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This is a review paper in which the authors summarise the transitioning of techniques detecting apoptosis from bench to bedside, along with the future possibilities they encase. Detection of Apoptosis in Retinal Cells (DARC) technology can be used as a surrogate end point biomarker. To date, it remains the only tool to have been tested in clinical trials in the fields of neuroscience, ophthalmology and drug development. It relies on the property that the cell membrane changes under stress and apoptosis. DARC uses intravenously-delivered fluorescently-labelled Annexin V (ANX776) as the marker of retinal cell apoptosis. This combines with phosphatidylserine (PS), a phospholipid which is normally found on the internal leaflet of the cell membrane; however, when a cell is stressed, PS flips to the exterior leaflet of the cell membrane and which show up as bright white spots in vivo images. The process of externalisation of PS is reversible, if appropriately and timely targeted therapeutically. DARC Phase 1 clinical trials investigated the tolerability and safety of ANX776. The study was carried out on eight healthy volunteers and eight patients affected by progressing glaucoma. The key finding of this trial was that there was a significant measure of efficacy, both with respect to apoptotic ganglion cell count being higher in glaucoma patients compared to healthy controls, but also with respect to predicting future glaucoma disease progression – in fact 18 months before changes in OCT and visual fields. This latter finding suggests that DARC could be a potential surrogate marker. The phase II non-randomised clinical trial of DARC used ANX766 and was designed to fully characterise the differences between the counts and distribution of DARC spots in patients (n=116), healthy volunteers, progressing glaucoma, age-related macular degeneration (AMD), optic neuritis and Down’s syndrome (with central nervous system (CNS) pathology similar to Alzheimer’s disease (AD)). The primary objective of this study is to assess the DARC count in different pathologies and further assess DARC’s potential in early diagnosis and predictive abilities. The authors also highlight other tests that have been used in experimental animal models to detect apoptosis. PSVue550 with a transitory labelling potential has been exploited for serial monitoring of retinal degeneration at different time points. Detection of ApoSense® the Amphipathic low-molecular weight of 300–700 Da that crosses apoptotic plasma membrane and accumulate in the cytoplasm of dying cell has been exploited in disease of AD, amyotrophic lateral sclerosis, melanomas, chemotherapy-induced enteropathy and reperfusion-induced damage. FLIVO is a family of fluorescent caspase inhibitors that allow visualisation of in vivo and in vitro apoptosis. These tracers are directly injected into the circulation and selectively accumulate in apoptotic cells. Being able to cross the blood-brain barrier, they have potential in the study of brain and ocular neurodegenerative diseases and have been exploited in oncology to develop new chemotherapeutic agents and cancer vaccines. In ophthalmology, it has been used to monitor the activity of diabetic retinopathy (DR), glaucoma retinitis pigmentosa blue-light induced retinal damage and AMD. Z-DEVD-Aminoluciferin is similar to the natural bioluminescence found in animals such as fireflies. Activated by caspases in the apoptotic cell, it emits bioluminescence which is exploited as a marker of apoptosis. This has been used in mice models of tumour xenografts. This tool has also been used in the drug development setting; however, its clinical field role remains unexplored. Several ocular and extraocular neurodegenerative diseases share the common feature of early and pathological death of retinal cells. If detected early they offer a window and possible means to delay and halt pathologic processes before it causes significant harm. This has implications not only in glaucoma and the prevention of blindness, but any condition associated with neuronal apoptosis, where findings can help enhance the clinical diagnosis and refine therapeutic intervention.

Detecting apoptosis as a clinical endpoint for proof of a clinical principle.
Zolleta P, Yapa TE, Cordeiro MF.
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Sofia Rokerya

MBBS MRCOphth FRCSI, King's College University Hospital, UK.

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