Introduction

We are developing CDX-301, our zeaxanthin pharmaceutical candidate, for macular degeneration. Our target initial indication for CDX-301 is Stargardt disease (STGD), a juvenile form of macular degeneration and potential orphan drug indication.

Stargardt disease is the most common inherited macular dystrophy in children and has an autosomal recessive mode of inheritance associated primarily with mutations in the ABCA4 gene. It is both clinically and genetically heterogenous, but the hallmark feature of the disease is progressive, irreversible central vision loss. There is currently no treatment for Stargardt disease, but genetic, stem cell, and pharmacological therapies are all being explored.

Zeaxanthin has a mechanism of action and excellent safety profile similar to astaxanthin, however, it accumulates in the human eye through uptake by a unique retinal receptor, providing protection against blue light, oxidative damage, and related inflammation that occurs in macular degeneration. Pre-clinical and clinical studies with zeaxanthin have demonstrated proof-of-concept for the treatment of macular disorders.

Cardax is exploring the therapeutic potential of the use of zeaxanthin in preventing the oxidative damage seen in Stargardt disease. The company is awaiting orphan drug designation from the U.S. FDA for the treatment of Stargardt disease.

Image from a Stargardt disease patient showing a central macular scar, pigmentary changes, and surrounding perimacular flecks (NIH.gov).

About Stargardt Disease

Stargardt disease (STGD) is the most common inherited macular dystrophy in children and has an autosomal recessive mode of inheritance associated primarily with mutations in the ABCA4 gene. It is both clinically and genetically heterogenous, but the hallmark feature of the disease is progressive, irreversible central vision loss.

Despite the clinical and genetic heterogeneity of Stargardt muscular dystrophy, there is one common ailment for all persons affected with the disease: vision loss. Although there has been extensive research on the topic, there is still no definitive treatments. Patients need new options to stall the accumulation of lipofuscin and degeneration of the macula.

Stargardt disease, also known as Stargardt’s macular dystrophy or juvenile macular degeneration, is an inherited disorder of the retina – the tissue at the back of the eye that senses light. The disease causes progressive damage to the macula, a small area in the center of the retina that is responsible for sharp, central vision. The disease typically causes vision loss during childhood or adolescence, although, in some forms, vision loss many not be noticed until later in adulthood (NEI, Stargardt Disease).

Stargardt disease has an estimated prevalence of approximately 1 in 8,000 to 10,000 individuals (NIH, Stargardt Macular Degeneration). While the progression of the disease is highly variable, the hallmark clinical feature is bilateral central vision loss. Macular atrophy and yellow-white flecks at the level of the retinal pigment epithelium (RPE), a cell layer essential for retinal integrity and photoreceptor survival, is commonly seen on ocular exam (Tanna, Br J Opthalmol, 2017). Patients may notice gray, black, or hazy spots in the center of their vision. Some may complain of sensitivity to bright lights or trouble adjusting their eyes to the lighting in different environments. Many patients develop color blindness later in the disease (NEI, Stargardt Disease).

In most cases, STGD is caused by autosomal recessive mutations in the ABCA4 gene. The high clinical variability in the presentation of Stargardt disease is due, in part, to the genotypic heterogeneity associated with the disease. Over 900 mutations in the ABCA4 gene are associated with STGD (Tanna, Br J Opthalmol, 2017 ; Lu, Graefes Arch Clin Exp Ophthalmol, 2017). To a lesser degree, mutations in the ELOVL4 gene have also been associated with the condition (NIH, Stargardt Macular Degeneration).

The ABCA4 genes provide instructions for making the ABCR protein, a transport protein in photoreceptors cells (the light-sensing cells in the retina). These proteins allow for normal recycling and degradation of enzymes in the phototransduction cycle, the process by which light is converted into signals interpretable by the brain in the retina of the eye.

Vitamin A is the compound most impacted by defective ABCR. Mutations in ABCR prevents removal of the toxic byproducts of vitamin A degradation. This biproduct, A2E, accumulates in the retinal pigment epithelium, and ultimately leads to the build-up of lipofuscin (Lu, Graefes Arch Clin Exp Ophthalmol, 2017; Tanna, Br J Opthalmol, 2017). Lipofuscin is a lipopigment formed by lipids, metals, and misfolded protein, and it cannot be degraded. Lipofuscin accumulation is a recognized hallmark of aging cells, but rapid accumulation can be tied to increased oxidative stress (Brunk, Free Radic Biol Med, 2002). The accumulation of excessive lipofuscin leads to atrophy of the retinal pigment epithelium followed by degeneration of the photoreceptors, especially in the macula. This cell death ultimately leads to the vision loss characteristic of STGD (Lu, Graefes Arch Clin Exp Ophthalmol, 2017).

Not only is there no definitive cure for Stargardt disease, but there is also no standard treatment paradigm to prevent or slow vision loss. Patients are offered low-vision aids to maximize use of their peripheral and paracentral vision and are advised to use photoprotection to delay the accumulation of lipofuscin and progression of vision loss. Patients are also asked to make other lifestyle changes like avoiding cigarette smoking and excess Vitamin A supplementation (NEI, Stargardt Disease; Lu, Graefes Arch Clin Exp Ophthalmol, 2017). The disease may be treated with anti-VEGF inhibitors if there is proliferation or leakage of blood vessels in the retina, but the treatment does not seem to ensure visual improvement or prevent macular atrophy (Battaglia, Br J Opthalmol, 2015).

STGD is subject to more clinical trials than any other inherited retinal disease currently being studied. Gene replacement, stem cell therapy, and pharmacological approaches are all being explored (Tanna, Br J Opthalmol, 2017). The target of gene therapy for patients with Stargardt disease is the introduction of functional ABCA4 gene to the retina, with the intended result being sustained levels of normal, active transporter in the photoreceptor cells and prevention of disease progression. Different viral vectors are currently being explored for the delivery of the ABCA4 gene to ocular tissue (Lu, Graefes Arch Clin Exp Ophthalmol, 2017). In the realm of stem cell therapy, some researchers are exploring ways to derive RPE from embryonic stem cells and transplant these cells subretinally into patients with advanced STGD. Pharmacological options aim at either reducing the formation of toxic byproducts of the phototransduction cycle (either by reducing the delivery of Vitamin A or inhibiting enzymes) or directly targeting the toxic metabolites created by defective ABCA4 (Tanna, Br J Opthalmol, 2017). While this research holds great promise, it has yet to yield any significant benefit for patients.

Zeaxanthin and Lower Oxidative Stress

The adult human retina is believed to lack regenerative capacity. Therefore, the RPE cells, which possess significant levels of antioxidants and antioxidant enzymes, are thought to be the key regulators of cellular health of the retina (Cai, Prog Retin Eye Res, 2000; Simó, J Biomed Biotechnol, 2010; Strauss, Physiol Rev, 2005).

These cells are under significant oxidative stress from the toxic byproducts generated in STGD. It is hypothesized that in STGD individuals, an increased rate of lipofuscin accumulation leads to depletion of the retinal antioxidant defenses. The increased stress and decreased defenses lead to RPE dysfunction, increased RPE cell death, and ultimately contributes to photoreceptor death and loss of vision.

Given that oxidative stress is directly involved in the generation of the toxic byproduct A2E and accelerates visual loss in patients with STGD, researchers at Cardax are currently exploring pharmacologic opportunities to reduce oxidative stress. One promising therapeutic opportunity is zeaxanthin, a xanthophyll carotenoid.

Carotenoids are natural pigments which occur ubiquitously in all organisms capable of conducting photosynthesis and they support animal health and vitality. Although not synthesized by animals, they can be obtained through diet and are present in blood and tissues. Carotenoids are important sources of vitamin A and are known to have strong antioxidant properties. Carotenoids have been shown to have potent anti-inflammatory activity and support mammalian longevity. Research suggests that carotenoids can play a protective role in a number of disorders caused by oxidative stress including cardiovascular disease, certain types of neurological disorders and cancers, and eye-related disorders (Fiedor, Nutrients, 2014).

Zeaxanthin is a carotenoid pigment that protects the macula against blue light and oxidative damage. It is known to preferentially accumulate in the human macula and peripheral retina. Studies have shown a positive relationship between increased zeaxanthin intake and lower oxidative stress (Bernstein, Prog Retin Eye Res, 2016; Kvansakul, Opthalmic Physiol Opt, 2006).

Preclinical Evidence Supporting Zeaxathin in Macular Degeneration

Zeaxanthin’s protective effects come, in part, from its ability to absorb blue light. Photo receptors are susceptible to damage by light, particularly in the blue visible spectrum. Blue light greatly enhances oxidative stress, the formation A2E, and ultimately the buildup of lipofuscin (Boulton, J Photochem Photobiol B, 1993; Sparrow, Vision Res, 2003). Studies have demonstrated that zeaxanthin is protective, in part, due to its ability to prevent the build-up of the toxic metabolites of Vitamin A after blue-light induced photoreceptor damage (Kim, Exp Eye Res, 2006; Bhosale, Arch Biochem Biophys, 2009). A 2002 in vivo study of quails (animals who similarly have high concentrations of zeaxanthin in the macula), supported this finding by demonstrating that retinal zeaxanthin dose-dependently reduced light photoreceptor apoptosis in the macula (Thomson, Invest Opthalmol Vis Sci, 2002).

Higher levels of macular carotenoids have also been associated with improved outcomes in certain forms of macular degeneration, especially age-related macular degeneration (Kim, Exp Eye Res, 2006; Chew, Opthalmology, 2013).

Some studies have demonstrated an inverse relationship between macular zeaxanthin levels and STGD risk (Zhao, Arch Opthalmol, 2003; Kvansakul, Opthalmic Physiol Opt, 2006). The severity of STGD has been shown to relate to macular pigment depletion in the fovea (Tomas, Invest Ophthalmol Vis Sci, 2009), as patients with higher levels of zeaxanthin are found to have better maintenance of peripheral vision compared to controls.

An in vitro study on the ability of zeaxanthin to protect the RPE cell line against oxidative insults was conducted by Cardax. Using a human retinal-pigmented epithelial line, the researchers assessed the ability of zeaxanthin to protect RPE cells by preparing a control cell line and a cell line incubated with a zeaxanthin formulation (CDX-380). They then challenged these cell lines with different oxidant insults (hydrogen peroxide, 4-HNE, and SIN-1, peroxynitrite). Across the forms of oxidative insult studied, the researchers found decreased cell death in all cell lines pre-treated with CDX-380. They were also able to show that after short-term hydrogen peroxide challenge, zeaxanthin was associated with decreased intracellular signaling in pathways normally activated by oxidative stress. These findings indicate that zeaxanthin is protective against RPE cell death under oxidative stress.

Given that zeaxanthin is associated with lower oxidative stress and improved outcomes in SGTD and findings that dietary supplementation increases serum and macular levels of zeaxanthin (Bone, Exp Eye Res, 2000 ; Hammond, Invest Ophthalmol Vis Sci, 1997; Kvansakul, Opthalmic Physiol Opt, 2006), even in patients with diseased retina (Koh, Exp Eye Res, 2004), it follows that pharmacological supplementation could benefit patients with STGD.

Cardax is currently pursuing orphan drug designation for its zeaxanthin preparation, CDX-301, for the treatment of Stargardt disease. CDX-301 is formulation of high purity, synthetic zeaxanthin. The preparation is designed to provide a stable, oral formulation of the carotenoid with optimal bioavailability for patients. With the success of earlier generations of CDX-380 in preclinical studies, Cardax believes the treatment has the potential to effectively treat this disabling disease.