Using ocular fluids and blood for precision diagnostics and the development of a potential new therapy for age-related macular degeneration
by Lei Zhou, Senior Principal Research Scientist, Ocular Proteomics Platform Head, Singapore Eye Research Institute, Singapore, and Gemmy Cheung Chui Ming, Senior Consultant, Retina Department, Singapore National Eye Centre, Singapore
“One of the problems is that you find yourself sitting doing nothing, and thinking well, it’s frustrating. There’s nothing you can do because you need your eyes to do things. You feel very insecure out of doors. You can’t see the edges of pavements. People whizzing past you on bicycles on the pavement are a nightmare… Also crossing a road, no matter how used to it you are, when you get to the other side your heart is racing because of a sort of mini panic. You can’t help tensing up. You feel very vulnerable. It takes away the individuality of being you… You’ve got to rely on somebody else to do the things that you’ve always done.”1*
Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries, affecting millions of people and costing $343 billion (USD) worldwide.2-4 As the name suggests, the condition affects older people and with aging populations, these numbers are only going to grow. AMD involves the loss of one’s central vision.2,5 The disease destroys the macula, the part of the retina at the back of the eye that provides sharp, central vision needed for seeing objects clearly. The progressive impairment of visual acuity and central vision can rob people of their ability to read, drive, watch television, use a computer, recognize facial expressions, or perform other activities of daily living (see Figure 1). The cause of AMD is not well understood and unfortunately, natural aging, the main risk factor, is not modifiable.2
At present, there is no cure for AMD.6,7 Current medical interventions serve only to delay the progression of the disease; such treatments include the use of intravitreous drugs, lasers, photodynamic therapy (PDT), and, sometimes, surgery.7 Until about 15 years ago, the main treatments for AMD was PDT or focal laser.6 But these therapies are of limited effectiveness and only suitable for a subset of patients.6,8 Wet AMD, also known as exudative or neovascular AMD, occurs when abnormal blood vessels form under the retina and bleed or leak fluid into the macula, thus causing visual impairment. This impairment is so severe that wet AMD is always considered to be an advanced stage of the disease.4,5 Although wet AMD accounts for only 10 percent of all AMD diagnoses (the other 90 percent being dryor other forms of AMD), it results in 90 percent of the cases of legal blindness.4,5
In the mid-2000s, a new class of drug was introduced, which has been so effective, it has since become standard of care for patients with wet AMD.6-9,11 Vascular endothelial growth factor (VEGF), secreted by the damaged retina in AMD, is a signalling protein that promotes the growth and leakage of new blood vessels. Anti- VEGF drugs injected into the vitreous of the eye controls exudation from the neovascularization of the retina and delays further central vision loss in up to 90 percent of treated eyes.10,11 However, despite its overall efficacy, there remains a broad range of patient responses, with reports of over 50 percent of patients still having bleeding or leakage from the abnormal blood vessels in their eyes after 3 months of treatment.9,12 This indicates that there are other disease mechanisms at play that are not predominantly VEGF-driven, and which could present novel targets for treatment, especially in patients who do not respond optimally to anti-VEGF therapies. Furthermore, the improved understanding of these mechanisms could also reveal new biomarkers for predicting anti-VEGF treatment response more precisely than current markers, allowing physicians and patients to make better informed decisions about management options.8
Shared risk factors between AMD and many systemic disorders suggests that AMD may be a manifestation of systemic disease.14 To understand more comprehensively what is going on in the body of patients with wet AMD, researchers at the Ocular Proteomics Platform at Singapore Eye Research Institute (SERI, Singapore), in collaboration with clinicians and researchers at the Singapore National Eye Centre (SNEC), the National University of Singapore (NUS, Singapore), the Agency for Science, Technology and Research (A*STAR, Singapore), and the University of Colorado (CU, USA), investigated the serum metabolomic profiles of patients with wet AMD.8,13 Metabolomics is the systematic study of all the metabolites present within a biological system.15 This means the large-scale qualitative and quantitative analyses of hundreds of thousands of small, biologically active molecules from complex biological samples.16-18
Metabolomic studies require advanced analytical techniques, particularly mass spectrometry (MS), which provides the necessary combination of analytical sensitivity and selectivity. The researchers at SERI, NUS, A*STAR and CU applied MS methods coupled with liquid chromatography (LC) using innovative technologies and instruments such as the SCIEX TripleTOF 5600 systems with data-independent acquisition (DIA) to detect comprehensively all the metabolites present in the samples, no matter how low or high their abundance.8,13 Metabolic profiles are examined using different types of samples, such as tear fluid and blood serum. Metabolomes were compared between individuals with AMD and age-matched healthy controls to identify markers of AMD disease.13 The study showed that individuals with AMD contained higher concentrations of certain molecules in their sera than healthy controls did, including glycerophospholipids, some amino acids and omega fatty acids.13
Another study performed by the SERI research team and researchers at the Retina Department of the Singapore National Eye Centre (SNEC), Singapore investigated the differences in the serum metabolomes between individuals with wet AMD who responded to anti-VEGF therapy and those who were suboptimal responders.9 They identified metabolites that differed between the metabolic profiles of responders and non-responders. Pathway analysis was performed on these metabolites, which revealed that there were alterations in the glycerophospholipid metabolic pathway. Responders had lower levels of glycerophosphocholine, lysophosphatidylcholine (LysoPC) and phosphatidylcholine (PC) in their sera than the non-responders. LysoPC is a breakdown product of PC and higher levels of it have been linked to cardiovascular complications associated with atherosclerosis, ischemia and diabetes. LysoPC plays an important role in vascular development and may account in part for the poor response to anti- VEGF drugs. Non-responders’ higher levels of LysoPC may be indicative of the accumulation of LysoPC in the serum, which interferes with vascular modelling and causes oxidative stress, leading to the poor response to anti-VEGF agents.9
Based on the findings of these and other studies that identified markers from serum samples, SERI scientists and other collaborators are now developing a novel clinical test based on an LC-MS MRM assay that detects metabolomic biomarkers in serum to predict whether an individual with wet AMD is likely to respond to anti-VEGF treatment. By differentiating responders and non- responders early in their treatment pathway, non-responders may be switched early on to a different therapy, which may be more effective for them. This is particularly relevant as several new drugs are being developed to treat AMD, using different mechanisms of action. The SERI team is also working with collaborators to develop a new treatment option.
By examining the proteomic profiles of patients with wet AMD using samples from the vitreous of their eyes, a new pathway has been discovered that contributes to the disease mechanism, but which does not involve VEGF. By examining the other molecules involved in this pathway, the researchers identified a novel potent anti-angiogenic factor. The active domain of this protein factor was identified and then synthesized to produce the candidate drug (a collaborative project with Associate Professor Wang Xiaomeng, Nanyang Technological University, Singapore). This investigational medicine is now being evaluated for efficacy and safety in animal models as well as in tissues and cell-based in vitro and ex vivo assays. These preclinical studies include comparisons with anti-VEGF drugs used as standard of care treatments. The drug will also be assessed to see whether it is effective for patients with wet AMD who are non-responders to current anti-VEGF treatments. As these and other studies are underway, the very early data suggests that this new drug may work as either an alternative or complement with anti-VEGF therapy to treat patients with wet AMD.
The research program described is a prime example of a potential end-to-end precision medicine solution. With a companion diagnostic test based on metabolomic biomarkers to predict treatment response, individual patients can be identified and stratified into streams for treatment using either the existing standard of care options or more appropriate novel treatments. This would be particularly good should one of the new treatments target precisely the disease mechanism indicated by the diagnostic test. Not only should the stratification of patients for treatment lead to improved clinical outcomes, it should also help alleviate the economic burden posed by better allocating patients for healthcare resource utilization. This is especially pertinent in light of our aging societies.
These research projects and their results to date bring hope for many who have or may go on to develop AMD. It seems that we may look forward to a brighter future – with earlier and more informative diagnoses and optimized treatment trajectories that could dramatically reduce the impact of wet AMD on sight loss and the activities of daily living.
Dr Lei Zhou is a Senior Principal Research Scientist and heads the Ocular Proteomics Platform in Singapore Eye Research Institute (SERI), Singapore. He was trained as a bio-analytical chemist and joined SERI after he completed his PhD in the Department of Chemistry, National University of Singapore in 1998. He applies cutting-edge mass spectrometry and proteomics/ metabolomics technologies for eye research. His research interests include discovering new biomarkers for ocular diseases, understanding the pathogenesis of these diseases, ocular metabolomics using LC-MS/MS, developing new peptide antibiotics for eye infections, and ocular drug pharmacokinetics.
Professor Gemmy Cheung is currently a Professor at the Duke-NUS Medical School, National University of Singapore, and Head of the Medical Retina Department, Singapore National Eye Center. She also heads the Retina Research Group at the Singapore Eye Research Institute. Her research focus on Asian retinal diseases, specifically age-related macular degeneration (AMD), polypoidal choroidal vasculopathy (PCV) and myopic macular degeneration.
The studies mentioned in this article are supported by the SERI-IMCB Programme In Retinal Angiogenic Diseases (SIPRAD).
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*Quote is a composite of multiple patient quotes