Real-time imaging of single neuronal cell apoptosis in patients with glaucoma
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Author(s)
Type
Journal Article
Abstract
Retinal cell apoptosis occurs in many ocular neurodegenerative conditions including glaucoma—the major cause of irreversible
blindness worldwide. Using a new imaging technique that we have called DARC (detection of apoptosing retinal cells), which until
now has only been demonstrated in animal models, we assessed if annexin 5 labelled with fluorescent dye DY-776 (ANX776)
could be used safely in humans to identify retinal cell apoptosis. Eight patients with glaucomatous neurodegeneration and evidence
of progressive disease, and eight healthy subjects were randomly assigned to intravenous ANX776 doses of 0.1, 0.2, 0.4 and
0.5 mg in an open-label, phase 1 clinical trial. In addition to assessing the safety, tolerability and pharmacokinetics of ANX776, the
study aimed to explore whether DARC could successfully visualize individual retinal cell apoptosis in vivo in humans, with the
DARC count defined as the total number of unique ANX776-labelled spots. DARC enabled retinal cell apoptosis to be identified in
the human retina using ANX776. Single ANX776-labelled cells were visualized in a dose-dependent pattern (P5 0.001) up to 6 h
after injection. The DARC count was significantly higher (2.37-fold, 95% confidence interval: 1.4–4.03, P = 0.003) in glaucoma
patients compared to healthy controls, and was significantly (P = 0.045) greater in patients who later showed increasing rates of
disease progression, based on either optic disc, retinal nerve fibre layer or visual field parameters. Additionally, the DARC count
significantly correlated with decreased central corneal thickness (Spearman’s R = 0.68, P = 0.006) and increased cup-disc ratios
(Spearman’s R = 0.47, P = 0.038) in glaucoma patients and with increased age (Spearman’s R = 0.77, P = 0.001) in healthy controls.
Finally, ANX776 was found to be safe and well-tolerated with no serious adverse events, and a short half-life (10–36 min).
This proof-of-concept study demonstrates that retinal cell apoptosis can be identified in the human retina with increased levels of
activity in glaucomatous neurodegenerative disease. To our knowledge, this is the first time individual neuronal apoptosis has been
visualized in vivo in humans and is the first demonstration of detection of individual apoptotic cells in a neurodegenerative disease.
Furthermore, our results suggest the level of apoptosis (‘DARC count’) is predictive of disease activity, indicating the potential of
DARC as a surrogate marker. Although further trials are clearly needed, this study validates experimental findings supporting the
use of DARC as a method of detection and monitoring of patients with glaucomatous neurodegeneration, where retinal ganglion
cell apoptosis is an established process and where there is a real need for tools to non-invasively assess treatment efficacy.
blindness worldwide. Using a new imaging technique that we have called DARC (detection of apoptosing retinal cells), which until
now has only been demonstrated in animal models, we assessed if annexin 5 labelled with fluorescent dye DY-776 (ANX776)
could be used safely in humans to identify retinal cell apoptosis. Eight patients with glaucomatous neurodegeneration and evidence
of progressive disease, and eight healthy subjects were randomly assigned to intravenous ANX776 doses of 0.1, 0.2, 0.4 and
0.5 mg in an open-label, phase 1 clinical trial. In addition to assessing the safety, tolerability and pharmacokinetics of ANX776, the
study aimed to explore whether DARC could successfully visualize individual retinal cell apoptosis in vivo in humans, with the
DARC count defined as the total number of unique ANX776-labelled spots. DARC enabled retinal cell apoptosis to be identified in
the human retina using ANX776. Single ANX776-labelled cells were visualized in a dose-dependent pattern (P5 0.001) up to 6 h
after injection. The DARC count was significantly higher (2.37-fold, 95% confidence interval: 1.4–4.03, P = 0.003) in glaucoma
patients compared to healthy controls, and was significantly (P = 0.045) greater in patients who later showed increasing rates of
disease progression, based on either optic disc, retinal nerve fibre layer or visual field parameters. Additionally, the DARC count
significantly correlated with decreased central corneal thickness (Spearman’s R = 0.68, P = 0.006) and increased cup-disc ratios
(Spearman’s R = 0.47, P = 0.038) in glaucoma patients and with increased age (Spearman’s R = 0.77, P = 0.001) in healthy controls.
Finally, ANX776 was found to be safe and well-tolerated with no serious adverse events, and a short half-life (10–36 min).
This proof-of-concept study demonstrates that retinal cell apoptosis can be identified in the human retina with increased levels of
activity in glaucomatous neurodegenerative disease. To our knowledge, this is the first time individual neuronal apoptosis has been
visualized in vivo in humans and is the first demonstration of detection of individual apoptotic cells in a neurodegenerative disease.
Furthermore, our results suggest the level of apoptosis (‘DARC count’) is predictive of disease activity, indicating the potential of
DARC as a surrogate marker. Although further trials are clearly needed, this study validates experimental findings supporting the
use of DARC as a method of detection and monitoring of patients with glaucomatous neurodegeneration, where retinal ganglion
cell apoptosis is an established process and where there is a real need for tools to non-invasively assess treatment efficacy.
Date Issued
2017-06-01
Date Acceptance
2017-02-13
Citation
Brain, 2017, 140 (6), pp.1757-1767
ISSN
1460-2156
Publisher
Oxford University Press (OUP)
Start Page
1757
End Page
1767
Journal / Book Title
Brain
Volume
140
Issue
6
Copyright Statement
This is an open access article distributed under the terms of the Creative Commons CC BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
License URL
Sponsor
St Marys Development Trust
St Marys Development Trust
Identifier
https://academic.oup.com/brain/article/140/6/1757/3755374
Grant Number
CFT/15/4003
CFT/15/4003
Subjects
Science & Technology
Life Sciences & Biomedicine
Clinical Neurology
Neurosciences
Neurosciences & Neurology
apoptosis
retinal imaging
glaucoma
real-time visualization
OPEN-ANGLE GLAUCOMA
RETINAL GANGLION-CELLS
DEATH IN-VIVO
VISUAL-FIELD
INTRAOCULAR-PRESSURE
PROGRESSION
NEURODEGENERATION
DISEASE
MODEL
EYES
Publication Status
Published
Date Publish Online
2017-04-26