It can take years or even decades to properly understand a pathway. But when a certain threshold is passed, new therapeutic opportunities can be revealed at an exciting rate. The apolipoprotein E (apoE) pathway has passed that threshold, and VC-backed start ups to exploit this new understanding are springing up like mushrooms.
As early as the mid-1980s, human genetics identified the importance of the apoE protein: a common allelic variant (apoE4) was associated with increased risk of Alzheimer’s Disease, while another variant (apoE2) was associated with longevity. Even today, the sequence of the apoE gene remains the only really robust association with longevity in humans.
But there is another side to apoE. As the name indicates, it is a structural component of lipoproteins, and plays a key role in the clearance of triglyceride-rich particles associated with heart disease. In mice, deletion of the gene encoding apoE leads to a massive hypercholesterolemia and formation of severe vascular lipid lesions resembling human atherosclerosis. In humans, too, the allelic variants of apoE are associated with abnormal lipoprotein profiles.
How do you make sense of this complicated ‘split personality’ of apoE, with central roles in the seemingly unconnected worlds of neurodegeneration and lipid metabolism? New research is finally providing the answers, and demonstrates, at last, how to drug this target. The VC community has been quick to respond, supporting start-ups in this area – can they deliver first-in-class therapies in these blockbuster indications with vast unmet medical needs?
The key to understanding apoE comes, as so often in biology, from the study of very simple model organisms: in this case, small roundworms called nematodes. One species, Caenorhabditis Elegans has taught us as much about developmental biology as any organism on the planet.
DrugBaron envisages an impact on global health for the best apoE mimetics that matches or exceeds that of antibiotics and statins
During development, cells divide in a very specific pattern to form the structures of the worm’s simple body. Some of the progeny cells are retained, but others die through programmed cell death (apoptosis). The remnants of the dead cells are then taken up by their neighbours, partly to keep things clean and tidy, but also to recycle the building blocks they contain.
One particularly important building block in these worms is cholesterol, because unlike humans, C. Elegans is a cannot manufacture its own cholesterol – it needs to scavenge it from the environment. The availability of cheolsterol is often growth-limiting to the worms in nature.
Genetic analysis is very easy in the fast-breeding worms and midway through the last decade, a central gene involved in the resorption of apoptotic cell corpses was identified as CED1. The key insight came from the sequence of this gene: it is the worm version of a human gene called LRP1. LRP stands for LDL-Receptor Related Protein 1, and like the LDL-receptor it plays a key role in cholesterol metabolism in man.
This genetic analysis in two species, one a microscopic worm and the other human beings, shed a light on the earliest evolution of our complex lipoprotein system for managing levels of cholesterol and other fats. The corpses of dead cells were the first ‘lipoprotein particles’.
As multicellular organisms developed, and became more complex this simple reuptake of apoptotic cell bodies has been refined, with enzymes deliberately adding and subtracting different fats from the particles and the liver assuming a central regulatory role. But the evolutionary origin of lipoproteins explain the dual-role of apoE – as a regulator of cell debris clearance and as a structural component of lipoproteins.
ApoE retains the accolade of being the only gene in the human genome that is robustly associated with longevity
While the role in lipid metabolism was the most obvious function of apoE (apoE-deficient mice, for example, have massive hyperlipidemia and develop vascular lipid lesions resembling atherosclerosis), it may be the ancestral role in cell debris clearance that is the most important. The strongest genetic associations are with neurodegeneration, and in particular with Alzheimer’s Disease. Carriers of a particular allele of apoE, the apoE4 variant, are more than twice as likely to develop Alzheimer’s Disease before the age of 75 than wild-type individual, while those with another variant, apoE2, are further protected against neurodegeneration.
Since Alzheimer’s Disease is characterized by the development of extracellular amyloid plaques, which frequently seed around Lewy Bodies (themselves persistent apoptotic cell corpses), its is much more likely that the disease results from impaired cell debris clearance over a lifetime than from perturbations in lipoprotein metabolism.
Our own phenome-wide association study (designed to ask, in a non-hypothesis driven manner, which phenotypes are most strongly linked to variation in a particular gene) suggest that this neurodegeneration might have subtler effects earlier in life, decades before the cognitive decline associated with Alzheimer’s Disease first becomes evident. Major depression, one of the most prevalent diseases of middle-age, is powerfully associated with the presence of the apoE4 variant, suggesting that at least in a proportion of cases there is a neuroanatomical structural basis for depression that could in principle be treated with agents that stimulate cell debris clearance.
But the consequences of impaired cell debris clearance are not limited to the CNS. The uncleared debris constantly presents self-antigens, often in an immunogenic partially-denatured state, underpinning the development of autoimmunity. In lupus (SLE), other mutations that impair cell debris clearance are already known to cause the disease, while drugs that impair debris clearance in vitro (such as procainamide and hydralazine) cause SLE if used for an extended period. Constant stimulation of the immune system by such debris likely contributes to other autoimmune conditions as well.
Compounds that mimic the effects of apoE on cell debris clearance are candidates for the title “Elixir of Life”
Even beyond autoimmune disease, impaired clearance can contribute to chronic inflammation. In Crohn’s Disease, the normal mechanisms that lead to clearance of fecal debris from sites of damage in the gut wall seem to be impaired, according to the ground-breaking work of Prof Segal at London’s UCL. Paradoxically, a lack of activity by phagocytes, such as gut-wall macrophages and neutrophils leads to persistence of the debris, which in turns leads to a persistent, but ineffective, inflammatory response. Whereas current treatment, on the face of it sensibly enough, attempt to treat Crohn’s Disease with anti-inflammatory agents, its likely that such treatments merely ameliorate the detrimental side-effects of the chronic inflammatory response on the gut-wall but perpetuate the original cause of the problem.
The importance of this ancient cell debris clearance pathway involving apoE is so great that it retains the accolade of being the only gene in the human genome that is robustly associated with longevity. Individuals with two copies of the apoE2 variant are twice as likely as their wild-type colleagues to celebrate their 100th birthday, and four-times more likely to receive their congratulatory telegram than those with two copies of the apoE4 variant.
Extending the life of the longest-lived members of a species is no mean feat. Only one drug has been unambiguously shown to have a similar effect in mammals: rapamycin (although sirtuin activators, such as resveratrol, may also fall into this category, once the current debate around the published studies is concluded). Rapamycin, through its receptor mTor, also stimulates cell debris clearance, and in particular the removal of intracellular debris through a related pathway called autophagy.
Fitting these jigsaw puzzle pieces together throws the importance of the cell debris clearance pathways into a new sharp focus. Stimulating cell debris clearance has the potential to be the 21st Century elixir of life, not merely preventing or curing a wide range of diseases from depression to Alzheimer’s Disease, inflammatory bowel disease to autoimmunity, but actually extending life.
But how might cell debris clearance be stimulated? Rapamycin is not the answer – mTor controls a binary switch between cell proliferation and autophagy, so stimulation of autophagy is accompanied by a profound suppression of cell division that leads to immunosuppression.
Calorie restriction (which means, quite literally, a starvation diet sustained for a long period of time, not a casual slimming fad) can achieve a similar switch in mTor, promoting autophagy while leaving enough cell division to keep the immune system functioning. This works to extend the lifespan of experimental animals, but it is not a particularly palatable recipe for life extension in man.
New evidence suggests exercise is another, potentially more sensible, option. An elegant recent study in mice showed that deletion of a gene required for cell debris clearance prevented mice from obtaining the normal benefit of exercise in preventing diabetes. Equally surprisingly, it is becoming clear in humans that the health benefits of exercise can come from very short, but intense, bursts of exercise that are insufficient to improve conventional measures of cardiovascular fitness. What intense exercise does do is damage (though clearly reversibly) your muscle fibres. The debris that is generated provides a constant boost to your systemic debris clearance pathways. In the process of clearing up your muscle tissue after exercise, your macrophages also seem to spring-clean your other tissues, ranging from pancreas to brain.
But for those of us who lack the self-discipline to maintain the necessary exercise regime, which (despite decades of public health campaigns) remains the majority, or indeed those who it seems, for genetic reasons, do not gain the health benefits of exercise anyway (which is a fair-sized minority), can the pharmaceutical industry come to our rescue?
If the crop of new VC-backed companies targeting this pathway are any indication, the answer a decade from now may be a resounding ‘yes’
The most straightforward approach is that of Ohio-based ReXceptor. Their strategy is to boost endogenous expression of apoE, using agonists of the ligand-dependent transcription factor RXR-alpha. As their recent publication in the prestigious Science journal testifies, this is a promising approach: the RXR-alpha agonist bexarotene induced rapid clearance of amyloid plaques in a mouse model of AD – a result sufficiently impressive that it garnered attention of the mainstream world media.
The challenge for ReXceptor will be the specificity of their approach. RXR-alpha heterodimerises with other nuclear hormone receptors, and affects the transcription of numerous gene beyond apoE. As a result, bexarotene is associated with some fairly daunting side-effects, with hyperlipidemia and thyroid dysfunction prominent in more than half the subjects treated with the drug for cancer indications. The solution may lie in better-optimized ligands, or in a careful titration of the dose, hoping that the beneficial effects on apoE can somehow be separated from the side-effects.
The other approach, adopted by US start-up Cognosci and by Cambridge-based Epsilon-3 Bio (where DrugBaron is a member of the senior management team), is to mimic apoE signaling (or at least the part of apoE signaling that promotes cell debris clearance).
The challenge here is that the receptors and signaling pathway responsible for linking apoE to cell debris clearance are still poorly understood, precluding molecular screens. But peptides derived from the apoE sequence and small molecules identified through functional screening both offer hope of recapitulating the beneficial properties of apoE.
A third strategy, pioneered by Prof Mahley and his colleagues at the Gladstone Institute in San Francisco, and also by the Cambridge team behind Epsilon-3 Bio, is to convert the “risk” variant of apoE (apoE4) back to wild-type, using small molecule “structure converters”. These are compounds that bind to apoE in such a way as to induce a switch in conformation from the apoE4 conformation to the apoE3 conformation. With luck, such molecules might even be able to improve the function of the wild-type variant (in the way apoE2 does).
Each of these lead compounds is a candidate for the title “Elixir of Life”, offering extended lifespan, freedom from a slew of chronic inflammatory and degenerative diseases, and all the gain from exercise with none of the pain. Small wonder there has been a rush to found new companies to exploit this science. And with impressive results like the bexarotene paper and the phenome-wide association study linking apoE and depression reaching the scientific literature, providing ever stronger validation for the pathway, new entrants, big and small, are likely to throw their hats into the ring as well.
The prize may be very large indeed, for public health and for investors. The last company to hold out the promise of an “Elixir of Life”, Sirtris, was sold to GSK for $720million up-front in 2008. And although their lead compound, resveratrol, was discontinued due to side-effects, sirtuins remain a promising therapeutic target. But compounds that mimic the effect of apoE on cell debris clearance may be bigger yet. It’s a bold prediction, but DrugBaron envisages an impact on global health for the best apoE mimetics that matches or exceeds that of antibiotics and statins. You would expect nothing less from a tiny pill that packs in all the goodness of a daily exercise routine while you sit on the couch reading the latest from DrugBaron.
RxCelerate Ltd is an outsourced drug development platform based near Cambridge, UK. We specialize in delivering an entire road map of drug development services from discovery and medicinal chemistry through to formal preclinical development and clinical up to Phase IIa. In the last five years, we have witnessed dramatic changes in the drug development …