Fight Aging!

Karl Pfleger is one of the small community of angel investors and philanthropists who collectively initially supported the first rejuvenation biotechnology companies to emerge in this present generation of the longevity industry. Here he is performing the public service of publishing a curated list of biotechnology companies in the industry, startups that are either definitively or at least arguably working on a means to intervene in important mechanisms of aging, along with their targets and progress to date. That there are still fewer than 100 such companies indicates that this is very much an industry in its initial growth phase - but growth is certainly happening. The sizable pools of venture funding dedicated to the longevity industry, such as Juvenescence and Life Biosciences are attracting new entrepreneurs, and the scientific community is starting to realize that the prospects for advancing their research programs into clinical translation have greatly improved these past few years.

Chronic diseases of aging have over the past century taken over from infectious diseases as the predominant causes of death and suffering. The science of aging has shown over the past few decades that certain slow biological changes collectively underlie most (if not all) chronic diseases. The aging and longevity field, the understanding of these slow changes and how to interfere with them, is currently a small part of the overall medical, healthcare, and biotech spaces, but the efficiency of targeting the underlying causes of multiple diseases will rapidly cause aging to grow to become a much larger portion.

The aging and longevity field has recently grown to the point where it is difficult to follow important developments, even for insiders. There are books, journals, and blogs, but few sources of structured information to refer to for broader context or to consult for targeted inquiries, particularly few focused narrowly on aging defined as the underlying molecular causes of multiple age-related diseases.

As an especially important example, the internet previously had no reasonably comprehensive and precise list of companies with therapies or diagnostics for underlying aging in the above sense. Some argue that aging will be a scientific, commercial, and cultural revolution to rival any others. What is even more certain is that the field is important and of interest to many, so concise ways to pay attention will be useful. This will be a living site with ongoing updates. Focus will be on content, not flashiness, with the goal being utility for the community, those interacting with it, and the wider interested public.

Link: http://agingbiotech.info/

We lose a third of our life to sleep. If we didn't need to sleep at all, then we would have the experience of living 50% longer, considered subjectively. We would accomplish much more, experience much more. There are, unfortunately, few useful ways to safely reduce the amount of time spent asleep, without reductions in the quality of life while awake. Here, researchers report on a rare human genetic variant that manifests itself in a family whose members with the mutation need comparatively little sleep to be fully rested, and who appear to be otherwise unaffected by this genetic difference.

This discovery may well prove to be the basis for enhancement treatments to reduce required sleep time in the years ahead. We should consider the caveats, however: sleep appears to be important in clearance of some aggregates from the brain, and it could be the case that individuals who sleep less have raised rates of neurodegenerative disease in late life, but these possible risks and associations have not yet been evaluated.

An understanding of the regulatory mechanism for sleep lays at the foundation for healthy living and aging. Sleep behavior has long been thought to be regulated by the interactions of circadian clock and sleep homeostasis pathways. In humans, variations of genetically inherited sleep features in the population have been recognized for a long time. Importantly, human sleep has unique features that are different from that of animal models. For example, human sleep is usually consolidated, whereas mice sleep throughout the 24 hour day. Drosophila sleep-like behavior is consolidated into one long period, but the level of similarity between the Drosophila and human molecular regulatory mechanisms remains unclear.

Previously, we identified a series of genetic variations that influence the timing of sleep in humans, and mouse models of these mutations mostly recapitulate the phenotypes. Timing of sleep is heavily influenced by the circadian clock, which has been intensely studied, and we now have a large and growing body of knowledge on how the clock is regulated at the molecular level. On the other hand, our understanding of sleep homeostasis regulation for human lags behind. We reported a mutation in the human DEC2 gene that causes mutation carriers to sleep 6 hours nightly for their entire lives without apparent negative effects. Another mutation in DEC2 was later reported in a single individual who is a short sleeper and resistant to sleep deprivation. Identification of additional genes participating in modulation of human sleep duration provides a unique way to expand our knowledge of genes and pathways critical for human sleep homeostasis regulation.

Noradrenergic signaling in the central nervous system (CNS) has long been known to regulate sleep. The network involving the noradrenergic neurons has been extensively studied, and most of the receptor subtypes have been genetically defined. In contrast to α1 and α2 adrenergic receptors (ARs), relatively little is known about the function of β receptors in the CNS. βARs within the brain were previously suggested to mediate the effect of norepinephrine (NE) for alert waking and rapid eye movement (REM) sleep. Clinically, β-blockers are widely used and can be associated with difficulty falling asleep and staying asleep, possibly due to reduced production and release of melatonin.

We report here a rare mutation in the β1AR gene (ADRB1) found in humans with natural short sleep. Engineering the human mutation into mice resulted in a sleep phenotype similar to that seen in familial natural short sleepers. We show that β1AR is expressed at high levels in the dorsal pons (DP). Neuronal activity measured by calcium imaging in this region demonstrated that ADRB1+ neurons in DP are wake and REM sleep active. Manipulating the activity of these ADRB1+ neurons changes sleep/wake patterns. Also, the activity of these neurons was altered in mice harboring the mutation. Together, these results not only support the causative role of this ADRB1 mutation in the human subjects but also provide a mechanism for investigating noradrenaline and β1AR in sleep regulation at the circuit level.

Link: https://doi.org/10.1016/j.neuron.2019.07.026

The lymphatic system a part of the broader circulatory system and has many similarities to the cardiovascular system of blood vessels. The lymphatic system is also a network of vessels, transporting various necessary cells and substances, and subject to processes of aging that degrade function and thereby cause issues. While circulation of fluid is an important function of the lymphatic system, and conditions such as lymphedema arise when it runs awry, the role of lymphatic vessels in the function of the immune system is arguably far more vital.

Immune cells must be able to rapidly travel the body and locally coordinate with one another in order to mount an effective immune response. The lymphatic system links repositories of immune cells, such as those of the spleen and thymus with tissues throughout the body. Lymphatic vessels also link the numerous lymph nodes of the body, where immune cells gather to secrete and accept signals necessary to the correct function of the immune response.

With aging, lymph nodes in particular degenerate and become fibrotic, making it ever harder for immune cells to mount an acceptable defense against pathogens and cancers. The presence of senescent cells and the chronic inflammation they produce is thought to be important in this process, but it is likely one of a number of contributing factors. Further, lymphatic vessels suffer problems arising from loss of control over permeability, regarding what can pass through the vessel walls, as well as many of the same issues with stiffening that are seen in aged blood vessels. This latter problem results from processes such as cross-linking that reduces elasticity by restricting the motion of the complex molecules making up the extracellular matrix, and dysfunction of the smooth muscle tissue responsible for contraction and dilation. The latter degeneration is also in part a consequence of senescent cells and chronic inflammation, but mitochondrial dysfunction is also implicated - as demonstrated by the ability of NAD+ enhancement to improve matters in older patients.

Pathophysiology of aged lymphatic vessels

The aging process induces changes in structure and function of lymphatic networks. Lymphatic-related diseases are prevalent in elderly, such as lymphedema. In 1960s, the specific "varicose bulges" in muscular lymphatic vessels were observed and this bulges were increased with age. Muscle cell atrophy, elastic elements destruction, and aneurysm-like formations were also found in aged lymphatic vessels. Aging associated alterations in lymphatic contractility decrease pump efficiency which result in excessive retention of tissue fluid within interstitial spaces.

Reduced responsiveness to inflammatory stimuli in aged lymphatic vessels decreases the normal capacity to react against foreign organisms. The occurrence of high permeability is caused by the loss of glycocalyx and the dysfunction of junctional proteins. In addition, increased caspase-3 activity, the dissociation of the VE-cadherin/catenin complex and the low expression of actin cytoskeleton that occur in aged blood vessels may also be seen in aged lymphatic vessels.

The lymphatic endothelial cell surface is covered by the glycocalyx layer on the lumen side. The glycocalyx functions as a barrier between lymphatic fluid and the endothelium to prevent immune cells and pathogens from adhering to the endothelium. A significant loss of glycocalyx with a reduction in thickness and destruction in continuity occurs in lymphatic endothelial membranes from aged rat. This observation was in contrast with the intact, continuous layer covering cell membranes from adult lymphatic vessels. The global proteomic analysis of ultrastructural changes of glycocalyx composition also demonstrated a dramatic difference between the adult and aged groups. The thin glycocalyx layer is impaired in its ability to limit certain pathogens from adhering to the endothelial cell membrane and becomes hyperpermeable in the lymphatic vessels from aged rats. Thus in aged lymphatic vessels, pathogens could escape more easily from the collectors into surrounding tissue, along with an increased leakage of lymph fluid and immune cells.

The effect of aging-related hyperpermeability is also observed in blood vessels. Adherens junctions consisting of vascular endothelial cadherin (VE-cadherin) and β-catenin maintain intercellular permeability in both blood vessels and lymphatic vessels. β-catenin, is an intracellular protein that links cadherin with the actin cytoskeleton. Studies have found that aging process may affect all of the adherens junctional proteins. First, global proteomic profiling of the lymphatic vessels from aged rats revealed a significant decrease in cadherins. The downregulation of cadherins expression results in a decreased number of adherens junction complexes. In contrast, β-catenin is a key regulator of barrier integrity and a known substrate for caspase 3, which is an effector caspase in the apoptotic signaling pathway.

Recent research found that increased activity of the intrinsic apoptotic signaling pathway in aged vessels leads to high expression of proapoptotic members (Bak, Bax). Caspase 3 is activated by Bak and mediates barrier dysfunction through the disruption of β-catenin. This series of reactions eventually causes dissociation of the VE-cadherin/β-catenin complex and results in vascular hyperpermeability.

In addition to adherens junctions, tight junctions are an equally important determinant of vascular permeability of blood vessels and lymphatic vessels. As part of the tight junction, occludin and claudin-5 showed significantly low expression level in senescent endothelial cells. We hypothesize that the mechanism of intercellular hyperpermeability caused by the disruption of endothelial cell-cell junctions in aged blood vessels may also exist in aged lymphatic collectors. Further investigations are needed to delineate the detailed mechanisms related to impaired barrier function in aged lymphatic vessels.

It is well known that wealth correlates with greater life expectancy and related measures such as improved health in later life. The question has always been why this is the case. Wealth correlates with a range of other factors such as education, intelligence, and so forth, all of which can be reasonably expected to influence health via lifestyle choices. In the case of intelligence, there is also some evidence to suggest that more intelligent people are also more physically robust, but the mass of evidence of genetics and longevity also suggests that effect size for genetic variation is small in comparison to that for lifestyle choice.

Genetics, lifestyle and environment are all factors that somehow influence when and how we all age. But the financial situation is also important. Now, researchers have found that four or more years with an income below the relative poverty threshold during adult life make a significant difference as to when the body begins to show signs of ageing.

To learn more about the context, the researchers have tested 5500 middle-aged persons, using various ageing markers: physical capability, cognitive function and inflammatory level. The results were then compared with the participants' income throughout the 22 years leading up to the test. An annual income of 60% below the median income is considered relative poverty. In this way, the researchers found that there is a significant correlation between financial challenges and early ageing.

The participants have been through both physical and cognitive tests, each of which is an expression of general strength and function. Among other things, the researchers measured the participants' grip strength, how many times they could get up from and sit on a chair in 30 seconds, and how high they could jump. The cognitive tests have e.g. been tasks of memorising sequences. The results show, among other things, that the financially challenged group, relative to the comparison group, can get up and sit down two times less per 30 seconds, and that their grip strength is reduced by 1.2 kilos.

In addition, the researchers have measured the inflammatory level of the participants - i.e. an inflammatory state that comes from within and is measured in the blood. A high inflammatory level is a sign that the body is in a state of alert and can likewise be used as a marker for illness and ageing. The study shows that the financially challenged also had higher inflammatory levels.

Link: https://healthyaging.ku.dk/news/repeated-periods-of-poverty-accelerate-the-ageing-process/

Researchers here report on genetic manipulations that alter the function of microtubules in the cells of nematode worms and thereby cause increased longevity. Microtubules help to support cell structure, and act as pathways to allow movement of structures and molecules around the cell. The effect on longevity appear to be mediated via improved or altered function of neurons, leading to alterations in metabolism of a sort already known to influence life span in this species. The degree to which this is relevant to aging in higher organisms remains to be established, but as a general rule one should expect ever smaller effect sizes for this sort of thing as one moves to test in larger and longer-lived species. Short-lived species have lifespans that are very plastic in response to metabolic stress and environmental change, while long-lived species do not.

Microtubules (MTs) play fundamental roles in cellular functions including cell division, cell shape, and intracellular transport. Abnormal MT regulation has been linked to age-related disorders and diseases, and MTs often serve as targets for disease therapies. MT regulation is particularly important for neurons, since their polarized morphologies dictate heavy reliance on MT function for their maintenance. MT regulation is involved on several levels in neuronal function and maintenance of neuronal structure, and also appears to be a general downstream indicator and effector in age-dependent neurodegeneration.

The nervous system modulates lifespan in various species. It detects sensory cues from the environment and internal signals from the animal, and coordinates organismal metabolic homeostasis and energy balance. Different neuron types respond to distinct cues to extend or shorten lifespan through activating distinct neuronal signals and signaling pathways, including the insulin/insulin-like growth factor-1 signaling (IIS) pathway, which plays an evolutionarily conserved role in regulating lifespan. Despite the essential role of MTs in neuronal function and the central role of the nervous system in regulating longevity, MT regulation has not been directly linked to lifespan modulation.

We have recently reported that mutations in MT regulators can affect lifespan in a DAF-16 dependent manner. We found that loss of EFA-6, a protein promotes MT catastrophe, increased mean lifespan of C. elegans and delayed age-associated changes in neuronal integrity, such as axon blebbing and branching as well as mislocalization of synaptic vesicles to dendritic structures within touch sensory neurons. The effects of the loss of EFA-6 opposed those effects seen with the loss of PTL-1, the worm homolog of human Tau protein that stabilizes MT, in previous research as well as experiments conducted in our study.

These results suggest that shifting the balance of neuronal microtubule regulation towards stabilization rather than destabilization without completely abolishing microtubule destabilization processes facilitates the maintenance of neuronal structure and promotes organismal longevity in C. elegans. The efa-6 loss-of-function mutants also maintained greater touch sensitivity and motor function during aging compared to wild-type worms. These differences in neuronal integrity and functional ability were time/aging-dependent with no significant differences between the efa-6 loss-of-function mutants and wild-type worms at day 1 of adulthood but significant differences between the groups by days 9 and 10 of adulthood. In addition, the expression of EFA-6 in neurons, but not in muscle, rescued the phenotypic changes seen in the loss-of-function mutants, suggesting that the regulation of microtubules within neurons is what contributes to the regulation of lifespan.

Link: https://doi.org/10.18632/aging.102224

The Forever Healthy Foundation's Rejuvenation Now program is engaged in the production of detailed analyses of risk and reward for presently available treatments that may act to slow or reverse aging. I think this is helpful, as a great deal of information exists, but is very scattered, and there is far too much uninformed hype out there. Putting all of the facts together in one place, coupled to a sober assessment of what those facts mean, is a good use of resources. This is particularly true given that senolytic therapies presently exist, and, to the degree to which these treatments successfully clear senescent cells with minimal side-effects, should be expected to produce sizable benefits to health for all old people who use them. Those old people just need to be told, so that they can make an informed choice about their own health.

One of the potential senolytic therapies of interest is a cheap and readily available supplement, fisetin. This is interesting because animal data shows it to be about as good as the dasatinib plus quercetin combination. One might ask how a supplement can be readily available for years, and yet no-one noticed that if you take the whole bottle at once, it significantly reverses inflammatory age-related conditions. Perhaps that is in fact the case, but as the analysis from the Forever Healthy Foundation notes, we just don't know. Human trials are ongoing, and we might expect initial publications from the research groups involved over the next year. This will clarify whether or not fisetin happens to be unusually effective in mice only.

In a sense, either outcome would be surprising. The important parts of the biochemistry of senescent cells, when it comes to the operation of senolytic drugs, are essentially the same between mice and humans. The dasatinib and quercetin combination has been shown to work in humans much as it does in mice when it comes to destroying these cells. Yet fisetin has been readily available and widely used as a supplement for a while, without the sort of attendant murmuring one might expect given the sizable benefits it should produce if it is as senolytic in humans as it is in mice. We shall see what the story is when the clinical trial data for fisetin emerges. Meanwhile, many more self-experimenters are using fisetin than are using the far more proven dasatinib and quercetin combination, given that fisetin is much more easily obtained.

Fisetin Senolytic Therapy Risk-Benefit Analysis

Senolytics are agents that selectively induce apoptosis of senescent cells. Fisetin is a flavonoid polyphenol found in many types of fruits and vegetables that is believed to act as a senolytic in addition to its numerous other known benefits. Although natural senolytics are less potent, compared to the targeted senolytics, they have lower toxicity and are thus, likely to be more readily translatable to clinical medicine. This risk-benefit analysis focuses on the risks and benefits of using fisetin as a senolytic rather than its more common use as a supplement.

There are currently three phase 2 clinical trials underway and the first data is expected to be reported in about a year. The data from the phase 1 trials has not been published. All trials are being conducted by the same investigators at the Mayo clinic using the same treatment protocol. Only two papers directly related to the use of fisetin as a senolytic were identified, neither of which were conducted in humans. The other 5 studies included in the table relate to pharmacokinetics, risk, and lifespan extension.

To the best of our knowledge, there haven't been any studies published on the pharmacokinetics of fisetin in humans. Only one animal study on fisetin has reported any form of toxicity from fisetin use and the authors concluded that the elevations in ALT/AST levels (indications of liver toxicity) were in large part due to the vehicle used to administer the fisetin (DMSO). However, the fisetin + vehicle group showed significantly higher elevations than the vehicle alone group indicating that high doses of fisetin may additionally burden the liver because of its poor bioavailability. At a lower dose (112 mg/kg), fisetin didn't significantly increase apoptosis or lead to liver toxicity. Intermittent dosing and use of a form of fisetin with increased bioavailability are likely to mitigate the risk of liver toxicity.

Fisetin has been shown to decrease senescent cell biomarkers as well as the numbers of senescent cells in a variety of tissues, including ex vivo, human adipose tissue as well as in vivo, in mice. The primary risk mitigation strategy is to wait to commence therapy until clinical trial data has been published that describes the possible benefits and adverse effects. At the current time, the only form and dose that has been tested in phase 1 clinical trials is the so-called "Mayo Protocol". The Mayo Protocol consists of taking 20 mg/kg of oral fisetin on two consecutive days and repeating the same dose, one month later.

To what degree does early life development impact the trajectory of late life aging? Plenty of evidence suggests a connection, much of it from epidemiological studies that, unfortunately, given little insight into possible mechanisms. Of other work, applying reliability theory to aging can only fit the observed mortality data if we are born with a non-zero level of damage. Further, early exposure to cytomegalovirus, a persistent infection that is corrosive of immune function over the long term, may explain links between socioeconomic status in childhood and pace of late life aging. These are two of many studies to provide hypotheses and at least some supporting evidence.

A diverse set of mechanisms could transform episodic or recurrent early exposures in utero, perinatally, and during infancy and early childhood into delayed impacts on adult illness, disability, and mortality. The mechanisms are associated with organ-specific embryo and fetal cell growth and differentiation, epigenetic changes, exposure to and contraction of early childhood diseases and sustained inflammation, and experiences with stressful conditions and environments. Furthermore, a large and influential body of empirical research documents the long-lasting impact of early nutritional status on adult health and mortality. Finally, there is widespread empirical evidence demonstrating that more diffuse exposures, such as poverty and severe deprivation in infancy and early childhood, can also have lasting impacts.

The mechanisms identified above are the focus of various strands of theories with unique histories and distinct disciplinary foundations. Different as they may be, however, they share an important trait as all invoke perturbations during critical periods of the development of a phenotype triggered by insults before conception, during embryonic and fetal life, perinatally, and across early stages of physical and cognitive growth. These early insults may then lead to disruptions in processes of organ growth, differentiation and function, immune response, neurological development, metabolic regulation, and even the formation of adult preferences and behaviors. After variable but usually prolonged latency periods, the disruptions could manifest themselves as increased susceptibility to adult chronic illness.

This shared feature emphasized by multiple variants of developmental effects on aging coexists with important differences that segregate them into distinct classes associated with different outcomes. For example, mechanisms that depend on epigenetic changes, such as methylation of CpG islands caused by nutrient deficiencies, disrupt metabolic function, and are associated with child and adult obesity. Others involve organ damage caused by an immune overresponse to bacteria, as in the case of acute rheumatic heart fever that increases the risk of adult heart valve stenosis. And yet others depend on misfiring of the hypothalamic-pituitary-adrenal (HPA) axis, an adaptation among some mammals to chronic stress, as when early abuse triggers adult depression, aggressiveness, and anxiety. The differences between these mechanisms range over a number of domains including nature and timing of insults, critical and sensitive periods, chronic illnesses, critical ages after which the damage is manifested, and interactions between initial insults and individuals' lifetime exposure to different environments.

Link: https://doi.org/10.4054/DemRes.2019.40.41

Platelets are essentially structured chunks of cytoplasm shed by the megakaryocyte cells responsible for producing them, released into the blood stream. They are important in blood clotting and the innate immune response. Inappropriate blood clot formation known as thrombosis occurs more readily in later life, but it is unclear as to the degree to which age-related changes in platelets, versus other systems, are important to this process, or where platelets sit in the complex chains of cause and effect. The open access paper noted here reviews what is known of the aging of platelets and related mechanisms, a topic that is not as well investigated as other aspects of cardiovascular aging.

Platelet count is inversely associated with age. A large study based on the Third National Health and Nutrition Examination Survey including 12,142 American subjects showed a significant decrease of 10 × 10^3 platelets/μL in individuals in the 60-69 year age group as compared with those between the ages of 20-59 years, and of 20 × 10^3 platelets/μL in patients aged over 69 years of age, after adjusting for many covariates such as nutritional deficiencies, medication, inflammatory conditions, autoimmune or viral illnesses, and consumption of alcohol and tobacco. This suggests that the drop in platelet count with age is part of the biological aging process per se and not only due to environmental factors.

One of the most documented changes in platelet function during aging is platelet hyperactivity. Bleeding time decreases significantly in aging, denoting a faster clot formation and indirectly an enhanced platelet activity in the elderly. Furthermore, platelets from older men and women have a greater sensitivity to aggregation induced by classical agonists. Furthermore, β-thromboglobulin and platelet factor 4 (PF4), two proteins secreted from platelets α-granules, are both found at a significantly higher level in plasma of older compared with younger subjects. This is consistent with the hyperaggregability observed in elderly individuals since platelets release their granule content during activation.

The mechanisms of this age-related platelet hyperactivity remain unclear. Researchers have tested the hypothesis that modifications in phosphoinositide turnover, an important signaling mechanism of platelet activation, may be responsible for platelet hyperactivity in aging. They have found that platelet phosphoinositide turnover is enhanced in aging and correlates positively with platelet aggregation and plasma β-thromboglobulin levels. It has also been suggested that there could be a functional or expressional change in platelet α and β-adrenoreceptors, however the reported literature is conflicting.

Link: https://doi.org/10.3389/fcvm.2019.00109

Setting aside the mice genetically engineered to destroy senescent cells, the combination of dasatinib and quercetin is the oldest of the senolytic treatments used in animal studies. Senolytic therapies are those that selectively destroy senescent cells in old tissues in order to produce rejuvenation, turning back the progression of numerous age-related conditions. Unusually for early stage research, these initial senolytics are actually quite effective, considered in the grand scheme of things. Thus they have moved directly to human trials in some cases. The first data on their ability to produce the same outcomes in humans as in mice emerged this year, and more data will continue to roll out over the next few years as the first trials run and complete. The results reported in today's open access paper provide an important demonstration for those not yet convinced that the animal data is relevant.

Meanwhile, the older members of the self-experimentation community have been using dasatinib, quercetin, and a few other senolytics for a few years now on the strength of the animal data and the whisper network of positive outcomes. Further, groups such as the Age Reversal Network are attempting to build physician networks and support for off-label use of senolytics.

While senescent cells are important in a range of beneficial processes, from cancer suppression, to the Hayflick limit, to wound healing, their presence is temporary in all such cases. Near all are destroyed, either by programmed cell death or by the immune system. Lingering senescent cells, on the other hand, are an entirely different story: they are in fact an important cause of aging and age-related dysfunction. They secrete a potent and inflammatory mix of signals that rouses the immune system to destructive chronic inflammation, degrades surrounding tissue structure and function, and encourages other cells to also become senescent.

The more lingering senescent cells present in the body, the worse the outcome for health, and their numbers steadily and inexorably grow with age. The silver lining here is that senescent cells actively maintain a degraded, aberrant state of metabolism, regeneration, and tissue function. If they are removed, rejuvenation takes place quite rapidly. In some cases, surprising levels of rejuvenation, with features of aging that might have been thought irreversible, and that no existing medicine can much affect, vanishing as the tissue environment becomes less aged and dysfunctional. The world at large will begin to wake up to the true potential here as trials continue, but it remains a tragedy that hundreds of millions of people worldwide could have their quality of life significantly improved overnight, if they only knew. But it isn't happening. Dasatinib and quercetin are both mass produced and cheap, easily obtained, easily used. Too little is being done to bring this treatment to the masses who could benefit.

Mayo researchers demonstrate senescent cell burden is reduced in humans by senolytic drugs

In a small safety and feasibility clinical trial, researchers have demonstrated for the first time that senescent cells can be removed from the body using drugs termed "senolytics". The result was verified not only in analysis of blood but also in changes in skin and fat tissue senescent cell abundance. Senescent cells are malfunctioning cells that accumulate with aging and in organs affected by chronic diseases. Senescent cells can remain in the body and contribute to multiple diseases as well as features of aging, ranging from heart disease to frailty, dementias, osteoporosis, diabetes, and kidney, liver, and lung diseases.

For three days the nine participants received a combination dose of dasatinab and quercetin. Though the drugs cleared the body in a couple of days, effects on reducing senescent cells were evident for at least 11 days. The researchers say this shows the senolytic drug combination significantly decreases senescent cell burden in humans. Senescent cells are characteristic in end-stage kidney failure as well as diabetes-related kidney disease. By removing the cells from mice, researchers had previously found that senolytics alleviate insulin resistance, cell dysfunction, and other processes that cause disease progression and complications. While more research is needed on the impact of senolytics on diseases and disorders of aging, the researchers say the results of occasional dosing reduces risks from having to give drugs continuously.

Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease

By definition, the target of senolytics is senescent cells, not a molecule or a single biochemical pathway. The first senolytic drugs, Dasatinib (D) and Quercetin (Q), were discovered using a mechanism-based approach instead of the random high-throughput screening usually used for drug discovery. Because senescent cells can take weeks to months to develop and do not divide, and because even eliminating only 30% of senescent cells can be sufficient to alleviate dysfunction in preclinical studies, D + Q is as effective in mice if administered intermittently, for example every 2 weeks to a month, as continuously, even though D and Q have elimination half-lives of only 4 and 11 hours, respectively. This is consistent with the point that, since the target of senolytics is senescent cells, these drugs do not need to be continuously present in the circulation in the same way as drugs whose mechanism of action is to occupy a receptor, modulate an enzyme, or act on a particular biochemical pathway, at least in mice. Intermittently administering D + Q effectively circumvents any potential off-target effects due to continuous receptor occupancy or modulation of an enzyme or biochemical pathway.

Based on the many promising studies of D + Q in mice, the experience gained from the use of D in humans for over 20 years, and the fact that Q is a natural product present in many foods such as apples, we initiated clinical trials of these agents. The first-in-human clinical trial of senolytics was a brief course of D + Q for patients with idiopathic pulmonary fibrosis, which resulted in statistically significant improvements in physical function in 14 subjects with this relentlessly progressive, debilitating, and ultimately fatal cellular senescence-driven disease. Although alleviation of this cellular senescence-related phenotype was demonstrated in that pioneering trial in tandem with trends for decreased senescence-associated secretory phenotype (SASP) factors, and others found decreased SASP factors in a trial of continuous D administration in the skin of subjects with systemic sclerosis, so far, there has been no direct demonstration of senescent cell clearance by senolytic drugs in peer-reviewed published human clinical trials.

To test whether intermittent D + Q is effective in targeting senescent cells in humans, we administered a single 3 day course of oral D + Q and assayed senescent cell abundance 11 days after the last dose in subjects with diabetic kidney disease, the most common cause of end-stage kidney failure and which is characterized by increased senescent cell burden. We found D + Q alleviates insulin resistance, proteinuria, and renal podocyte dysfunction caused by high fat diet-induced or genetic obesity in mice. We also found that even Q alone can prevent high fat diet-induced increases in markers of senescence, renal fibrosis, decreases in renal oxygenation, and increased creatinine in mice, although Q alone did not prevent insulin resistance. Diabetes and chronic kidney disease (CKD) in humans therefore represent conditions that may benefit from D + Q therapy-induced alleviation of tissue dysfunction and disease progression, which is being tested as the clinical trial reported here continues. In this interim report of findings from that trial, we found the single brief course of D + Q attenuated adipose tissue and skin senescent cell burden, decreased resulting adipose tissue macrophage accumulation, enhanced adipocyte progenitor replicative potential, and reduced key circulating SASP factors.

Chimeric antigen receptor (CAR) T cell therapies are used to treat cancer, engineering T cells to be more aggressive towards cancer cells. The approach has proven quite effective in comparison to past treatments for a number of cancer types. In principle this CAR-T immunotherapy can be used to target any cell population that has distinct surface markers, not just cancer cells. Here, researchers demonstrate the ability to destroy the fibroblasts responsible for generating fibrosis in the aging heart. Fibrosis is a form of dysregulated tissue maintenance, in which cells build up scar-like deposits of collagen that degrade tissue structure and function. It is interesting to compare this with work on clearing senescent cells in heart tissue, which also reverses fibrosis. Senescence is clearly one of the factors driving fibroblasts to become overactive, most likely via the inflammatory, pro-growth signaling produced by senescent cells, rather than via fibroblasts becoming senescent in large numbers.

Heart disease is the leading cause of death in the United States, and excessive cardiac fibrosis is an important factor in the progression of many forms of heart disease. It develops after chronic inflammation or cardiac injury, when cardiac fibroblasts - cells that play an important role in the structure of the myocardium, the muscular middle layer of the heart's wall - become activated and begin to remodel the myocardium via extracellular matrix deposition. Research has shown that the removal of activated cardiac fibroblasts can reduce heart stiffness, making it easier for the ventricles to relax. However, there are no therapies that directly target excessive fibrosis, and very few interventions have shown the ability to improve heart function and outcomes among patients with impaired cardiac compliance.

As a first step, researchers launched a genetic proof-of-concept experiment using mice that can express an artificial antigen (OVA) on cardiac fibroblasts. The mice were treated with agents to model hypertensive heart disease, a condition associated with left ventricular hypertrophy (enlargement or thickening of the heart walls), systolic and diastolic dysfunction (pumping of blood in and out of the heart), and widespread cardiac fibrosis. To selectively target the OVA proteins expressing cardiac fibroblasts, the team treated one cohort of mice with engineered CD8+ T cells that express a T-cell receptor against the OVA peptide. At the four-week mark, the mice who were treated with the reengineered cells had significantly less cardiac fibrosis, whereas the mice in the control groups still had widespread fibrosis.

After establishing the feasibility of this approach, researchers sought to identify a protein specifically expressed by activated fibroblasts that they could program the genetically modified T cells to recognize and attack. Using an RNA sequence database, the team analyzed gene expression data of patients with heart disease and identified the target: fibroblast activation protein (FAP), a cell surface glycoprotein. Researchers then transferred engineered FAP CAR T-cells into mice at the one and two week marks, aiming to target and deplete FAP-expressing cardiac fibroblasts. Within a month, researchers saw a significant reduction of cardiac fibrosis in the mice that were treated with the engineered cells, as well as improvements in diastolic and systolic function.

Link: https://www.pennmedicine.org/news/news-releases/2019/september/car-t-cell-therapy-may-be-harnessed-to-treat-heart-disease

In Silico Medicine specializes in the application of computational methods to speed up the process of screening small molecule drugs, while reducing the costs, an advance in infrastructure technology that is very much in favor these days. Most of the large entities in medical development still proceed exactly has they have done for decades in the matter of developing new therapies: find a molecular target, then find a small molecule that influences that target, then iterate over variations to try to increase efficacy and reduce side-effects. Thus numerous groups work in this part of the field, trying to cut presently sizable costs and improve the presently poor odds of success. In this paper, the In Silico Medicine team demonstrates that they can very rapidly identify candidate small molecule senolytic drugs, capable of clearing the senescent cells that contribute to aging and age-related disease.

A team of researchers has succeeded in using Artificial Intelligence to design, synthesize and validate a novel drug candidate in just 46 days, compared to the typical 2-3 years required using the standard hit to lead (H2L) approach used by the majority of pharma corporations.

By using a combination of Generative Adversarial Networks (GANs) and Reinforcement Learning (RL), the team of researchers behind this study (documented in a paper published this month) have succeeded in validating the real power that AI has to expedite timelines in drug discovery and development, and to transform the entire process of bringing new drugs to market from a random process rife with dead ends and wrong turns to an intelligent, focused and directed process, that takes into account the specific molecular properties of a given disease target into account from the very first step.

Researchers have long advocated for the extreme potentials that AI has in terms of making the process of discovering and validating new drugs a faster and more efficient process, especially as it pertains to aging and longevity research and the development of drugs capable of extending human healthspan and compressing the incidence of age-related disease into the last few years of life. While this is the newest in a long line of steps and accomplishments aiming to turn the theoretical potentials of AI for longevity research into practice, it is also the largest step made thus far, and goes a very long way in terms of proving that potential via hard science.

Link: http://bg-rf.org.uk/press/ageing-research-to-accelerate-with-experimental-validation-in-ai-powered-drug-discovery

Cells communicate with one another constantly, and a large portion of that communication is not carried by individual secreted molecules, though there are certainly a lot of those, but rather takes the form of small membrane-bound packages of diverse molecules known as extracellular vesicles. Cells generate and secrete extracellular vesicles of various sizes, and other cells ingest them. Two important areas of active research into cell signaling are the way in which young tissue can restore the function of old cells, and the way in which senescent cells change the activity of surrounding cells for the worse. In both cases, extracellular vesicles are important in this process of communication and influence.

There is a substantial faction in the research community focused on potentially beneficial effects that derive from young blood, emerging from the study of heterochronic parabiosis in which the circulatory systems of a young and old mouse are linked. The old mouse benefits and shows some signs of reversal of the consequences of aging, the young mouse exhibits accelerated signs of aging. Is this in fact due to beneficial signals in young blood? There is good evidence that strongly supports the case that benefits result from a dilution of harmful factors in old blood, and that beneficial factors in young blood are not important. Nonetheless, there is further independent evidence in which factors or extracellular vesicles derived from young blood have been used to produce benefits in old mice. There are also a number of failures to show meaningful benefits from blood or plasma transfusion, in mice and humans. It is an interesting field, in which conflicting evidence abounds.

Extracellular vesicles circulating in young organisms promote healthy longevity

In the late 1950s, parabiosis experiments provided some scientific consistency to these beliefs. Indeed, a shared circulatory system was sufficient to increase bone weight and density of old mice when joined to younger ones. The same experimental design was applied to demonstrate a lifespan-enhancing effect of young blood. Many years later, elegant reports demonstrated a rejuvenation-promoting effect of young blood in a wide variety of cells and tissues, e.g. stem cells, muscle, brain, and the heart. However, the pursuit of the circulating factors responsible for such effects did not achieve the same success. In fact, the suggested pro-regeneration role of growth differentiation factor 11, a member of the TGFβ superfamily, has been questioned.

Extracellular vesicles (EVs) are membrane-coated nanoparticles actively released by almost all cell types. Increasing evidence indicates that both are able to shuttle and deliver functional proteins and nucleic acids in a paracrine and systemic manner. Blood contains a heterogeneous mixture of EVs of different origins, which are currently being characterized for therapeutic and diagnostic purposes. The effects of EVs are now attracting intense interest also in the context of ageing and age-related diseases (ARDs).

In particular, senescent cells (SCs) are emerging as major drivers of ageing and key contributors to inflammaging, the age-associated pro-inflammatory drift that promotes the development of ARDs. Recent evidence suggests that EVs are also central constituents of the SCs secretome. In particular, SCs secrete an increased amount of EVs, excreting pro-inflammatory DNA and possibly spreading pro-ageing signals. Conversely, a seminal paper suggests that a 4-month injection of small EVs derived from hypothalamic neural stem cells and rich in specific miRNAs into the hypothalamic third ventricle is sufficient to ameliorate some age-associated detrimental outcomes in C57BL/6 mice, including hypothalamic inflammation and the drop in physical activity.

These and other observations prompted the hypothesis that EVs are central mediators of the circulating communicosome fostering inflammaging. In that framework, we hypothesized that the chronic administration of EVs purified from a young healthy mouse to an old one should ameliorate some age-associated phenotypes. This experimental approach appeared to be enough feasible and robust to demonstrate a tangible role of EVs in the ageing process. Researchers have now shown a clear pro-longevity role for EVs isolated from young mouse plasma. Indeed, they injected EVs isolated from 4-to-12-month-old mice into 26-month-old female mice once a week until sacrifice and observed an increase of 10.2% and of 15.8% in median and maximal lifespan, respectively, in mice receiving the treatment vs. vehicle-treated mice of the same age.

The study here shows that given a population of individuals with hypertension, those who manage to control their high blood pressure go on to suffer lesser degrees of cognitive decline. Numerous mechanisms may link hypertension to structural damage in the brain: degeneration of the blood-brain barrier, allowing inappropriate molecules and cells into the brain, leading to neuroinflammation and other effects; rupture of capillaries causing microbleeds, effectively tiny strokes; outright pressure damage in tissue very close to small vessels that directly harms brain cells; and so forth. This damage adds up, but note that it is a set of physical issues that stem from increased pressure rather than the biochemistry that causes that increased pressure. Therefore these downstream issues can be suppressed by any method that reduces blood pressure consistently, even though that will leave the underlying damaged biochemistry to continue to cause other issues.

High blood pressure appears to accelerate cognitive decline among middle-aged and older adults, but treating high blood pressure may slow this down, according to a preliminary study. According to the American Heart Association's 2017 Hypertension Guidelines, high blood pressure affects approximately 80 million U.S. adults and one billion people globally. Moreover, the relationship between brain health and high blood pressure is a growing interest as researchers examine how elevated blood pressure affects the brain's blood vessels, which in turn, may impact memory, language, and thinking skills.

In this observational study, the researchers analyzed data collected on nearly 11,000 adults from the China Health and Retirement Longitudinal Study (CHARLS) between 2011-2015, to assess how high blood pressure and its treatment may influence cognitive decline. High blood pressure was defined as having a systolic blood pressure of 140 mmHg or higher and a diastolic blood pressure of 90 mmHg or higher, and/or taking antihypertensive treatment. According to guidelines of the American Heart Association, high blood pressure is defined as 130 mmHg or higher or a diastolic reading of 80 mmH or higher.

Researchers interviewed study participants at home about their high blood pressure treatment, education level, and noted if they lived in a rural or urban environment. They were also asked to perform cognitive tests, such as immediately recalling words as part of a memory quiz. Among the study's findings: (a) Overall cognition scores declined over the four-year study; (b) Participants ages 55 and older who had high blood pressure showed a more rapid rate of cognitive decline compared with participants who were being treated for high blood pressure and those who did not have high blood pressure; (c) The rate of cognitive decline was similar between those taking high blood pressure treatment and those who did not have high blood pressure.

Link: https://www.mailman.columbia.edu/public-health-now/news/high-blood-pressure-treatment-may-slow-cognitive-decline

Researchers here show that lipid turnover in fat tissue decreases with age, and suggest that this mechanism explains some fraction of the tendency to gain weight with age. Everyone of a certain age recognizes that it takes ever more effort to evade or get rid of excess fat tissue. It remains an open question as to which underlying mechanisms cause this change in lipid turnover, though given progress in rejuvenation research we are at the point of being able to test hypotheses such as chronic inflammation resulting from senescent cells, or mitochondrial dysfunction. We shall see what new data on this topic emerges in the years ahead.

Scientists studied the fat cells in 54 men and women over an average period of 13 years. In that time, all subjects, regardless of whether they gained or lost weight, showed decreases in lipid turnover in the fat tissue, that is the rate at which lipid (or fat) in the fat cells is removed and stored. Those who didn't compensate for that by eating less calories gained weight by an average of 20 percent, according to the study.

The researchers also examined lipid turnover in 41 women who underwent bariatric surgery and how the lipid turnover rate affected their ability to keep the weight off four to seven years after surgery. The result showed that only those who had a low rate before the surgery managed to increase their lipid turnover and maintain their weight loss. The researchers believe these people may have had more room to increase their lipid turnover than those who already had a high-level pre-surgery.

"The results indicate for the first time that processes in our fat tissue regulate changes in body weight during ageing in a way that is independent of other factors. This could open up new ways to treat obesity." Prior studies have shown that one way to speed up the lipid turnover in the fat tissue is to exercise more. This new research supports that notion and further indicates that the long-term result of weight-loss surgery would improve if combined with increased physical activity.

Link: https://news.cision.com/karolinska-institutet/r/new-study-shows-why-people-gain-weight-as-they-get-older,c2899205

Here Matthew O'Connor of the SENS Research Foundation talks about the research that led to founding of Underdog Pharmaceuticals, a biotech startup incubated by the foundation to commercialize a means of targeting 7-ketocholesterol in atherosclerosis and other conditions. Oxidized cholesterols, and largely 7-ketocholesterol, are the primary cause of dysfunction in the macrophage cells normally responsible for preventing the build up of fatty plaques in blood vessel walls. That dysfunction is the cause of atherosclerosis, and the fact that the presence of oxidized cholesterols increases with age is one of the reasons why atherosclerosis is an age-related disease, and why young people don't exhibit the plaques that narrow and weaken blood vessels.

A sufficiently effective way of selectively clearing 7-ketocholesterol from the body should go a long way towards preventing and reversing atherosclerosis - and possibly other conditions as well. As noted here, there is evidence for 7-ketocholesterol to accumulate in other tissues and contribute to age-related conditions in other ways. The particular approach taken by the SENS Research Foundation scientists is to find a non-toxic molecule that selectively binds to the toxic molecule that one would like to remove, 7-ketocholesterol in this case, and deliver it to the body in volume. The bound molecules are then processed and excreted in the normal way. It will be interesting to see how the Underdog project progresses in the years ahead.

Matthew O´Connor presenting at Undoing Aging 2019

Thank you everybody. It is a real honor and a pleasure to be here today, speaking in a last minute slot. Don't volunteer to take over to speak for someone who didn't show up, because then you have to stay up all night, working on your slides. This slide is the SENS team from this last summer, a combination of the MitoSENS team and the team working on 7-ketocholesterol that Aubrey de Grey was just referring to, that we're calling the Underdog team. You can see me as the chief Underdog there.

I want to just spend a minute, Aubrey had mentioned that this is the tenth anniversary of SENS Research Foundation, and it is my 9th year with SENS Research Foundation. It has been a privilege to have started so early, to help build the lab, and I wish that I could show a time lapse photography of where it started and where it has gone, and also to be involved with some of these amazing interns. So many of these projects, including this one, are very intern driven. Regarding the MitoSENS team, people may not realize that all the progress Aubrey was talking about, publishing papers all of a sudden, didn't happen until after we hired Amutha, so you can see why that project got taken away from me and given to her. She is just a phenomenal mitochondrial biologist.

So this project that I'm about to tell you about involves a story about 7-ketocholesterol. I hope that a lot of you have heard about it. It is kind of one of the classic bad guy molecules that Aubrey has been talking about for a long time. My history with it goes back even further than SENS Research Foundation, as the foundation and Methuselah Foundation before it have been funding work on trying to get rid of this really nasty molecule 7-ketocholesterol for at least ten years, with work that we've funded in various places. I actually heard Aubrey give a talk about this, one of the best talks I've ever seen, at Rice University something like 15 years ago when I was in graduate school. So I've been thinking about it ever since then.

This project kind of started out as some ideas about how to think about this problem and how to get rid of this nasty molecule a different way. It started out as something that we were sort of dabbling in, and graduated from being my 20% project to something that is all-consuming now. On this slide, you can see causes of death worldwide, which probably looks familiar to you many of you. As Aubrey was just saying, atherosclerosis is the world's biggest killer. If you risk-adjust all of these diseases for what their underlying cause is, atherosclerosis is believed by world health organizations to kill about 44% of everybody. This molecule, 7-ketocholesterol, an oxidized form of cholesterol, is thought to be one of the earliest stages leading to atherosclerosis. But not everyone simplistically believes in that model; the classic model is that you eat too many hamburgers and you get too much cholesterol in your bloodstream, and that just sort of stochastically builds up, and eventually you get plaques, and eventually they rupture and you have heart attacks and strokes, and you die.

However 7-ketocholesterol is many, many, many times more toxic than LDL cholesterol is - so I call it the really bad cholesterol. It is by far the most common product of the reaction between a free radical and cholesterol, and so more often than not you get this stable 7-ketocholesterol which is extremely toxic. It has no useful purpose in your body. It can accumulate in the lysosomes of macrophages and can be an early step in the progression towards becoming a foam cell. Foam cells build up as a layer in atherosclerotic plaques, and 7-ketocholesterol is found inside them, as well as in the necrotic core of the plaque.

Let me change gears now and reveal the class of drug, the class of molecules, that we have been playing around with for the last few years. They are called cyclodextrins. You may or may not have heard of them, but they are a huge industry, and they have a huge variety of applications. The medical applications, despite the fact that cyclodextrins have been studied and applied for many decades, are only just starting to be realized. They come in three basic flavors, alpha, beta, and gamma, which are three different sizes, and they have six, seven, or eight different sugar rings that they are made out of. There are different forms of them, probably thousands of different ones that have been invented. They are extremely customizable and modifiable. Any one of these hydroxyl groups you can stick just about anything you want on it, doing synthetic chemistry.

The slide here shows just a few examples of common cyclodextrins that are used for various industrial purposes. Medically, they are mostly now only used as excipients, meaning as carrier molecules for small hydrophobic drugs. They are used in food: alpha-cyclodextrin is approved in the European Union as a bulk fiber supplement, so maybe you ate some of it this morning. There are versions of these that are extremely low toxicity household items, like Febreze. People can use cyclodextrins, use different formulations of them mixed with other guest molecules that stick into the cyclodextrin cavity, to engineer all kinds of materials such as self-healing gels. They look like jello, and you cut it in half and put it back together and there is no seam any more. Somebody in Japan built a car out of cyclodextrins, and there is a German scientist who is making self-healing paint for cars.

Now let me summarize a bit the history of using cyclodextrins themselves as the active component of drugs. That is what we're trying to do here, to engineer them to be drugs to target 7-ketocholesterol directly. The history here goes back to the 1990s, where this Australian group had a lot of foresight; they already knew that 7-ketocholesterol was a really toxic atherogenic molecule back then, this isn't a new discovery. There has been tons of evidence for that for decades. Cyclodextrins were just starting to be explored in the 80s and 90s as cholesterol binding drugs; different versions of them bind cholesterol well. So they went looking for a modified cyclodextrin that can specifically bind 7-ketocholesterol with a hypothesis, and they found hydroxpropyl-beta-cyclodextrin, which I will tell you about. After they published a bunch of papers, however, they abandoned it and never went into any animal studies.

However, there was a group in Texas that seems to have picked up on this idea, and there was an orphan disease called Niemann-Pick type C, which is a lysosomal storage disease, that hyperaccumulates 7-ketocholesterol. These patients, almost always children, are very sick and die very young. There are good mouse and cat models for this disease, and if you dose them with very high doses of hydroxpropyl-beta-cyclodextrin, you can rescue the small animals. People have started to look at this for atherosclerosis because hydroxpropyl-beta-cyclodextrin is so safe, but there is some debate as to whether you are targeting 7-ketocholesterol, and I'll present some evidence as to why I'm strongly in favor of one hypothesis over the other. For the Niemann-Pick studies, it is going into clinical trials, it is entering phase II, there is a lot of funding going into this now. Presumably because they've got their eyes on atherosclerosis rather than just Niemann-Pick, which is an extraordinarily rare disease.

This slide shows some older data on cyclodextrin, and this came from the Australians. They are soluablizing 7-ketocholesterol, and here is the effect that it has on cholesterol, which is to say nothing, while it does a great job soluablizing 7-ketocholesterol. I'm going to talk a lot about this assay, so if you don't understand it, hold tight and I'll explain it in more detail. 7-ketocholesterol is very toxic, so with increasing doses you kill cells in culture and this was work we funded at Rice University, kind of as a scientific control for the experiments that they were doing. They were playing with cyclodextrin; as I said, it has a lot of different functions. They were using it as a carrier molecule, and found that it was working better than anything else that they were using, while we were working on the earlier LysoSENS enzymatic process to rescue the cells from 7-ketocholesterol toxicity. Then more recently, work in that same lab led to a paper and a patent on the idea of using that same cyclodextrin to prevent and reverse lipofuscin from forming in cells.

So I'll leave that there as history and show you this happy and sad video of cats with Neimann-Pick disease. All three of these cats have this disease and their phenotype is pretty similar to that of the humans. It is a devastating disease. All of these cats have it, one of these cats has been treated with hydroxpropyl-beta-cyclodextrin at a very high dose. Apparently this video is famous in the FDA for how you get something fast tracked into humans by showing a heart-tugging video like this. Because it clearly is working very dramatically to help these cats.

This slide shows the assay that we use. It is a really simplistic assay to screen through many different compounds, the many different cyclodextrins, many different modifications to cyclodextrins that we've made. It is a simple turbidity assay that we've automated. If you dump most sterols that are not water soluable into an aqueous solution, they get cloudy. Then if you managed to add something to the solution that can soluablize the sterols, then the solution turns clear. That is an indication that they are binding to your target.

So we've automated it, you run many of these plates, read them in a plate reader, and you get data that looks like this next slide. I'm just reporting the percent turbidity, so start at 100% and then go down, or in some cases it becomes more cloudy. So these are some cyclodextrins that have been studied for Neimann-Pick disease, as I keep talking about, hydroxpropyl-beta-cyclodextrin is one of our favorites. It doesn't bind cholesterol, even at ridiculously high concentrations. With 7-ketocholesterol it has nice specificity. There's another one, sulphobutyl, that has also been tested somewhat in animal models for Neimann-Pick disease but hasn't really gone further than that, and doesn't seem to be as effective as hydroxpropyl-beta-cyclodextrin.

The safety profile for some cyclodextrins such as hydroxpropyl-beta-cyclodextrin is just phenomenal. It is much less toxic than aspirin, or something like that. So you can dose humans or animals with grams of it and they are fine. As you can see, hydroxpropyl-gamma-cyclodextrin, a bigger one, doesn't do anything in the assays.

We've checked many different cyclodextrins and run them through our screening process. This slide shows a bunch more from the catalogs. Some of them are better and some of them are worse. The point of showing this to you is to show how we gathered a ton of data about which cyclodextrins were interacting with which targets in which ways, and I deleted a whole bunch of data because I was practicing this this morning, and I had too much in the presentation. But we also screened a bunch of other targets, other sterols that you can order from the Sigma catalog. That helps us look at off-target effects.

We can also do it computationally. On this slide you see the former intern that Aubrey was bragging about before. She had the idea when she was an intern - we were already working on this project, and making some progress, and she said well why don't you just model them, you can learn so much about them. She said I'll figure it out. So now she's one of the world's experts in modeling cyclodextrins. Which is a very niche field. Only a few people in the world know how to do this.

What you see here is a molecular dynamic simulation of just a basic beta-cyclodextrin. We've done hundreds of different simulations on different tweaks and different cyclodextrins, mostly with just cholesterol and 7-ketocholesterol, trying to optimize selectivity binding. Also to optimize the affinity. So beta-cyclodextrin, just the core molecule, is known as a good cholesterol binding molecule, but we can learn additional things here. If you stick it in this way, both cyclodextrin and cholesterol are asymmetrical molecules and if you start with it the wrong way, it will go in the way that it prefers. It seems kind of nitty-gritty to talk about, but that kind of information is what helped us figure out all the different subtle ways in which cyclodextrins are binding their targets.

Now on this slide is a model of hydroxpropyl-beta-cyclodextrin binding 7-ketocholesterol extremely tightly and with specificity. To really get the lowdown on this, and this is all unpublished data, we'll have a publication coming out soon, that we're furiously writing now. Go see the posters at this conference, and grill me on this, because the author is brilliant, she's great at explaining it. So we started optimizing cyclodextrins, making little tweaks to them, and came up with some tricks on how to improve the affinity for them, starting out with hundreds of these models. This slide shows the kind of data you'll get from it, the geometry of how they are interacting, how closely in space they are interacting. Then the information, that I think is the most dramatic, about the affinity.

So when you find one that is actually going to grab on super-tightly, then you massively increase the free energy of interaction, or decrease it. You can see that this also corresponds with super-close, tight, and stable interaction between the two molecules. Those kinds of simulations are extremely high resolution. We do them for a whole microsecond, which is an eternity in terms of nanosecond resolution simulations of molecules. The chief way to do lots of simulations is to do molecular docking, and so using this method - this slide is, I think, 28 different simulations that are done quickly, doing just small little tweaks to a particular family of cyclodextrins. We look for areas such as here where you seem to get separation between cholesterol and 7-ketocholesterol.

That is what led us to the step shown on this slide, which is that we synthesized some new cyclodextrins and checked to see if they could increase their affinity and maintain specificity for 7-ketocholesterol. Up at the top here you have hydroxpropyl-beta-cyclodextrin, which doesn't bind cholesterol, it binds 7-ketocholesterol a little bit. However, note the change in scale down here, we're using lower dosing ranges. The modifications that we're making to these that I'm showing you here today, they all work phenomenally well to massively increase the affinity for our target 7-ketocholesterol. However, all of them also increase their affinity for cholesterol. You probably can't see all of them in this slide, but some of them are maintaining better specificity than others, and those are the ones we're most excited about.

So there are a couple of different families of new cyclodextrins that we've invented that have extremely high affinity for 7-ketocholesterol. One in particular that caught our eye that we're really excited about - again, hydroxpropyl-beta-cyclodextrin up here at the top of the slide, some nice specificity for 7-ketocholesterol. Down here, one of the new cyclodextrins that we made has an extremely high affinity for 7-ketocholesterol, and good specificity.

Like I was saying before, these are exciting molecules to work on because they are really safe, the different versions are edible, breathable, or injectable, as in happening in the Neimann-Pick trials. A classic safety test before going in vivo is to collect some blood from whatever hapless CEO or intern happens to be wandering down the wrong time, and then treat their blood with something, and if it lyses it, then it is killing their blood cells, and if it stays clear then it is not. All of the new cyclodextrins that we've made seem to be non-toxic in the pharmacological range, and as you go above that you get some variation. For the one we're most excited about, that had the highest specificity for 7-ketocholesterol, we couldn't find a dose at which we would kill any blood cells.

I'm going to try to make the case that animal models for atherosclerosis are useless, and that we should skip straight to humans. My argument is that mice, rodents, don't get atherosclerosis naturally, and when you do give it to them by knocking out their ability to metabolize cholesterol, they are just not able to take up cholesterol, to clear it, so that it just builds up into artificial plaques. We don't think that is a good model. So once again we're working in humans, we're stealing their blood, and then treating blood with the drug in concentrations that we think are realistic and for time periods that we think are realistic. We know a lot - so much work has been done on safety for cyclodextrins over the years. We know a lot about how quickly it is cleared from circulation in different animal systems and even humans.

Then what we're doing is instead of trying to measure serum 7-ketocholesterol levels, which basically don't exist, as 7-ketocholesterol isn't transported in HDL or LDL, and that is part of what makes 7-ketocholesterol so toxic, as it can't be transported out of cells. We're measuring our ability to release 7-ketocholesterol into the serum, and measuring it by mass spectrometry. The early data on our new cyclodextrins is looking good. In this slide here is hydroxpropyl-beta-cyclodextrin that can remove some 7-ketocholesterol from human blood cells, and the first new cyclodextrin that we made can do a lot more, a lot better, and with blood from multiple donors.

So to recap, there are some big players in this area that are excited about being able to treat an orphan disease like Neimann-Pick disease. They probably have their eyes on going after bigger indications like heart disease, but they think that they are treating cholesterol, which I think is crazy. I think that they are actually treating 7-ketocholesterol, and that they are having the success that they are having because of this. However our drug blows it away in terms of affinity for the target by something like tenfold.

Beyond atherosclerosis, 7-ketocholesterol is implicated in a number of diseases, as shown on this slide. It is one of those bad guys that accumulates in different tissues with age, and we don't even know yet everything that it is implicated in. Just as for senescent cells now that people are finding ways to kill them, all of a sudden you are finding aspects of aging that are being reversed when you kill senescent cells, and I predict that someday we'll see the same thing with 7-ketocholesterol. However, atherosclerosis and heart failure I think are great indications. Of course familial hypercholesterolemia is just an exaggerated way to get atherosclerosis. There is Neimann Pick disease, and we think are drug would work better than the ones that are out there now.

I don't want to try to claim that 7-ketocholesterol causes everything that has ever made everyone sick, but it does accumulate in macular degeneration and some cells in Alzheimer's disease. Our tools that we've developed to make new cyclodextrins, to test them, to model them, I think that this is a great platform. There are other toxic oxysterols that we could be going after that we haven't starting working on yet, but are good targets for this kind of technology. Basically any small hydrophobic molecule that is bioaccumulating is a potential target. So the space in this area looks crowded because there are tens of thousands of patents on cyclodextrins, however when you get down into using cyclodextrins as drugs themselves, then you are down into single digits, like four or five patents that are out there. So we think that we have pretty strong data and protections that will get there.

Because cyclodextrins have such a good regulatory profile and are so well established in how you can use them safely, industrially, in food, in medicine, we think that the pathway to turning this into an actually effective drug - that we can write a plan, and have it be realistic is good. We're talking to experts on formulation and manufacturing, and what you see on this slide are realistic timelines. We're developing some more assays that we think will be more effacious; assays that will demonstrate that we can reverse a foam cell phenotype for example, or remove 7-ketocholesterol from plaque samples that we're working on getting from human patients or cadavers. So we have a detailed month by month plan on how we think that we could get a drug to clinical trials in three years.

As Aubrey said, we're working and making great strides on turning this into a SENS Research Foundation spinout, wholly incubated and owned at the foundation. We're calling it Underdog Pharmaceuticals.