxicity. It is possible
that because methanol toxicity happens quite quickly, the mediating effects of
light exposure would not be observed. There is typically a latent period between
ingestion and symptom initiation followed by ocular symptoms that accompany the
systemic symptoms of methanol toxicity within 48 hours.
92
ERG effects of methanol are not the same as those observed in this study due to
vigabatrin. To understand what type of metabolism might turn vigabatrin into a
compound to sensitize retinal cells to oxidative damage, it could be useful to
understand the structure and property of other molecules that demonstrate similar
ERG effects. One other toxin, D-alpha aminoadpic acid (D-α AAA), which is a
glutamate analogue, although not subject to retinal metabolism, shows a similar
pattern of ERG changes. When D-α AAA is intravitreally administered to carp
retina, a reduction in glutamine synthetase activity, which is exclusively localized
to Muller cells, occurs within hours (Kato, Sugawara, Matsukawa, & Negishi,
1990). Of note, of the three isomers of α AAA (D, DL, L), D caused the least
reduction (28% vs. 45-65%) in GST activity. D-α AAA also caused the least
difference in the protein profile of the retina compared to L and DL. D-α AAA
caused a reversible decrease in the ERG b-wave and an increase in the a-wave
(Kato et all, 1990). In further experiments in Mudpuppy retinae, 5 mM of D-α AAA
preferentially reduced the d-wave (versus b-wave) of the ERG and the off
response of the Muller cells, but did not cause Muller cell damage (Zimmerman &
Corfman, 1984). L-α AAA however, caused preferential reversible b-wave and on
response reduction and was accompanied by sustained histological damage to
Muller glial cells. It is believed that D-α AAA may act as an antagonist to synaptic
receptors in the “off” pathway. As this xenobiotic selectively reduced the d –wave
93
(and also causes an increase in the a-wave, similar to the increase in maximum
response in this study), it is interesting to consider the structure, see figure 20.
D- alpha amino adipic acid vigabatrin
Figure 20. Structure of D- alpha amino adipic acid (left) and vigabatrin (right).
Bipolar cells primarily have two ionotropic glutamate receptors: AMPA which is
suppressed by AMPA antagonist GYKI 52466 (a 2,3-benzodiazepine) and Kainate
which is suppressed by SYM2081 (4-methyl glutamic acid), see figure 21.
4-methyl glutamic acid GYKI 52466
Figure 21. Structure of 4-methyl glutamic acid (left) and GYKI 52466 (right).
Given that D-AAA is more similar to 4-methyl glutamic acid than GYKI 52466, and
this structure is also much more similar to vigabatrin vigabatrin (or its metabolite)
might suppress the kainate receptor of bipolar cells.
94
Retinal metabolism may occur by CYP1A1/1A2, CYP4A, MOA A and B and retinal
specific enzymes ‘retinal amine oxidase’ (RAO) and xanthine oxidase (oxidative
stress in retinopathy). RAO is a human retinal specific enzyme, encoded by the
gene ACO2, and known to have an alternatively spliced variant which can lead to
different isoforms (Imammura et al., 1998), converts amines to aldehydes and
ammonia.
R–CH2–NH2 + O2 + H2Oà R–CHO + H2O2 + NH3
This could convert vigabatrin to 4-oxo 5-hexenoic acid and simultaneously create
hydrogen peroxide. While this step in itself would not lead to a structure similar to
those of D-AAA or 4-methyl glutamic acid, further metabolism of vigabatrin could
do that, with the initial RAO metabolism stage causing oxidative damage, and
further metabolism leading to bipolar cell signal blockade.
RAO is localized in mouse models to retinal ganglion cells (Imammura et al., 1998)
though no ganglion cell staining was detected when similar techniques were used
in human retinal cells. Authors noted as that when assessing AOC-2-like SSAO
activity, the retinal samples exhibited dramatic individual variation (Kaitamieni et
al., 2009).
The initial insult may be at the level of the cone off bipolar cell due to retinal
metabolism by RAO (or other retinal specific enzymes), and photoreceptor damage
results more from oxidative damage (as photoreceptors are very sensitive to this)
95
after sensitization. This would potentially make cone bipolar cells an earlier marker
of VGB toxicity. More research is needed to investigate this potential theory.
11.1 Clinical Implications
This study highlights several important issues for clinicians treating children with
Infantile Spasms on vigabatrin. The first is that abnormalities appear in these
children as early as three months of age. It was also demonstrated that some
children did not show an abnormal test result until coming off vigabatrin. These
two findings give further support to our current protocol of testing within three to
four months after the baseline test, and testing after the drug was discontinued. It
is important to assess each child as early as possible after an abnormal test.
Currently, because of the variability in the flicker response, clinicians must wait
until two tests have been conducted, at an average of four months apart; until
they can confidently say the child has an abnormal ERG. Ideally, we would see
children with an abnormal ERG within one month after the first abnormal test. This
however is unrealistic given the requirements for a sedated ERG (orthoptist, ERG
time, sedation nurse, physician assessment etc.). In this study, there were no
cases where the cone off response was abnormal and went on to be normal again.
If it can be confirmed that the cone off response is a valid marker of toxicity then
clinicians would not need to wait for two tests (four months) in order to diagnose
vigabatrin attributed retinal toxicity. It is however still unclear if stopping the drug
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after an abnormal cone off response test would lead to a reversal, or stop
progression of damage due to vigabatrin and therefore further research is needed.
11.2 Problems and Considerations
One of the major difficulties in using the cone off response as a marker is the
propensity for infants and children (if not sedated) to blink over the course of the
200 ms light stimulus. A blink would result in a different waveform, which might
alter the amplitude of the response. To use the cone off response as a valid
marker, the average waveform should return to the level of the a-wave to ensure
that the amplitude of the off response is most accurate (Horn et al., 2011). In our
data, even in those cases in which the waveform did not return to the level of the
a-wave at 200 ms, a d-wave could still be easily identified, however these tests
were still excluded. As a result, the number of useable recordings was
approximately halved. The reduction in useable tests because of blink artifacts
could present a problem if this response was used as a marker in these infants. In
this study, we only performed 3 repetitions of the cone off response. The minimal
amount of repetitions had to be used in order to comply with ethical standards in
the hospital of minimizing the time a child is sedated. If this response were added
to the clinical protocol for vigabatrin monitoring, it would be prudent to allow more
than 3 repetitions to ensure that some recordings could be made without blinks.
As well, it may be worth moving the step earlier in the photopic sequence, as the
infants eyes may be less tired or aversive to the light, and less likely to blink.
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Another problem with the cone off response as a potential marker is that it thus
far appears to identify non-reversible changes due to vigabatrin, as after one
abnormal test, the cone off response remains abnormal and does not recover after
the drug has stopped. It is noted that the longest time after drug cessation that a
child may be seen in our clinic is 1.5 years, it is possible that after this point some
recovery may be seen. More importantly, while the changes may not be
permanent, early identification may be able to stop the progression of these
changes. If the functional changes are as a result of damage by free radicals, early
identification could signal the need for treatment with anti-oxidants and / or
vigabatrin cessation. This may halt further structural changes.
Lastly, the study was hindered by the use of a convenience sample. Our recruiting
pool existed of all IS patients referred to SickKids for ERG testing. We were unable
to ask children to come in for extra visits to monitor them more frequently, nor
were we able to recruit from outside of SickKids. This was true as well for normal
controls, where we could only approach those patients who were already
scheduled to be seen for ERG testing to participate. This is primarily due to the
risk and cost associated with sedation, which is necessary for the procedure. These
IS patients included in this study will continue to be studied to gain more
longitudinal data. This study supports a need for seeing children, especially those
who have an abnormal test, more regularly than every 3-4 months.
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11.3 Future Directions
This study provides preliminary data to support the use of the cone off response as
a marker of retinotoxic changes due to vigabatrin. Further studies at our centre
and others, are needed to confirm in a larger population if cone off response is
indeed a preferable marker to 3.0 flicker amplitude.
Another step in validating the cone off response as a marker of changes due to
vigabatrin is to recall these study patients when they are four years of age and
older. These children would undergo repeat ERG’s and visual field testing when
they are old enough to complete behavioural visual field tests. This would allow
correlation between visual field loss and ERG results and give information as to
whether ERG dysfunction can predict visual field dysfunction.
It would also be prudent to investigate changes in the cone off response in adults
with CPS taking vigabatrin. In these patients, ERG responses could be serially
measured with visual fields to see whether cone off response or cone sensitivity
dysfunction correlate with visual field loss.
Optical coherence tomography could also be used to investigate early structural
changes in IS patients. Early damage to middle retinal layers on OCT would help to
validate the cone bipolar cells as a marker. There are similar difficulties in testing
infants with OCT as with visual field testing. One group has used a technique in
which sedated infants are held up to the OCT for testing, however this is not
99
commonly performed and results did not correlate entirely with ERG results (Mets
et al., 2011).
Further support could be garnered for this theory if it were established that
vigabatrin was metabolized by retinal enzymes and by investigating where these
enzymes are localized. Local retinal metabolism should be studied in animal
models. Some enzymes have been found only in human retinas and this would
make the problem more difficult to investigate. Histological studies of deceased
vigabatrin users may help to discover which enzymes might be involved.
Identification of specific enzymes in either animal or human specimens could lead
to a target for genetic screening. It is clear that there is often genetic variation in
enzymes, including drug metabolizing enzymes. Genetic polymorphisms can be
identified using directed screens of different genes. Genetic polymorphisms may
either be responsible for a patient developing toxicity, or may make them more
susceptible to damage. It could be any combination of specific isoforms of an
enzyme, a certain threshold of light exposure, reduced taurine intake and
increased vigabatrin doses that are related to developing toxicity. Until it is clear
whether retinal enzymes play a role in vigabatrin metabolism and whether they
are susceptible to genetic variation, this theory cannot be confirmed.
Lastly, controlled studies in IS patients investigating the effects of taurine
supplementation and decreased light exposure on the development of vigabatrin
100
associated retinal dysfunction would help to understand the mechanism of toxicity
in humans.
In the interim, it is important to continue monitoring flicker response, cone off
response and sensitivity.
101
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Rey, E., Pons, G., Richard, M. O., Vauzelle, F., D'Athis, P., Chiron, C., . . . Olive, G. (1990). Pharmacokinetics of the individual enantiomers of vigabatrin (gamma-vinyl GABA) in epileptic children. British Journal of Clinical Pharmacology, 30(2), 253-257.
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Riikonen, R., & Donner, M. (1980). ACTH therapy in infantile spasms: Side effects. Archives of Disease in Childhood, 55(9), 664-672.
Riikonen, R. S. (2000). Steroids or vigabatrin in the treatment of infantile spasms? Pediatric Neurology, 23(5), 403-408.
Roberts, J. E. (2001). Ocular phototoxicity. Journal of Photochemistry and Photobiology.B, Biology, 64(2-3), 136-143.
Roccella, M., Parisi, L., & D’Iapico, N. (2001). Analisi del campo visivo nei bambini con epilessia parziale in monoterapia con Vigabatrin. Boll Lega It Epil, 114, 243–244.
Roubertie, A., Bellet, H., & Echenne, B. (1998). Vigabatrin-associated retinal cone system dysfunction. Neurology, 51(6), 1779-1781.
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Appendices (if any)
Appendix 1. List of current lab protocol
For all steps: Background colour: White Stimulus colour: White
Stimulus Intensity
Sweeps/result
Time between results
Background Intensity
Adaptation Time
Unit cd*s/m2 ms s cd*s/m2 s
1 0.00039 8 2 0p
2 0.00151 8 2 0p
3 0.00245 10 2 0p
4 0.00632 8 2 0p
ISCEV (1)
5 0.01578 6 5 0p
6 0.04 6 6 0p
7 0.097 6 10 0p
ISCEV (2)
8 2.291 6 15 0p
9 7.6 4 15 0p
10 10 3 30 0p
ISCEV (4)
11 2.291 15 2 29 600
12 4.1 20 2 29
ISCEV (5) -
flicker
13 2.291 30 0, continuous phase
29
14 10 8 2 29
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Appendix 2. Vigabatrin and Infantile Epilepsy Subject Consent Form
THE HOSPITAL FOR SICK CHILDREN
Department of Ophthalmology
Visual Electrophysiology Unit
Phone (416) 813-6516
Hospital for Sick Children (SickKids)
RESEARCH CONSENT FORM (For Parents of Subjects Prescribed Vigabatrin)
Title of Research Project: Vigabatrin and Infantile Epilepsy
Investigators
Director of Electrophysiology: Dr.Carol Westall 416-813-6516
Responsible Individual: Dr. Carol Westall 416-813-6516
The drug vigabatrin is used to help control seizures. In some people the drug might cause problems with vision. This might be related to some small changes of the retina. The retina is the inner lining of the eye that makes a picture of what we see (like film in a camera).
We want to better understand what is happening to the eyes in children on vigabatrin. The following tests may be performed before, during vigabatrin therapy and after its withdrawal.
The electroretinogram (ERG) is an electrophysiological test to measure the electrical response of the retina. ERGs are a routine clinical test used to assess retinal function when a retinal disease is suspected or known. We want to better understand what is happening to the eyes in people undergoing vigabatrin treatments.
Description of the research:
The following tests will be performed once your child’s neurologist or ophthalmologist has referred them to the Visual Electrophysiology Unit and an appointment has been made. Sedated ERGs are clinically indicated for children with Infantile Spasms taking the antiepileptic vigabatrin to find out whether any changes to the retina have taken place. The tests will take one hour to be performed.
Electroretinogram (ERG): This study will involve the addition of two extra steps to clinic protocol. These extra steps are intended to isolate the response from a specific part of the retina and will extend the testing time by approximately 1 minute.
Patient’s health records will be reviewed for purposes of this study for information about drug history, co-morbidities etc. Standard clinic intake tests may be performed including vision, colour vision, refractive error, and / or ophthalmoscopy.
Potential Harms (Injury, Discomforts or Inconvenience):
There are minimal harms associated with participation in this study. Under exceptional circumstances there is a slight risk that your child may receive a minor scratch to the front of her/his eye. This scratch would feel similar to having a piece
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of sand in your eye and the discomfort may last for 2-3 days. We will check for this and provide any necessary treatment. The eye drops cause slight stinging, but this resolves within 10 seconds. The drops which we use to dilate your child’s pupils may cause his/her vision be blurred up close for 4-8 hours. The risks involved in this study are no greater than those for normal clinic protocol.
Potential Benefits:
To the individual: Your child may not benefit directly from participating in this study. Ophthalmological and neurological care will continue whether your child continues in this study or not. Our research team will send you a letter detailing our findings when the study is completed.
To Society: Knowledge gained from this study will hopefully allow physicians to optimize vigabatrin therapy to ensure patients receive the maximum benefit whilst minimizing visual toxicity. Better control of the risks associated with this powerful therapy will make its use in a wider patient population more feasible.
Confidentiality:
We will respect you and your child’s privacy. No information about who you are will be given to anyone or be published without your permission, unless the law makes us do this.
For example, the law could make us give information about you • If a child had been abused • If you have an illness that could spread to others • If you or someone else talks about suicide (killing themselves), or • If the court orders us to give them the study papers
Sick Kids Clinical Research Monitors or the regulator of the study may see your health record to check on the study. By signing this consent form, you agree to let these people look at your records. We will put a copy of this research consent form in your patient health record and give you a copy as well.
The data produced from this study will be stored in a secure, locked location. Only members of the research team (and maybe those individuals described above) will
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have access to the data. Following completion of the research study the data will be kept as long as required by the Sick Kids “Records Retention and Destruction” policy. The data will then be destroyed according to this same policy.
Reimbursement
Compensation will be provided at a rate of $20.00 for each testing session in recognition of your time and effort. If you stop taking part in the study, you will be compensated for those tests your child has undergone up until that point.
Participation:
Participation in research is voluntary. Your child is likely to be refered for testing every 3-6 months once they are on vigabatrin. If you do choose to participate you will only be asked to sign the consent form once. By signing this consent form you agree to have testing extended by 1 minute every time your child comes in for testing. You can withdraw your child from the study at any time. The care you get at Sick Kids will not be affected in any way by whether you take part in this study.
New information that we get while we are doing this study may affect your decision to take part in this study. If this happens, we will tell you about this new information ask you again if you still want to be in the study.
During this study we may create new tests, new medicines, or other things that may be worth some money. Although we may make money from these findings, we cannot give you any of this money now or in the future because you took part in this study.
We will give you a copy of this consent form for your records.
In some situations, the study doctor or the company paying for the study may decide to stop the study. This could happen even if the medicine given in the study is helping you. If this happens, the study doctor will talk to you about what will happen next.
If your child becomes ill or harmed because your child took part in this study, we will treat your child for free. Your signing this consent form does not interfere with your child’s legal rights in any way. The staff of the study, any people who gave money for the study, or the hospital are still responsible, legally and professionally, for what they do.
Sponsor / Funder of the study
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The sponsor of this research is Sick Kids Hospital. The funder of this research is Lundbeck Pharmaceuticals.
Conflict of Interest
Some of the people doing this study may have a conflict of interest. That means that they may benefit personally, financially, or in some other way from this study .
Dr. Westall (Principal Investigator) has received or may receive for research related to the present study money, or one or more of the following other benefits: speaker's fees, travel assistance, industry-initiated research grants, investigator- initiated research grants, consultant fees, honoraria, gifts, intellectual property rights such as patents, etc. from sponsor(s) that have activities related to the present study.
Consent
•By signing this form, I agree that:
1) You have explained this study to me. You have answered all my questions.
2) You have explained the possible harms and benefits (if any) of this study.
3) I know what I could do instead of having my child take part in this study. I understand that I have the right to refuse to let my child take part in the study. I also have the right to take my child out of the study at any time. My decision about my child taking part in the study will not affect my child’s health care at SickKids.
4) I am free now, and in the future, to ask any questions about the study.
5) I have been told that my child’s medical records will be kept private. You will give no one information about my child, unless the law requires you to.
6) I understand that no information about my child will be given to anyone or be published without first asking my permission.
7) I have read and understood pages 1 to 5 of this consent form. I agree, or consent, that my child ________________________ may take part in this study.
The drug vigabatrin is used to help control seizures. In some people the drug might cause problems with vision. Your child does not have seizures and is not taking vigabatrin.
To better understand what is happening to the eyes of people taking the drug vigabatrin, we need to record these tests in normal children like your child who do not have visual abnormalities due to medication. The following tests may be performed to find out how much these responses change in children with normal retinas.
The electroretinogram (ERG) is an electrophysiological test used to measure the electrical response of the retina. Sedated ERGs are a routine clinical test used to assess retinal function when a retinal disease is suspected or known. Sedated ERGs are clinically indicated for children with suspected idiopathic nystagmus. We want to better understand what is happening to the eyes in people undergoing certain drug treatments.
Description of the research:
The following tests will be performed once your child’s neurologist or ophthalmologist has referred them to the Visual Electrophysiology Unit and an appointment has been made. These tests are clinically indicated to find out whether any changes to the retina have taken place. The tests will take one hour to be performed.
Electroretinogram (ERG): The ERG will be administered according to standard clinic protocol with the addition of two extra steps. These extra steps are intended to isolate the response from a specific part of the retina and will extend the testing time by approximately 1 minute.
124
Patient’s health records will be reviewed for purposes of this study for information about drug history, co-morbidities etc. Standard clinic intake tests may be performed including vision, colour vision, refractive error, and / or ophthalmoscopy.
Potential Harms (Injury, Discomforts or Inconvenience):
There are minimal harms associated with participation in this study. Under exceptional circumstances there is a slight risk that your child may receive a minor scratch to the front of her/his eye. This scratch would feel similar to having a piece of sand in your eye and the discomfort may last for 2-3 days. We will check for this and provide any necessary treatment. The eye drops cause slight stinging, but this resolves within 10 seconds. The drops which we use to dilate your child’s pupils may cause his/her vision be blurred up close for 4-8 hours. The risks involved in this study are no greater than those for normal clinic protocol.
Potential Benefits:
To the Individual: Your child will not benefit directly for participating in this study. Ophthalmological and neurological care will continue whether your child continues in this study or not. Our research team will send you a letter detailing our findings when the study is completed.
To Society: Knowledge gained from this study will hopefully allow physicians to optimize vigabatrin therapy to ensure patients receive the maximum benefit whilst minimizing visual toxicity. Better control of the risks associated with this powerful therapy will make its use in a wider patient population more feasible.
Confidentiality:
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We will respect you and your child’s privacy. No information about who your child is will be given to anyone or be published without your permission, unless the law makes us do this.
For example, the law could make us give information about you • If a child had been abused • If you have an illness that could spread to others • If you or someone else talks about suicide (killing themselves), or • If the court orders us to give them the study papers
Sick Kids Clinical Research Office Monitor or the regulator of the study may see your child’s health record to check on the study. By signing this consent form, you agree to let these people look at your child’s records. We will put a copy of this research consent form in your patient health records.
The data produced from this study will be stored in a secure, locked location. Only members of the research team (and maybe those individuals described above) will have access to the data. Following completion of the research study the data will be kept as long as required by the Sick Kids “Records Retention and Destruction” policy. The data will then be destroyed according to this same policy.
Reimbursement
Compensation will be provided at a rate of $20.00 for each testing session in recognition of your time and effort. If you stop taking part in the study, you will be compensated for those tests your child has undergone up until that point.
Participation:
Participation in research is voluntary. If you choose to participate in this study you can withdraw your child from the study at any time. The care you get at Sick Kids will not be affected in any way by whether you take part in this study.
New information that we get while we are doing this study may affect your decision to take part in this study. If this happens, we will tell you
126
about this new information. And we will ask you again if you still want to be in the study.
During this study we may create new tests, new medicines, or other things that may be worth some money. Although we may make money from these findings, we cannot give you any of this money now or in the future because you took part in this study.
We will give you a copy of this consent form for your records.
In some situations, the study doctor or the company paying for the study may decide to stop the study. This could happen even if the medicine given in the study is helping you. If this happens, the study doctor will talk to you about what will happen next.
If your child becomes ill or is harmed because they took part in this study, we will treat your child for free. Your signing this consent form does not interfere with your child’s legal rights in any way. The staff of the study, any people who gave money for the study, or the hospital are still responsible, legally and professionally, for what they do.
Sponsor / Funder of the study
The sponsor of this research is Sick Kids Hospital. The funder of this research is Lundbeck Pharmaceuticals.
Conflict of Interest
Some of the people doing this study may have a conflict of interest. That means that they may benefit personally, financially, or in some other way from this study .
Dr. Westall (Principal Investigator) has received or may receive for research related to the present study (money, or one or more of the following other benefits: speaker's fees, travel assistance, industry-initiated research grants, investigator- initiated research grants, consultant fees, honoraria, gifts, intellectual property rights such as
127
patents, etc.) from sponsor(s) that have activities related to the present study.
Consent
“By signing this form, I agree that:
1) You have explained this study to me. You have answered all my questions.
2) You have explained the possible harms and benefits (if any) of this study.
3) I know what I could do instead of having my child take part in this study. I understand that I have the right to refuse to let my child take part in the study. I also have the right to take my child out of the study at any time. My decision about my child taking part in the study will not affect my child’s health care at SickKids.
4) I am free now, and in the future, to ask questions about the study.
5) I have been told that my child’s medical records will be kept private. You will not give anyone information about my child, unless the law requires you to.
6) I understand that no information about my child will be given to anyone or be published without first asking my permission.
7) I have read and understood pages 1 to 5 of this consent form. I agree, or consent, that my child___________________ may take part in this study.
_________________________________
Printed Name of Subject & Age Subject’s signature & date (if applicable)
Printed Witness’ name (if the subject/legal guardian Witness’ signature & date
does not read English
If you have any questions about this study, please call Julianna Sienna at (416)-813-7654 ext. 3606.
If you have questions about your rights as a subject in a study or injuries during a study, please call the Research Ethics Manager at (416) 813-5718.
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Appendix 4. Patient report form.
130
131
Appendix 5. R software for Hood and Birch model
library(tcltk)
loadWaves<-function(folder,eye=NA){
#load rod waves, file is expected to be a csv #each column represents a wave #supporting file (*.info) contains start time, sample frequency, intensities if(is.na(eye)){ inp<-readline('Which eye (l/r)?') }else{ inp<-eye }
legend('bottomright',paste(data$info$Intensity,'(',c(1,2,3),')'),col=rainbow(ncol(data$data)),lwd=2) # inp<-tolower(substr(readline('Do you wish to zero these waves (y/n) ?'),1,1)) inp<-'y' if(inp=='y'){
prestim<-window(data$data,end=0)\ vals<-apply(prestim,2,mean) for(iloc in 1:ncol(data$data)){
#continue<-tolower(substr(readline('Accept (y/n) ?'),1,1)) continue<-'y’ while(continue=='n'){ inp<-readline('Enter the waves to be zero\'d (indexes separated by ,) or 0 for all:') inp<-as.integer(strsplit(inp,',')[[1]]) if(inp[1]==0){ inp<-seq(from=1,to=ncol(data$data)) } if(max(inp)>ncol(data$data) | min(inp)<1){ cat('Wave not found',sep='\n') next } else{ targets<-inp } inp<-readline('Enter the new zero factors (comma seperated list, or times separated by a colon):')
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if(length(grep(':',inp))>0){ cat('Using new window for 0\n') inp<-as.numeric(strsplit(inp,':')[[1]]) prestim<-window(data$data,start=inp[1],end=inp[2]) vals<-apply(prestim[,targets],2,mean) } else{ vals<-as.numeric(strsplit(inp,',')[[1]]) if(length(vals)<length(targets)){ cat('Must provide a factor for each wave to be zeroed\n') next } }
for(iloc in 1:length(targets)){ newData$data[,targets[iloc]]<-data$data[,targets[iloc]]-vals[iloc] }
findTimeIdx<-function(times,target){ #support function to find nearest time to a target minIdx<-max(which(times<target)) maxIdx<-min(which(times>target))
selectIntensities<-function(intensities){ #cat(paste(' (',c(1:3),') ',intensities,'\n',sep='')) #inp<-readline('Which intensities should be used in the model (enter index numbers seperated by comma, or 0 for all):') #ans<-as.integer(strsplit(inp,',')[[1]]) #if(ans[1]==0){ # ans<-1:length(intensities) # } #if(max(ans)>length(intensities)){ # cat('Unidentified intensity') # stop() # } ans<-c(1,2,3)
times<-seq(from=0,to=(window[2]*10)+1) out<-matrix(ncol=length(intensities),nrow=(length(times)+(Td*10))) #convert flash intensities to correct units (Td.ms)
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intensities<-(10^intensities)*0.0001^2 for(iloc in 1:length(intensities))