Longevity Cookbook: Pharmacological Extension of Lifespan
Here is a teaser from the Longevity Cookbook project. The first chapter is on pharmacologic enhancement of lifespan. This chapter includes different ways of trying to develop pharmaceuticals to combat aging. What are the different research avenues? Are there special considerations when trying to develop treatments for aging? What are the pitfalls? It also includes an overview of the field as it stands today. Different drugs or substances that are especially promising or interesting are discussed. Can we meaningfully impact healthy lifespan through pharmacological means?
The second part is an ambitious proposal for testing a mixture of promising compounds for their effect on the health and lifespan of mice. This includes seven compounds tested individually side by side for their effects on longevity and a variety of health-span measures. We will also try to determine the optimal dose and see if the compounds together have an additive or even possibly synergistic effects on lifespan.
We hope you enjoy it, and if you do, there is much more coming in the Longevity Cookbook.
Pharmacological Extension of Lifespan
As we age, our risk of disease and death increase dramatically. This, of course, is something that we want to postpone, reduce or even eliminate. There are different ways of approaching aging. In this chapter, we will discuss what we know today about pharmacological interventions.
Can we impact lifespan with pharmaceuticals? The average lifespan of humans has increased unevenly throughout history, but since the beginning of the industrial revolution it has taken rapid and steady strides upward. Much of this increase has been due to better sanitation, nutrition and living standards. Improvements in medicine, such as vaccines and antibiotics, have been very successful in combating infectious disease. This used to be the main cause of death for humanity but we have been so successful in combating them (world wide) that the main causes of death are now chronic age-related disease and cancer. These are what we hope to tackle now. The risk of death increases exponentially from about age 35. But it is not only the risk of death and age related diseases we hope to combat, but also the general frailty that accompanies old age. This is not recognized as a disease but is still a very undesirable part of aging. The closest thing to frailty that is a recognized disease might be osteoporosis. This will be discussed further when we look at drugs that may affect bone density.
We will not cover all of the compounds that have been studied to extend lifespan, but those that I find most promising or most interesting. We will also discuss compounds that possibly work through different mechanisms. This could mean that we are neglecting some potentially important compounds, so we will try to explain what we think constitutes good evidence for a compound being promising. Extending lifespan in a model organism is certainly an important factor. If the compound extends lifespan in multiple model organisms, that strengthens the case, making it less likely that effect is idiosyncratic to that specific organism or strain. The type of organism is also important where organisms closely related to humans or data from humans being given larger weight. The magnitude of the extension is also important. Large effects make it more likely that aging is really affected and not some marginal effect on metabolism. Another consideration is the effect on median and maximum lifespan. Some would argue that aging is not truly affected unless there is an effect on median as well as maximum lifespan. Another possible consideration is whether the compound can be added late in life or needs to be started early, since we are not going to start treating babies but more likely people who have already reached middle age.
In this chapter we will be exploring how we discover potential lifespan and healthspan extending compounds. The compounds we discuss as promising candidates should not be taken as recommendations. The gold standard, large randomized placebo controlled clinical trials for aging have not been done on these compounds but we hope this type of research will get done. It should be noted that although compounds are available as supplements, the supplement industry is largely unregulated and while there are ethical suppliers out there, there are also many that will sell you pills with reduced or no active ingredient, or even worse, unlisted ingredients with pharmacological properties, or even real drugs. The spot checks that have been done do not look promising [1, 2].
Considerations for identifying lifespan-extending compounds
There are many lessons that can be learned from studies in humans such as treating patients with certain drugs for one disease and noticing this confers protection from other diseases, or reduces all cause mortality. This is certainly a very promising avenue of research that we think will yield results in the near term; however, in order to not be limited by only testing existing drugs we need other models we can work with. What about using cells or tissues? Ideally we would know enough about the biology and the aging process that we could rationally design therapies that could be tested on cells or tissues. Unfortunately, these types of models are not the same as a living organism where different parts of the body influence each other and work together. It is also very hard to assess effects on lifespan or healthspan on cells. Just because a cell lives long or is immortal does not mean that will be beneficial for the organism. Cancer cells are immortal in the sense that they can keep dividing forever, but that is not very helpful for their host. There is no one ideal model organism. Each one comes with different strengths that help us learn what treatments will work to combat the aging process.
One criticism of how model organisms are used is that they lack genetic diversity. Generally, animals are inbred strains that are almost like identical twins. This increases the chance that the intervention you are testing only works because of some quirk of the specific strain you are working with. There are ways to try to get around this, but they are rarely used. We will discuss this more below.
Yeast, nematodes, fruit flies, mice, and rats are the most commonly used model organisms for aging studies, especially when testing compounds. Other species can be informative when comparing the aging process between different species. Generally, larger animals live longer, but there are exceptions that are interesting. For example, naked mole rats live about 30 years and they weigh about as much as a mouse, which can live 3 years. They also show very few signs of aging as they get older . Then there are extremely long-lived animals such as clams that can be over 400 years old. Some animals might not age at all, such as Hydra, which some argue show no age-based increase in mortality.
All model organisms have their strengths and weaknesses. Mice are of course closer to humans than yeast or nematodes, but they are expensive and lifespan experiments take a very long time. In yeast and nematodes large screens (more on this in the section on screens) can be conducted, optimal doses can be tested in a matter of weeks, and more can be learned about the biology by rapidly employing genetic tools and using lifespan as an outcome. This is very slow in mice. One way to use the relative strengths of the organism is to triage compounds in less expensive and less labor-intensive organisms before moving them up the ladder to organisms more related to human physiology. One way at attempting this is the Caenorhabditis intervention testing program (CITP). Caenorhabditis is a genus of nematode or roundworm. The most studied species is Caenorhabditis elegans (C. elegans). The CITP is divided to three testing centers across the United States where each center does the same experiment as the other sites to ensure reproducibility. They also introduce genetic diversity by having 22 different strains over three different species of nematode. If the compound can extend lifespan robustly across such genetic diversity, it is more likely to target conserved mechanisms, and therefore be more likely to work in mammals or even humans. Clearly there are mechanisms in human aging that cannot be addressed by looking at yeast or nematodes, but there are many commonalities also.
Pharmaceutical companies have had a hard time developing treatments for the diseases of aging. Perhaps it is because the models that they use are lacking the underlying causes. Young animals are generally used to model the diseases of aging. These models can be created by introducing mutated genes or by chemically disrupting cells or tissues. This causes the animals to develop symptoms similar to human diseases early. These animals did not get their disease for the same reasons as humans do. It seems being old is the largest risk factor for getting age related diseases. Perhaps more studies should be done on old animals.
The intervention testing program (ITP) is a consortium under the National Institute on Aging (NIA) that use mice. It treats mainly old animals, and measures their lifespan. This is done across three centers and great care is taken to standardize procedures. To introduce genetic diversity, they employ a four way cross, so that genes from four different strains end up in the mice that are receiving the treatment. Genetic diversity is important if you want to know that the treatment is more generally applicable. The ITP has had some successes, with rapamycin being the most prominent. If you have a good candidate with some promising data to back it up, they will consider your compound for testing .
A screen is basically when you start with a large number of compounds and try to find one that works. When preforming target-based screens, the target needs to be known beforehand. The screen can then proceed in two basic ways: either through a biochemical assay which measures how strongly the compound binds to the target, or through a cell-based assay in which the protein is overexpressed in a mammalian cell line and some kind of output of its activity can be measured. The screen then proceeds by taking a large compound library, and testing all the compounds against the target. Compound libraries can come in many different forms. They can be natural products, derived from plants and animals. They can be enriched for compounds that look “drug like”. They can be variations of known compounds. The common denominator is that it is a lot of compounds, thousands, or even millions. Once a hit compound is found, it is then tested to see if it has the desired effect in a live organism.
Unbiased phenotypic screens don’t concern themselves with a particular target. Instead what you screen for is the effect on some visible phenotype. Such a phenotype could be many things, but preferably it should be easy to score. For example, if you have marked aggregating proteins with fluorescent labels you could visibly score for protein aggregation. The most straightforward measurement in a screen for lifespan extending compounds, so called “geroprotectors “, is lifespan. For this, very few organisms are practical, such as yeast or the nematode C.elegans. This is due to the fact that they can live out their life in a 96 well plate format. Different compounds are put in each well, and after a period of time the wells are then checked to see if there are any survivors. If the well with the compound has more survivors than the control wells, this would be considered a hit.
After the hits are independently validated, the process to find the target begins. While it is not strictly necessary to know the target for the drug to work, it is highly valued in industry and in academia. There are still many drugs for which the targets are unknown, but methods are being developed that make target identification easier. These include new ways to fish out the target chemically, and in silico (computational) ways of finding binding partners.
Using phenotypic screens like this targeted on aging, we can find promising compounds and also learn more about the aging process. If we can find the target, we can uncover new ways to combat the aging process that we might not have thought of using a target-based approach.
When a disease is well characterized, with known targets, targeted screens may be preferred. Since the 80’s, targeted screens have generally been favored by pharmaceutical companies. Some credit this preference with the failure to produce new first-in-class drugs[7, 8]. With aging, it is a mixed bag: there are known targets we would like to modulate, but also a lot of unknown biology, so there is certainly room for both targeted and phenotypic screens.
Mining existing compounds
Drugs that are already approved for human use have some huge advantages. Since they have been used by thousands of people, they are better understood. In order to be used in humans, they have passed important toxicity tests and they have some biologically important effect. Many times a drug is developed for one indication and as we learn more about it and realize that it is an even better treatment for something else. This type of research is certainly relevant for geroprotectors, and there are quite likely compounds already on the market that could be used to prolong the lifespan of healthy individuals.
One way to do this is through epidemiological studies that follow large groups of people, taking certain drugs and looking at their mortality or risk of certain diseases. This can certainly be very informative, and several drugs have shown promise through these types of studies.
One criticism of this approach is that we should already be seeing people living for a very long time if this were true. The fact is that life expectancy has risen by 6.2 years worldwide from 1990 to 2013 and it is mostly good healthy years being added. 5.4 healthy years are being added and 0.8 years with disability . We would certainly like this to rise much faster. 2.7 years per decade falls short of 1 year per year, which would be nice. Still, progress is being made, and the fact that it is mostly healthy years being added is very hopeful to us.
So can only modest gains be had from adapting drugs that are already on the market? Not necessarily. Different dosing regimes, or new formulations of the drugs, could ameliorate the side effects and increase the potency. Combinations of these drugs might prove effective. Likely though, we do not have drugs on the market that combat every aspect of the aging process. New research on new classes of geroprotectors is certainly needed.
Back in the middle of the 19th century, bisphosphonates were synthesized and used in industries as a water softener, to prevent calcium carbonate precipitation. This was important in the textile, fertilizer and oil industries. Much later, in the 1960’s, some researchers noticed that pyrophosphate, a naturally occurring analog to bisphosphonates, inhibited calcium phosphate dissolution a test tube (in vitro), but not in a live animal (in vivo). The lack of effect in vivo was probably due to the drug being broken down. The search was on for analogs resistant to biological breakdown, and bisphosphonates were the perfect candidate. The fact that these compounds could prevent dissolution of calcium phosphate led researchers to believe that they would prevent bone resorption. This turned out to be true, but as is often the case, the way in which they did this turned out to be quite different from the simple chemical inhibition that was first envisioned. The bisphosphonates indeed bind to the bone. In the bone they interact with the cells of this tissue - mainly with the osteoclasts, which are the cells that break down bone. They inhibit the recruitment and activity of these cells, and also kill them through programmed cell death called apoptosis. These are the mechanisms now thought to be most important for the protection of bone[10, 11].
Even though weakened bone is a biomarker of aging, it is not this that makes bisphosphonates an interesting candidate to interfere with aging. The more interesting findings are that in several studies, it actually seems to lower mortality in the elderly. One study reported a lowering of mortality by 28% in patients with hip fracture. Patients with hip fracture have an increased mortality rate. Another study looking at survival showed a 76% percent reduction in mortality, even when eliminating the patients with fractures from both the treated and non-treated group. This means the reduction in mortality was not related to fractures. Why mortality is reduced in the treatment groups is not clear, but a likely candidate is an effect on cardiovascular and cerebrovascular events. Recently another mechanism was suggested. Mesechymal stem cells were protected from radiation through increased DNA repair. This increased DNA repair was mediated by mTOR inhibition. mTOR signaling an important longevity/aging pathway that will be more discussed in the section on rapamycin.
Why these are promising compounds is that the effect size is quite large and it is in humans. We would however like to see data from larger cohorts. Survival data from multiple animal species are lacking, but it does increase the lifespan of some prematurely aging mice. However, if larger randomized controlled studies could be carried out on humans we could deem it a longevity drug without the mechanistic insight that could be gleaned from animal studies.
Far out in the middle of the South Pacific Ocean lies Rapa Nui, or Easter Island. Famous for its Moai statues of giant heads, it is one of the most remote places on earth. When Europeans first discovered it in 1722, it had already been inhabited for a thousand years. It baffles the mind to think of how the Polynesian people made it there originally. However in 1965 it was visited by a group of researchers collecting soil samples. On a certain hill on Easter Island they took some soil samples containing the bacteria Streptomyces hygroscopicus. This sample was then shared with Ayerst’s Research Laboratories in Montreal, which was later to become Wyeth Laboratories Research inc. The soil bacteria produced Sirolimus or Rapamycin, which was first hoped to become an anti-fungal agent. However, this was abandoned when it was found that rapamycin stopped cells from dividing and suppressed the immune system. When Ayerst closed its Montreal laboratories it had deemed rapamycin unviable and ordered it destroyed. Dr. Sehgal, who worked with the compound, was too intrigued by it to have it destroyed. He brought it home and put it in his freezer. Years later, when Ayerst was bought by Wyeth, Sehgal convinced his new bosses to let him continue his work, and in 1999 rapamycin was approved for use in transplant patients to reduce rejection of the donated organ.
Meanwhile in Switzerland, Dr. Michael N. Hall cloned the target of rapamycin in yeast, naming it just that, TOR (target of rapamycin). Soon after that, the mammalian version of the gene was found and named mTOR (mammalian target of rapamycin). mTOR is part of the nutrient sensing apparatus of the cell. When nutrients are abundant, mTOR is turned on and cells grow and divide, whereas when mTOR is turned off, cells become more stress resistant. This is similar to what happens in caloric restriction, which extends lifespan in many different organisms.
Turning down TOR signaling, it turned out, extended the lifespan in many different model organisms. Worms, flies and yeast were the start. What really grabbed the headlines was when in 2009 a paper was published in Nature showing that rapamycin increased the lifespan of both male (9%) and female (14%) mice across 3 testing sites. This study was done in the Interventional Testing Program under the umbrella of the National Institute on Aging. It was the first compound to show such robust results and in both sexes. What made people very excited was that the treatment was started late in the life of the mice, and still produced such an effect. Since then, there has been a flurry of research around rapamycin.
Since choosing one concentration is likely to underestimate the level of lifespan extension, the ITP repeated the study with a higher and a lower concentration. This showed a dose dependent effect on lifespan, with the higher concentration extending lifespan by 23% in males and 26% in females. There is even some hints that even higher concentrations may have beneficial effects. A dose 27 times higher than that shown to extend lifespan in normal mice, increased the survival of mice with mitochondrial disease without obvious signs of side effects.
Lifespan studies are ongoing in dogs in the Dog Aging project headed by dog lovers Daniel Promislow and Matt Kaeberlein at the University of Washington in Seattle. The goal is to extend the lifespan of household dogs, especially the healthy years. It is also a way study rapamycin’s effect on lifespan in non-laboratory conditions. They argue that household dogs are ideal since they share the same environment as humans. These trial conditions might be more similar to what would be seen in a human trial. In addition, rapamycin and its derivatives (rapalogs) are currently in clinical trials for a range of diseases, with cancer studies being most numerous.
Mitochondrially targeted ubiquinone and plastoquinone
Long ago on the primordial earth, two very different cells came together and learned to live together. One of them lived inside the other and helped it produce lots of energy. This was what would become the mitochondrion. The other took care of making most of the proteins for its new guest; this was the eukaryotic cell. Eukaryotic cells are the type of cells we and all other plants and animals are made of. The bargain that was struck came with a price. As the mitochondria produce useful energy for the cell, they also produce reactive oxygen species that can damage the cell. Defenses were developed, and we learned to use these toxic byproducts to fight off invaders, and even as signaling molecules.
In order to produce this useful energy, the mitochondria use something called the respiratory chain. In this chain, the mitochondria pass electrons between different protein complexes in the inner mitochondrial membrane. This is part of cellular respiration; the process the cell uses for combusting fuels stepwise to carbon dioxide and water. Ubiquinone is part of the respiratory chain in the mitochondria. It can accept electrons so it becomes semiquinone then ubisemiquinone and finally ubiquinol. Its ability to easily accept or donate electrons comes in handy in the electron transport chain, and is also why it can work as an antioxidant. At your local pharmacy you can buy this amazing molecule, Q10. The problem with this Q10, is that it won’t go where it is needed — your mitochondria.
Because so very little Q10 actually gets to your mitochondria, mitochondrially targeted ubiquinones have been developed. The first one was developed by Murphy and Smith and called MitoQ. It is a ubiquinone molecule with a lipophilic cation stuck to it. This lets it accumulate in the mitochondria 1000-fold compared to the rest of the cell. It is pulled in by the mitochondrial membrane potential. In the mitochondria, it is able to accept and donate electrons. This makes it a rechargeable antioxidant- although at high concentrations it can actually act as a pro-oxidant and damage the cells[24, 25]. MitoQ has been selected by the ITP to be tested for effects on the lifespan of mice.
There is a similar molecule, called SKQ, developed in the lab of Vladimir Skulachev. Whereas MitoQ is based on ubiquinone, SKQ is based on the chloroplasts’ analog, plastoquinone[26, 27]. This has some effect on lifespan especially in cancer prone mice and mice with an increased amount of mutations in mitochondrial DNA.
Both mitoQ and SKQ are currently in a couple of clinical trials. MitoQ has shown some promise in protecting the liver in patients diagnosed with hepatitis C.
Galega officinalis, or goat’s-rue, is a 3 foot tall perennial herb with white, blue or purple flowers. In ancient times it was used in folk medicine to increase milk production. There is also some indication it was used for symptoms of type II diabetes. At the turn of last century, it was shown that it was rich in guanidine, which was later found to cause hypoglycemia (low blood sugar) in animals. This, however, was too toxic to be used clinically. Several synthetic forms of guanidine were later synthesized and better tolerated. Metformin, which was brought to market under the name Glucophage (glucose eater), is dimethyl biguanidine, or two guanidine molecules stuck together, with two methyl groups in one end.
In 1995, the UK Prospective Diabetes study found, after following 3867 patients for 10 years, that metformin reduced the risk of myocardial infarction (heart attack) and all-cause mortality. This was independent of its glucose lowering effects, as those were achieved in other patients taking insulin or sulfonylurea (another diabetes drug). The insulin or sulfonylurea groups did not show this same reduction in myocardial infarction or all-cause mortality. A 2014 large retrospective study has looked at 78,241 patients taking metformin and compared them to age matched controls. This study showed that patients taking metformin actually had a 15% reduced mortality compared to the age-matched healthy individuals not taking metformin. It also extends the lifespan of several animal models[32, 33].
Metformin is often called a glucose sensitizer. Likely the main effect of metformin is to reduce glucose production by the liver. The liver stores glucose in the form of glycogen. The liver produces glucose by a process called gluconeogenesis. Metformin inhibits gluconeogenesis by inhibiting the mitochondria in the liver. This changes the energy state of the cell and makes producing glucose harder.
Since reduced insulin sensitivity is a side effect of rapamycin, and metformin increases insulin sensitivity, a combination therapy of the two might make a lot of sense. This study is actually underway at the ITP at the moment.
Since Metformin has shown such promising effects on mortality, the hope is now to try it out in healthy individuals. A project has been proposed. The TAME trial (targeting aging with metformin), spearheaded by Dr. Nir Barzilai of the Albert Einstein College of Medicine, hopes to treat 3,000 healthy individuals with metformin and study their health and the occurrence of age related disease. If this study becomes a reality, it would be a game changer for how we think about treating aging and age-related disease. FDA has recently approved this trial, so if the necessary amount of money is raised, we will be able to know whether metformin delays the onset of numerous age-related pathologies by slowing down aging processes. [31, 32]
Since ancient times, plants containing salicylic acid have been used to reduce pain or fever. Willow bark was described by Galen and Herodotus, and used by the ancient Greeks, Egyptians and even the Sumerians. In 1828, Johan Buchner purified the compound and named it salicilin, which means, “willow” in Latin. The purified compound started to be prescribed to relieve pain, but had some negative effects on the stomach.
In 1897 Felix Hoffman, a chemist at Bayer, modified salicylic acid to create acetylsalicylic acid, which was named aspirin. The company initially dismissed the drug, in order to focus on their very promising new cough remedy — heroin. It was, however, eventually tested and found to be much more tolerable for the stomach than salicylic acid. And thus the wonder drug was born.
Aspirin is considered a non-steroidal anti-inflammatory drug, or NSAID. Like most NSAIDs, it inhibits the action of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). This leads to a decrease in eicosanoid synthesis and reduces pain, fever, blood clotting and inflammation.
It is well known that aspirin has effects on cardiovascular disease. It decreases the chance of heart attack and reduces your risk of dying if you have one. What has only recently been found is that it also strongly reduces your risk of developing several different types of cancers and dying from them[36–38]. There are many studies looking at this, and the numbers change somewhat, but they point in the same direction. The strongest effects are on colorectal, esophageal, and gastric cancers, where mortality is reduced between 30% and 50%,but it also shows benefits for lung cancer, prostate cancer and breast cancer, where mortality is reduced between 5–10%. The total reduction in mortality is about 4% over a 20-year period. The main risk with aspirin is increased bleeding, with gastrointestinal bleeding being most common. This is, however, rarely related to mortality. The most worrying side effect is related to stroke. It reduces the risk of stroke but increases mortality of strokes when they do occur. Overall this seems like a favorable risk benefit profile.
Aspirin was one of the first compounds tested by the ITP, and it did show positive effects on lifespan, at least in male mice. This difference between males and females could be due to how aspirin was metabolized. It seems like the females were more efficient in converting aspirin (acetasalicylic acid) to its metabolite salicylic acid. Salicylic acid is 100 times less efficient at inhibiting COX-1, and 2 times less efficient at inhibiting COX-2. This could be the reason for the gender discrepancy in its effect on lifespan. Currently, different doses of aspirin are being tried by the ITP. The initial study was feeding the mice 20 ppm, whereas the new trials are being done at 60 and 200 ppm. In humans, the effect of dose has varied from no effect on outcome to reducing cancer risk further. It will be exciting to see how the higher doses affect the mice.
Another interesting study comes from the company Techfields pharma. They devolped a different delivery method for aspirin. Using transdermal patches, they let an aspirin pro-drug aspirinamine be soaked up through the skin. They also did a lifespan study on mice in which the patches extended the lifespan of mice by 27% lifespan extension in mice.
Even if the effect of aspirin on lifespan is quite small, there might be ways to enhance this effect. One way could be to reduce the negative side effects on the stomach so that higher doses can be used. This could be accomplished by foregoing the oral route and use something like transdermal patches. A transdermal patch is like a Band-Aid that allows medication in through the skin. Other ways could be to use a second medication such as proton pump inhibitors or to eradicate the stomach bacterium Helicobacter.pylori before treatment. Proton pump inhibitors are used to treat peptic ulcers. Helicobacter.pylori is a common cause of peptic ulcers and eradication decreases the risk of developing new ones. If the risk of peptic ulcers could be reduced this would likely reduce the major side effect of aspirin, gastric bleeding. This is in fact being done by the Helicobacter Eradication Aspirin Trial (HEAT) trial that is currently recruiting patients. 
Just like aspirin ibuprofen is considered an NSAID. Ibuprofen use was associated with decreased risk of developing Alzheimer’s by about 40%. It also seems protective against Parkinson’s disease, where it lowers the risk by around 30–40% whereas aspirin does not have this effect [38, 41]. Interestingly, ibuprofen can eliminate the cardio-protective effect of aspirin when taken together.
A recent study looked at the effect on lifespan of ibuprofen on three model organisms: yeast, fruit flies and worms. It extended median lifespan in all three organisms, although quite modestly. It was a 10% extension in flies and worms, and 17% in yeast. The mechanism of extension in yeast was proposed to be decreased uptake of tryptophan .
Acarbose is an anti-diabetic medicine. After a meal there is usually a spike in blood sugar. Acarbose has been used clinically to lower this spike. It does this by inhibiting a-glucosidases in the intestine, slowing the breakdown of starches to glucose. This compound was tested by the NIA for its effect on longevity. It produced an increase in median lifespan by 22% in male mice and 5% in females. The big advantage of acarbose is that it is safe and well tolerated in most patients. One downside is that it produces flatulence in 78% of all patients. So you may lead a long but lonely life.
17-a-estradiol is a non-feminizing estrogen, shown to have neuro-protective and mitochondria-protective effects. The proposed mechanism is that it intercalates into lipid membranes. These membranes enclose the cell and compartments in the cell such as the mitochondria, nucleus, and endoplasmatic rectiulum. In these membranes it terminates lipid-peroxidation chain reactions thereby protecting the integrity of the membrane. This is very important in mitochondria, which can initiate apoptosis (programmed cell death) if their membrane is compromised. It does all this without binding strongly to the estrogen receptor so that there are no feminizing effects. The ITP studied this compound and found that there was a 12% increase in lifespan in male but not female mice, supporting the hypothesis that females already get these protective effects from estrogens.
Nordihydroguaiaretic acid (NDGA) has anti-inflammatory and antioxidant properties. It comes from the creosote bush and has been used in traditional medicine. It has been shown to increase the lifespan of mosquitoes and Drosophila (fruit fly). It was fed to mice in the ITP and lengthened median lifespan in male but not female mice. Other studies have shown increase in some tumor types at these doses. In humans the consumption of creosote bush extracts have been associated with liver toxicity.
STACs are short for sirtuin activating compounds. Sirtuins are a class of proteins that regulate metabolism. In 2001 Leonard Guarente and Heidi Tissenbaum reported in Nature that increasing the amount of SIR-2 (a worm sirtuin) extended the lifespan of C. elegans by 50%. In 2003 in Nature David Sinclair (a former postdoc of Leonard Guarente) reported that resveratrol activated sirtuins in yeast and extended its lifespan. Resveratrol is a molecule made by plants and is relatively abundant in certain grapes, especially the skin. This has earned it the nickname “the red wine molecule”. It has been proposed as the solution to “the French paradox”. The French paradox is named so because the French have relatively low levels of heart disease even though they have a diet rich in saturated fats. The idea is that the French consumption of red wine would give them enough resveratrol to offset the effects of their “risky” diet. It is however not clear that this is the case. Even if red wine is the major contributor of resveratrol to the human diet, the amount of resveratrol in wine is still quite low compared to what is used in most studies. It is not even clear there is a paradox at all. In 2004 Sinclair founded Sirtris, a company focused on targeting sirtuins. In addition to resveratrol the company developed other sirtuin activators. Resveratrol and other sirtuin activators are thought to act through mimicking caloric restriction. Also, in 2004 Sinclair’s group reported that resveratrol extended lifespan in C. elegans and Drosophila by the same mechanism. The compound went on to show 59% lifespan extension in a vertebrate in 2006, the short-lived fish Nothobranchius furzeri. This same year Sinclair’s group showed that resveratrol extended the health and lifespan of mice on a high fat diet. Resveratrol has not however been shown to extend the lifespan of mice on a normal diet, and it was tested by the ITP at two different concentrations. However another sirtuin activator from Sinclair’s company, SRT1720, extended lifespan in mice fed a normal diet.
The work around sirtuins and resveratrol has not been without controversy. One controversy is concerning whether overexpressing the sir-2 genes in C. elegans and Drosophila really extended their lifespans. It was found that when proper controls were used, the lifespan extension went away. This was countered by the initial group showing that there still was an extension, only smaller than originally reported. Another controversy exists over whether resveratrol actually targets sirtuins. It is a dirty compound that has many targets. One way it may activate sirtuins is indirectly through a protein involved in energy sensing in the cell.
However it works, it certainly has some interesting metabolic effects. In 2008 GlaxoSmithKline bought Sirtris for 720 million dollars and resveratrol formulations are in several clinical trials. Most of the trials have been quite small, but some have shown interesting effects such as increased insulin sensitivity and cardio protection.
Also, resveratrol seem to have an effect on Alzheimer’s disease. A well-known biomarker for the disease, called Aβ, is found at lower levels in the blood of Alzheimer’s disease patients compared to healthy individuals. Patients who received resveratrol had a reduced loss of circulating Aβ.
Alagebrium is an interesting example of something that would reverse age-related damage that has already occurred. As we age, our arteries lose their elasticity and become stiffer and stiffer. This can lead to complications such as high blood pressure. But what is the cause of this increased stiffness? One culprit is advanced glycation end products (AGEs) in the arteries. AGEs are formed in a non-enzymatic reaction between sugars and proteins. The aldehyde groups on the sugar react with the amino group of the protein. This forms something called a Schiff-base, which can then further react through a series of steps. This process can take years and culminates in irreversible crosslinks, AGEs. This happens during aging, especially to long-lived proteins. The process is accelerated in diabetes and Alzheimer’s disease. In addition to contributing to stiffness of the extracellular matrix, they can also activate the receptor for AGEs called RAGE. This in turn can raise the level of inflammatory cytokines especially NFκB. This leads to inflammation, which is useful for fighting infections but chronic inflammation as seen in aging is a major problem. This could be particularly problematic for the arteries.
There are also dietary sources of AGEs. These might also contribute to the increased inflammation through the RAGE receptor. Foods high in protein and fat have the highest levels, whereas carbohydrates contribute the least. Cooking temperature also plays a role. High cooking temperatures such as in frying or grilling increase AGEs compared to slow cooking in a crock pot.
Alagebrium was developed to break AGE crosslinks. There are many types of AGEs and alagebrium seems to be able to break the crosslinks in at least some of them. It showed promise in animal studies where it reduced arterial stiffness. Several clinical phase II trials have also been performed. Significant improvements have been seen in heart function and even quality of life[62, 63]. Other studies have shown negative results. The company has chosen not to develop it further for business reasons.
Another way to reduce AGEs is through amino guanidine, which can inhibit AGE formation. Interestingly, this molecule is related to metformin (dimethyl biguanidine) and it is hypothesized that metformin may also have this effect.
Although there are no data on effect on lifespan of alagebrium, it is quite interesting that it can reverse this biomarker of aging. Since it does not remove all AGEs, maybe not even the most abundant ones, it would be interesting to see what a more effective AGE breaker or a combination of AGE breakers could do. 
Lithium is a very strange drug. It is not a complex molecule but actually an element. Unlike other metals like iron, copper, zinc, magnesium, or manganese, it does not have a clear biological function. It is not needed for any protein to perform its action and does not perform signaling within the cell. However, there are some indications that some amount of lithium is beneficial. Animals kept on a low lithium diet have increased mortality and reproductive abnormalities. So far lithium deficiencies have not been described in humans, but, as we shall see, there are indications that some amount of lithium might be beneficial.
Mineral wells have long been popular. Some of these wells contained high levels of lithium. The crazy well in Texas for example was named after a “crazy” woman who would beg for water next to the well. The water purportedly had some beneficial effect on her condition. Water with lithium was and is still sold for its curative effects. Initially it was even included in 7up for its “invigorating effects on the body”.
As an element it was discovered in 1817 by Swedish chemist Johan August Arfwedson, working in the lab of Jöns Jakob Berzelius. Its name is derived from the Greek Lithos, which means stone. This was to reflect that it was isolated from a solid mineral. By the late 19th century John Aulde in America and Carl Lange in Denmark had reported its use for prophylactic treatment of recurrent depression. However, it fell into disrepute because of waters marketed by hucksters as a miracle cure for all sorts of diseases.
In modern clinical care, John Cade rediscovered it in 1949 when he described its impressive effect on 10 manic patients. He had gotten the idea when he saw that guinea pigs became lethargic when given lithium. This led him to believe it might be used to control mania.
After its initial success, lithium quickly suffered a major setback. In order to lower sodium, lithium was given as a salt substitute to heart patients in the US. This led to several overdoses and several fatalities and quickly established the impression of lithium as a dangerous drug . Subsequent studies in large groups of patients have shown that lithium can be a safe and effective drug if the correct doses are given and patients are monitored. When used as prescribed, lithium can still have some side effects such as polyuria (increased urination), hyperparathyroidism and hypothyroidism. The most serious side effect is renal failure, which occurs in about 0.5% of patients.
Lithium has shown neuroprotective properties in many different models. In rat and mouse models of stroke, lithium has reduced inflammation, protected cells from dying, induced neurogenesis, and helped angiogenesis (the growth of new blood vessels) the brain. In bipolar patients, lithium treatment has been associated with increased grey matter and thicker cortex and hippocampus compared to patients not on lithium. A recent clinical study has also shown that lithium might slow down cognitive decline. Another interesting study showed that the telomeres of white blood cells are longer in bipolar patients that have taken lithium. Lithium does not however seem to influence telomerase activity.
In 2008 the Lithgow lab discovered that lithium could extend the lifespan of the nematode C. elegans . Based on this, there was a study of lithium levels in tap water between different municipalities in Japan. They found that the more lithium there was in the tap water the lower the death rate was. This was excluding rates of suicide. Another study investigated the suicide rate and found it also to be lower in municipalities with high levels of lithium in the tap water . Even in the municipalities with the highest amount of lithium in the tap water it was only about 1/1000 of the level given to the nematodes or people with bipolar disorder. To see if this level had any effect they fed it to the worms and saw a slight increase in lifespan, even at these low levels .
Lithium can have some unwanted side effects at doses relevant for bipolar disorder. The protective effects however might not require these high doses. It could be interesting to examine if low dose lithium can be used as a neuroprotector in the aging process.
Glucosamine is naturally made in humans, and is part of the formation of cartilage. Glucosamine is popular among older adults. It is often used for joint health where evidence of its effectiveness is lacking. Interestingly, it may have an effect on mortality. A large self-report study found that people who reported taking glucosamine had a 20% reduction in mortality over the study period. It has also been shown to extend lifespan in nematodes and mice. The mechanism proposed was that it inhibits glycolysis and thereby causes an energy deficit. The cell then compensates for this deficit by making more mitochondria. Glucosamine also switches the cells fuel use from glucose to amino acids, leading to increased production of reactive oxygen species. These increased reactive oxygen species seem to be important for the longevity effect, since it can be eliminated by antioxidants. This compound is very safe, except for some allergic reactions and possibly insulin resistance. It would be interesting to see a proper randomized placebo controlled trial in humans for mortality or biomarkers of aging.
C60 Fullerenes are interesting molecules. They consist of 60 carbon atoms bound to each other to form the shape of a Bucky ball. Bucky ball is named after Buckminster Fuller, American architect and futurist who popularized the geodesic dome. If carboxyl groups are added to C60 Fullerenes, they become superoxide dismutase (SOD) mimetics . SOD is an important part of the antioxidant system in the cell. It catalyzes the conversion of the very reactive superoxide molecule into hydrogen peroxide. In 2008, these SOD mimetics were given in the drinking water to 1-year-old mice. This achieved a mean lifespan extension of 11% and retained neurological function longer than the control mice . This molecule has also shown some neuroprotection in a monkey model of Parkinson’s disease .
Another study, from 2012, looked at C60 Fullerenes alone, without any modifications . It seems the researchers were mainly interested in toxicity of the molecule. C60 Fullerenes were given to the rats in olive oil through oral gavage (administration of the oil through a tube down the throat). Treatment began at 10 months and continued for 7 months. Also in this study, they saw free radical scavenging properties of the Bucky-balls, even though they didn’t have any carboxyl group on them to act as a SOD mimetic. What is noteworthy is the magnitude of lifespan extension. They report a median extension of 90%. This would be the longest published extension of lifespan in any organism with a pharmaceutical intervention. It should be kept in mind that his was a very small study with only 6 mice per group. 100 mice is more in line with a normal lifespan study. Further doubt was cast on the study when people noticed some mistakes in the images that were later corrected. This significantly reduced the maximum lifespan of the treatment group.
Even though the 2012 study was way too small and raised some warning flags, it would be interesting to see it replicated with a large enough cohort, and see the treatment continued throughout the study, instead of being stopped after 7 months. A comparison between carboxyfullerenes and fullerenes without carboxyl groups would also be nice, although it might be harder to compare them head to head in vivo since the former is water soluble and the latter lipid soluble.
Telomeres are the protective end caps on our chromosomes. A consequence of how DNA replication works is that the last three bases on the end of the chromosomes are not copied. This and the fact that the tips of the chromosomes get damaged leads to a progressive shortening of the chromosomes with every cell division. When the telomeres get short enough the cells do not divide anymore. To get around this, cells have an enzyme called telomerase. Telomerase can lengthen the telomeres since it carries with it the template to the repeat sequence that make up the telomeres. However, not all cells express telomerase and it is not active all the time. This may be a good thing, since many cancers need to activate telomerase in order to keep dividing.
As we get older our telomeres get shorter, and that may not be a good thing. When the telomeres get critically short, they activate a DNA damage response and can make cells go into cellular senescence. These cells stop dividing. They sit around secreting inflammatory cytokines and they don’t die. We call them senescent cells. These are cells that probably have a role in wound healing where they can be removed with the scab, but are likely quite detrimental when hanging around in the body promoting inflammation.
It has been shown that lengthening of telomeres in mice extends lifespan and improves their health in several ways, such as increased bone density and retained insulin sensitivity at old age . These mice were engineered to express higher levels of telomerase from birth. They used cancer resistant mice since it caused normal mice to have more cancer. The same research group later preformed gene therapy on adult mice and showed an increase in lifespan by 24% in the group treated at 1 year of age, and 13% increase for the group treated at 2 years of age .
Another approach is to use compounds that activate telomeres. There are several compounds that claim to do this. One is TA-65 discovered by Geron Corporation. It is an herbal supplement based on Astragalus membranaceus. This supplement was given to middle aged mice. It did not, however, extend the lifespan of the mice. Some effects on healthspan where noted, such as improved glucose tolerance, reduced osteoporosis, and increased skin thickness. Somewhat alarming was the fact that the treated mice showed a trend toward more tumors. This was however not statistically significant.
Bill Andrews, whose team at Geron Corporation discovered the human telomerase, now runs Sierra sciences. They are currently working on developing more potent telomerase activators.
Two companies, Telocyte and Bioviva, want to use telomerase gene therapy directly on humans. Telocyte aims to cure Alzheimer’s with telomerase gene therapy. From what we can see, there is very little evidence that telomerase gene therapy would work specifically for Alzheimer’s disease, even if it shows benefits in other areas. The other company, Bioviva, will treat healthy but aging volunteers. They have in fact already treated their first patient, their CEO Elizabeth Parish.
Combinations of compounds
Combinatorial interventions are used for treating different diseases in the clinic. In cancer therapies, combinatorial treatments are used with success. The cancer can be attacked from different angles with multiple targeted therapies and cytotoxic agents. Perhaps the most famous example of combinatorial interventions is the anti-retroviral cocktail of reverse transcriptase inhibitors and protease inhibitors given to HIV patients. When it was introduced, it was just amazing to see how people on deaths door were brought back to a relatively healthy life. The death rate plummeted with the introduction of protease inhibitors to the cocktail. Going from a practical death sentence, HIV is now a manageable disease. Could such a combination therapy be found for aging?
So what is the difference between testing one compound and testing a combination of compounds? At some level a single compound may act as a combination of compounds. Single compounds can bind to many targets. But aren’t those off-target effects just creating negative side effects? Often that may be true, but some compounds might work well, precisely because they have multiple targets. The schizophrenia drug Clozaril probably works because it has many targets. The same can be said for many anti-cancer kinase inhibitors such as imatinib, sorafenib and sunitinib. Pharmaceutical companies are making a large effort to develop single-target drugs, but that may not always be the best choice.
If two compounds can extend lifespan separately, together they must be even better, right? Well, it might not be that simple. The only published combination of compounds that has successfully extended lifespan to my knowledge is valproic acid and trimethadione in C. elegans. On their own, valproic acid extended the mean lifespan by 35% and trimethadione extended it by 45% at optimal dose. Together they extended the mean lifespan by 61%. This same study from the Kornfeld lab also shows that combining two compounds that on their own extend lifespan, can produce toxic effects. This was the case when mixing ethosuximide with trimethadione . So why is this? One explanation can be that they are both acting on the same pathway. Too much of a good thing is not always good.
The opposite can also be true; compounds can cancel the effect of each other. Some compounds that could confer longevity seem to require ROS signaling (and so does caloric restriction). These effects could be cancelled out by taking an antioxidant, which might on its own be helpful in some cases. An example of this is glucosamine (which extends lifespan in mice and C. elegans) and the antioxidant N-acetyl-cysteine (NAC). NAC completely abolishes the lifespan extension effect of glucosamine in C. elegans .
So how do we know which compounds will work well together? The truth is we don’t really know. We can try to make educated guesses though. Perhaps targeting different pathways is the way to go. However, sometimes targeting the same pathway can yield very potent effects with genetics. The insulin/IGF1 signaling pathway can be targeted from different angles in C. elegans yielding very long lived worms. There is also the possibility that two compounds targeting the same pathway can maximize the beneficial outcome while minimizing the side effects. Targeting different pathways can also work very well. In C. elegans the TOR and insulin signaling pathway were targeted genetically to produce a worm with nearly 5 times its normal lifespan .
One hope is that if we know more about the biology it would be easier to make educated guesses. Biology however, remains dirty and interconnected. The only way to know for sure is to test it.
Rapamycin and metformin are two drugs that could potentially work well together to extend longevity. A side effect of rapamycin is insulin resistance but metformin improves this condition. A combination of rapamycin and metformin treatment was started by the ITP in 2011. No results are yet published, but rumor has it, it’s working.
One combination of compounds that was reported to be working in mice to combat one aspect of aging is quercetin and dasatinib. The combination is for killing senescent cells. They were each effective in killing senescent cells in different tissues but worked better together. No lifespan was measured, but health was improved. The mice were able to run farther on a treadmill.
Another interesting combination could be resveratrol and nicotinamide. Nicotinamide raises NAD levels. NAD is an important redox molecule. Resveratrol increases the activity of sirtuins, which in turn use NAD to carry out its activity. Both molecules have shown some promise in extending lifespan in some organisms and reducing biomarkers of aging in others. Perhaps these compounds could potentiate each other.
Screening for combinations
Testing different compounds in combination is certainly a daunting task. How many compounds do you want in your longevity cocktail? Lets say we want to test 100 promising geroprotectors in combination. Perhaps we are assaying lifespan of C. elegans in a 96 well format. If we are just doing pairwise combinations it is almost manageable. 10,000 combinations performed in triplicate make 30,000 wells to be assayed. Only very large effects will be noticed in these types of screens if we are to eliminate false positives. That means results that look positive but are not real. This happens a lot when dealing with large numbers. If we are combining 3 or 4 compounds together in our screen we need to assess 3,000,000 or 300,000,000 wells respectively. In this setup, we are only using one concentration per compound. We have seen that sometimes concentrations of drugs need to be lowered when used together with another drug. A way to get around some of the problem is to first do a pairwise screen and use your best combination as the base for the next screen. This way you can keep adding compounds to your cocktail with a manageable amount of screening. This will of course miss some combinations that would be in the massive 300,000,000 well screen but some compromises always have to be made. There are also algorithms that can be used to help the search of combinatorial therapies. In cancer research this has been explored somewhat. One way is to use many different complex mixtures once the results from your assay collected they are fed back into the algorithm. The algorithm iterates on the most successful ones and gives you new suggestions on what you can test in your assay. For cancer therapies success would be killing cancer cells but not healthy cells. Hopefully the next round of mixtures is more effective than the first (killing more cancer cells and less healthy ones). This can then go on until you see diminishing returns. This could possibly be used in organisms and measure their lifespan, however this could take a very long time, so measuring biomarkers of aging might be better for this kind of approach.
Other than having compounds interacting on the biological level, such as acting on the same pathway, they may also interact chemically. This can cause quite the headache when trying to test pairwise combinations or even worse, complex mixtures.
Regimes, doses and biorhythms
As important as getting the right compounds is administering them properly. All drugs have a therapeutic window when the drug is working but is not very toxic. Preferably, this window is as wide as possible so the chance of efficacy is maximized and the risk of toxicity is minimized. More is not always better, like with the mitochondrially targeted ubiquinone and plastoquinone: when used at low levels they work like antioxidants but at higher levels they start acting like pro-oxidants . When you have found a geroprotector it is important to know the peak effective dose. On short-lived animals this is easy. You simply try different doses so you get a dose-response curve. Lower doses produce a slight increase in lifespan whereas higher doses might produce toxicity. For longer-lived animals this is harder and seldom done. The ITP aims to do it for compounds that it finds effective. For example, the initial rapamycin trial produced a 9% increase in lifespan for male mice and a 14% increase for females. The higher dose subsequently produced an increase of 23% in males and 26% in females [21, 22]. No higher dose has been tried by the ITP so we don’t yet know its peak potential. Indeed, a recent study shows that a dose much higher (27 times) than that initially used to extend mouse lifespan, increased lifespan of a mouse model of Leigh Syndrome, in a dose dependent manner with no observed side effects . This suggests that the therapeutic window for rapamycin is much larger. It is important to remember that if a compound has shown a weak increase in lifespan, and higher doses have not been tried, we do not know its potential.
In addition to getting the dose right, some geroprotectors should likely not be taken all the time. Some side effects of rapamycin treatment include hyperglycemia and increased susceptibility to opportunistic fungal infections. A recent study using an intermittent dosing regimen with rapamycin showed that this dose was enough to suppress the ability of senescent fibroblasts to stimulate prostate tumor growth in mice . Regimens like this could allow us to reap the benefits of drugs like rapamycin without the side effects.
Biorhythms are naturally recurring changes in hormone levels. Biorhythms could therefore play a role in the effectiveness of drugs. For example, blood pressure can spike in the morning. A more stable blood pressure may be achieved if blood pressure medicine is taken in the evening . In the context of pharmacologic enhancement of lifespan, one study looking at melatonin supplementation found that it increased their lifespan only when given at night. However, the results of this study are hard to interpret since the sample size was very low (10 mice per group).
In addition, there may be cases when certain treatments are effective in certain phases of aging. For example, a high protein intake between the ages 50–65 was associated with a 75% increase in overall mortality and a 4-fold increased risk of cancer. On the other hand, the high-protein-intake group aged 66 and older showed a 28% decrease in all cause mortality . Proteins effect hormone levels of for example IGF-1. IGF-1 is a hormone that is strongly implicated in aging together with growth hormone (GH). Mice and other model organisms that have defective GH or IGF-1 signaling, live much longer. Even humans that have defective GH signaling are protected from cancer and diabetes. Since geroprotectors may target these kinds of pathways, it suggests that they may be beneficial for people in certain ages but not others. Very little research has been done on this, but it could possibly be quite important.
Clinical trials for age related disease
The FDA does not recognize aging as a disease; hence, targeting aging for clinical trials can be difficult. There is also the question of what to measure. Lifespan takes too long in all but the very old. All cause mortality is a decent proxy. A certain period of time is chosen and the number of people dying in each group is counted. The problem with this is that the time of the study must be very long or the sample size very large. Another method is to use occurrence of age related disease, such as cancer, heart disease, and neurodegenerative diseases. This last example is the method chosen by the metformin TAME trial. It will recruit patients to the study when they get an age related disease, and measure the time it takes to develop the next age related disease.
Another way to evaluate outcome from clinical trials on aging is measuring biomarkers. A biomarker is something that can be measured and used as a proxy for aging. Ideally it should have a functional significance and not only be correlated with increased age. Changing the biomarker should then change the outcome for the patient. Better biomarkers are being developed; recently, combinations of biomarkers have given quite good results. Unfortunately, most biomarkers are only known to correlate with aging, with little being known if changing the biomarker will increase life span. Walking speed for example, is a good biomarker for aging, but would an intervention making people walk faster work or is it only a measure of functional decline? Genome methylation can be used as a biomarker and can be very precise in determining a person’s age, but little is known about the effect of changing this methylation pattern. The Paolo Alto prize for homeostatic capacity attempts to affect a biomarker for aging in order to improve health. The biomarker is heart rate variability. A less variable heart rate is associated with dramatically increased mortality, at least in patients with previous heart attacks. The idea is that if you can increase heart rate variability to youthful levels you would be performing a kind of rejuvenation. If you can do this you will get 500,000 dollars.
Pitfalls and Limitations
Moving between organisms
There are many considerations when moving between organisms. The most obvious may be drug dose. There is no way to exactly calculate the dose from one organism to the other. It is especially hard when translating from invertebrate models, such as flies or nematodes, to mammals. When moving from one mammal to another, it is a little easier. There is of course no guarantee of how the drug will be metabolized, but a good rule of thumb is to scale the dose with the surface area of the animal. Scaling by mass looks obvious but the quicker metabolism of the smaller animals offsets some of what they lack in mass. Another consideration is if the drug will work in the new organism. We cannot yet say for sure whether a drug will bind to a certain target based on DNA or protein sequence similarity between two animals. It may bind to a target that looks less similar by sequence but still has the right shape. Many drugs however seem to work on wildly different organisms, all the way from yeast to humans.
Toxicity is a huge part of why drugs fail. In certain diseases, such as cancer, toxicity is tolerable. But when treating healthy people to prevent aging, toxicity is probably not going to be tolerated. These medications likely have to be very safe. It is ok to have some toxicity in animal studies if you still increase lifespan. In humans however, it is another story. Would you take a drug that increased your healthy lifespan by 50% but had a 5% chance of killing you now? Some might want to, but it is unlikely to get approved. Some compounds extend lifespan by hormesis. Essentially they cause a little toxicity in order for the organism to ramp up its defenses. This in turn leads to a longer life. Paraquat is one such compound. It is a toxin that can be used as an herbicide. It increases free radical production and prolongs lifespan in C. elegans by upregulating stress responses. These types of compounds might not be good candidates to move into clinical trials. However exercise, which is considered quite safe, produces beneficial results and is likely an example of hormesis. During and after exercise a large amount of reactive oxygen species are produced, signaling for repair and rebuilding you stronger than before.
Reliablity of data
One of the main problems with research in general is the reliability of the data. Many times a finding is not repeatable when tried by another lab. This is usually not due to fraud or anything as nefarious as that. It is mainly that research is hard and there are many ways to fool yourself. One way to easily fool yourself is to use too small a sample size. This is quite common. Using a small sample size when looking at lifespan increases the chance that a long-lived or short-lived population was picked by chance. Generally around 100 animals should give around 80% chance of detecting a 10% difference in lifespan. By detecting I mean the difference being statistically significant (if they indeed are). But not only large sample sizes and statistical significance are important. Large effects are generally more reproducible than small effects. What I like to see is a large effect with a large sample size with a good statistically significant result. It is always nice to see if can be replicated by other groups. However, many small badly designed studies do not equal one large well-designed study.
It is always good to look at the entire literature on the subject when determining if something is likely to be true. Even if data conflicts between groups it does not mean one group did not get the results they report, it means the effect is not robust to changes. Biology is noisy and small changes in protocol or random chance can make a difference.
Another sin is looking at too many parameters. The space of things that can be different between 2 treatment groups is very large. It can be expression of different genes, weight, movement, metabolism or protein aggregation. The list is almost endless. If you keep looking you are bound to find something that is different, just by chance. That is why in clinical trials you are forced to decide your primary endpoint in advance. That way you are locked in and cannot cheat. If you find other things of interest while doing your clinical trial, these can be reported on, but a new clinical trial will have to be done if you want those to be part of your indication. This type of research is seldom done in non-clinical research. It is however attempted by the ITP (mouse lifespan) and CITP (Worm lifespan) testing programs. Compounds tested are announced ahead of time and any result will be reported with lifespan being the primary endpoint. That and the fact that it is replicated across 3 different centers, increases the confidence in this data.
Human studies are of course great, but a lot of sources of error can creep in since humans cannot be experimented on the same way animals can. A lot of human studies are epidemiological. In these studies people are not randomly put in groups that receive treatment or not. That way there may be other differences in the group that explains the difference in outcome.
There are many challenges ahead but the field of pharmacological intervention in aging has matured a lot over the recent years. Both health span and lifespan can now be improved in a variety of species and possibly humans as well. What we need are several more good candidates and a clear road forward to translating them into treatments for humans. If we can get these treatments into humans and understand how they work together for a comprehensive treatment of the underlying causes of aging, there is no telling how far we can go.
We invite you to form a non-profit organization working on testing of different combinations of compounds. We believe that anti-aging drugs should be developed using an open non-commercial approach, not limited by IP protection and available for everyone.
And let us introduce you to our Longevity Drug Combination Project.
By Daniel Edgar and Mikhail Batin, illustrated by Olga Posukh
This is not intended as a guide to health and longevity. It is meant to illuminate some of the interesting findings in this field. We do not recommend any of these substances. You should consult your physician before trying any of this. Do not order these substances online from unverified sources there is no way to know what you are getting without substantial laboratory analysis. We would also not advise taking several substances in combination. Even less is known about the effects of combinatorial interventions. We hope to learn what types of therapies are effective and in the future be able to make recommendations.
1. Newmaster, S.G., et al., DNA barcoding detects contamination and substitution in North American herbal products. BMC Med, 2013. 11: p. 222.
2. Harel, Z., et al., The frequency and characteristics of dietary supplement recalls in the United States. JAMA Intern Med, 2013. 173(10): p. 926–8.
3. Buffenstein, R., Negligible senescence in the longest living rodent, the naked mole-rat: insights from a successfully aging species. J Comp Physiol B, 2008. 178(4): p. 439–45.
4. Martinez, D.E., Mortality patterns suggest lack of senescence in hydra. Exp Gerontol, 1998. 33(3): p. 217–25.
5. Aging, N.I.o. 2016 [cited 2016 4 Jan]; Available from: https://www.nia.nih.gov/research/dab/interventions-testing-program-itp.
6. Hughes, J.P., et al., Principles of early drug discovery. Br J Pharmacol, 2011. 162(6): p. 1239–49.
7. Frantz, S., Drug discovery: playing dirty. Nature, 2005. 437(7061): p. 942–3.
8. Hopkins, A.L., Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol, 2008. 4(11): p. 682–90.
9. DALYs, G.B.D., et al., Global, regional, and national disability-adjusted life years (DALYs) for 306 diseases and injuries and healthy life expectancy (HALE) for 188 countries, 1990–2013: quantifying the epidemiological transition. Lancet, 2015. 386(10009): p. 2145–91.
10. Russell, R.G., Bisphosphonates: from bench to bedside. Ann N Y Acad Sci, 2006. 1068: p. 367–401.
11. Francis, M.D. and D.J. Valent, Historical perspectives on the clinical development of bisphosphonates in the treatment of bone diseases. J Musculoskelet Neuronal Interact, 2007. 7(1): p. 2–8.
12. Lyles, K.W., et al., Zoledronic acid and clinical fractures and mortality after hip fracture. N Engl J Med, 2007. 357(18): p. 1799–809.
13. Center, J.R., et al., Osteoporosis medication and reduced mortality risk in elderly women and men. J Clin Endocrinol Metab, 2011. 96(4): p. 1006–14.
14. Misra, J., et al., Zoledronate Attenuates Accumulation of DNA Damage in Mesenchymal Stem Cells and Protects Their Function. Stem Cells, 2015.
15. Varela, I., et al., Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging. Nat Med, 2008. 14(7): p. 767–72.
16. Kunz, J., et al., Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell, 1993. 73(3): p. 585–96.
17. Brown, E.J., et al., A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature, 1994. 369(6483): p. 756–8.
18. Jia, K., D. Chen, and D.L. Riddle, The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span. Development, 2004. 131(16): p. 3897–906.
19. Kapahi, P., et al., Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol, 2004. 14(10): p. 885–90.
20. Powers, R.W., 3rd, et al., Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev, 2006. 20(2): p. 174–84.
21. Harrison, D.E., et al., Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature, 2009. 460(7253): p. 392–5.
22. Miller, R.A., et al., Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell, 2014. 13(3): p. 468–77.
23. Johnson, S.C., et al., Dose-dependent effects of mTOR inhibition on weight and mitochondrial disease in mice. Front Genet, 2015. 6: p. 247.
24. Gruber, J., et al., Mitochondria-targeted antioxidants and metabolic modulators as pharmacological interventions to slow ageing. Biotechnol Adv, 2013. 31(5): p. 563–92.
25. Kelso, G.F., et al., Selective targeting of a redox-active ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties. J Biol Chem, 2001. 276(7): p. 4588–96.
26. Skulachev, V.P., et al., An attempt to prevent senescence: a mitochondrial approach. Biochim Biophys Acta, 2009. 1787(5): p. 437–61.
27. Skulachev, V.P., Cationic antioxidants as a powerful tool against mitochondrial oxidative stress. Biochem Biophys Res Commun, 2013. 441(2): p. 275–9.
28. Gane, E.J., et al., The mitochondria-targeted anti-oxidant mitoquinone decreases liver damage in a phase II study of hepatitis C patients. Liver Int, 2010. 30(7): p. 1019–26.
29. CJ Bailey, C.D., Metformin: its botanical background. Pract Diab Int. 21: p. 115–117.
30. King, P., I. Peacock, and R. Donnelly, The UK prospective diabetes study (UKPDS): clinical and therapeutic implications for type 2 diabetes. Br J Clin Pharmacol, 1999. 48(5): p. 643–8.
31. Bannister, C.A., et al., Can people with type 2 diabetes live longer than those without? A comparison of mortality in people initiated with metformin or sulphonylurea monotherapy and matched, non-diabetic controls. Diabetes Obes Metab, 2014. 16(11): p. 1165–73.
32. Anisimov, V.N., et al., Metformin slows down aging and extends life span of female SHR mice. Cell Cycle, 2008. 7(17): p. 2769–73.
33. Martin-Montalvo, A., et al., Metformin improves healthspan and lifespan in mice. Nat Commun, 2013. 4: p. 2192.
34. Viollet, B., et al., Cellular and molecular mechanisms of metformin: an overview. Clin Sci (Lond), 2012. 122(6): p. 253–70.
35. Miner, J. and A. Hoffhines, The discovery of aspirin’s antithrombotic effects. Tex Heart Inst J, 2007. 34(2): p. 179–86.
36. Rothwell, P.M., et al., Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials. Lancet, 2011. 377(9759): p. 31–41.
37. Cuzick, J., et al., Estimates of benefits and harms of prophylactic use of aspirin in the general population. Ann Oncol, 2015. 26(1): p. 47–57.
38. Rees, K., et al., Non-steroidal anti-inflammatory drugs as disease-modifying agents for Parkinson’s disease: evidence from observational studies. Cochrane Database Syst Rev, 2011(11): p. CD008454.
39. Strong, R., et al., Nordihydroguaiaretic acid and aspirin increase lifespan of genetically heterogeneous male mice. Aging Cell, 2008. 7(5): p. 641–50.
40. Vlad, S.C., et al., Protective effects of NSAIDs on the development of Alzheimer disease. Neurology, 2008. 70(19): p. 1672–7.
41. Gao, X., et al., Use of ibuprofen and risk of Parkinson disease. Neurology, 2011. 76(10): p. 863–9.
42. He, C., et al., Enhanced longevity by ibuprofen, conserved in multiple species, occurs in yeast through inhibition of tryptophan import. PLoS Genet, 2014. 10(12): p. e1004860.
43. DiNicolantonio, J.J., J. Bhutani, and J.H. O’Keefe, Acarbose: safe and effective for lowering postprandial hyperglycaemia and improving cardiovascular outcomes. Open Heart, 2015. 2(1): p. e000327.
44. Harrison, D.E., et al., Acarbose, 17-alpha-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males. Aging Cell, 2014. 13(2): p. 273–82.
45. Dykens, J.A., W.H. Moos, and N. Howell, Development of 17alpha-estradiol as a neuroprotective therapeutic agent: rationale and results from a phase I clinical study. Ann N Y Acad Sci, 2005. 1052: p. 116–35.
46. Richie, J.P., Jr., B.J. Mills, and C.A. Lang, Dietary nordihydroguaiaretic acid increases the life span of the mosquito. Proc Soc Exp Biol Med, 1986. 183(1): p. 81–5.
47. Miquel, J., J. Fleming, and A.C. Economos, Antioxidants, metabolic rate and aging in Drosophila. Arch Gerontol Geriatr, 1982. 1(2): p. 159–65.
48. Spindler, S.R., et al., Nordihydroguaiaretic Acid Extends the Lifespan of Drosophila and Mice, Increases Mortality-Related Tumors and Hemorrhagic Diathesis, and Alters Energy Homeostasis in Mice. J Gerontol A Biol Sci Med Sci, 2015. 70(12): p. 1479–89.
49. Arteaga, S., A. Andrade-Cetto, and R. Cardenas, Larrea tridentata (Creosote bush), an abundant plant of Mexican and US-American deserts and its metabolite nordihydroguaiaretic acid. J Ethnopharmacol, 2005. 98(3): p. 231–9.
50. Tissenbaum, H.A. and L. Guarente, Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature, 2001. 410(6825): p. 227–30.
51. Howitz, K.T., et al., Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature, 2003. 425(6954): p. 191–6.
52. Wood, J.G., et al., Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature, 2004. 430(7000): p. 686–9.
53. Valenzano, D.R., et al., Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr Biol, 2006. 16(3): p. 296–300.
54. Baur, J.A., et al., Resveratrol improves health and survival of mice on a high-calorie diet. Nature, 2006. 444(7117): p. 337–42.
55. Miller, R.A., et al., Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice. J Gerontol A Biol Sci Med Sci, 2011. 66(2): p. 191–201.
56. Mitchell, S.J., et al., The SIRT1 activator SRT1720 extends lifespan and improves health of mice fed a standard diet. Cell Rep, 2014. 6(5): p. 836–43.
57. Dang, W., The controversial world of sirtuins. Drug Discov Today Technol, 2014. 12: p. e9-e17.
58. Park, S.J., et al., Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell, 2012. 148(3): p. 421–33.
59. Tome-Carneiro, J., et al., Resveratrol and clinical trials: the crossroad from in vitro studies to human evidence. Curr Pharm Des, 2013. 19(34): p. 6064–93.
60. Turner, R.S., et al., A randomized, double-blind, placebo-controlled trial of resveratrol for Alzheimer disease. Neurology, 2015.
61. Engelen, L., C.D. Stehouwer, and C.G. Schalkwijk, Current therapeutic interventions in the glycation pathway: evidence from clinical studies. Diabetes Obes Metab, 2013. 15(8): p. 677–89.
62. Zieman, S.J., et al., Advanced glycation endproduct crosslink breaker (alagebrium) improves endothelial function in patients with isolated systolic hypertension. J Hypertens, 2007. 25(3): p. 577–83.
63. Little, W.C., et al., The effect of alagebrium chloride (ALT-711), a novel glucose cross-link breaker, in the treatment of elderly patients with diastolic heart failure. J Card Fail, 2005. 11(3): p. 191–5.
64. Hartog, J.W., et al., Effects of alagebrium, an advanced glycation endproduct breaker, on exercise tolerance and cardiac function in patients with chronic heart failure. Eur J Heart Fail, 2011. 13(8): p. 899–908.
65. Schrauzer, G.N., Lithium: occurrence, dietary intakes, nutritional essentiality. J Am Coll Nutr, 2002. 21(1): p. 14–21.
66. Shorter, E., The history of lithium therapy. Bipolar Disord, 2009. 11 Suppl 2: p. 4–9.
67. Shine, B., et al., Long-term effects of lithium on renal, thyroid, and parathyroid function: a retrospective analysis of laboratory data. Lancet, 2015. 386(9992): p. 461–8.
68. Chiu, C.T., et al., Therapeutic potential of mood stabilizers lithium and valproic acid: beyond bipolar disorder. Pharmacol Rev, 2013. 65(1): p. 105–42.
69. Forlenza, O.V., et al., Does lithium prevent Alzheimer’s disease? Drugs Aging, 2012. 29(5): p. 335–42.
70. Martinsson, L., et al., Long-term lithium treatment in bipolar disorder is associated with longer leukocyte telomeres. Transl Psychiatry, 2013. 3: p. e261.
71. McColl, G., et al., Pharmacogenetic analysis of lithium-induced delayed aging in Caenorhabditis elegans. J Biol Chem, 2008. 283(1): p. 350–7.
72. Ohgami, H., et al., Lithium levels in drinking water and risk of suicide. Br J Psychiatry, 2009. 194(5): p. 464–5; discussion 446.
73. Zarse, K., et al., Low-dose lithium uptake promotes longevity in humans and metazoans. Eur J Nutr, 2011. 50(5): p. 387–9.
74. Bell, G.A., et al., Use of glucosamine and chondroitin in relation to mortality. Eur J Epidemiol, 2012. 27(8): p. 593–603.
75. Weimer, S., et al., D-Glucosamine supplementation extends life span of nematodes and of ageing mice. Nat Commun, 2014. 5: p. 3563.
76. Ali, S.S., J.I. Hardt, and L.L. Dugan, SOD activity of carboxyfullerenes predicts their neuroprotective efficacy: a structure-activity study. Nanomedicine, 2008. 4(4): p. 283–94.
77. Quick, K.L., et al., A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice. Neurobiol Aging, 2008. 29(1): p. 117–28.
78. Dugan, L.L., et al., Carboxyfullerene neuroprotection postinjury in Parkinsonian nonhuman primates. Ann Neurol, 2014. 76(3): p. 393–402.
79. Baati, T., et al., The prolongation of the lifespan of rats by repeated oral administration of fullerene. Biomaterials, 2012. 33(19): p. 4936–46.
80. Tomas-Loba, A., et al., Telomerase reverse transcriptase delays aging in cancer-resistant mice. Cell, 2008. 135(4): p. 609–22.
81. Bernardes de Jesus, B., et al., Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer. EMBO Mol Med, 2012. 4(8): p. 691–704.
82. Palmisano, L. and S. Vella, A brief history of antiretroviral therapy of HIV infection: success and challenges. Ann Ist Super Sanita, 2011. 47(1): p. 44–8.
83. Evason, K., et al., Valproic acid extends Caenorhabditis elegans lifespan. Aging Cell, 2008. 7(3): p. 305–17.
84. Chen, D., et al., Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans. Cell Rep, 2013. 5(6): p. 1600–10.
85. Zhu, Y., et al., The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell, 2015. 14(4): p. 644–58.
86. Laberge, R.M., et al., MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation. Nat Cell Biol, 2015. 17(8): p. 1049–61.
87. Zhao, P., et al., Evening versus morning dosing regimen drug therapy for hypertension. Cochrane Database Syst Rev, 2011(10): p. CD004184.
88. Pierpaoli, W. and W. Regelson, Pineal control of aging: effect of melatonin and pineal grafting on aging mice. Proc Natl Acad Sci U S A, 1994. 91(2): p. 787–91.
89. Levine, M.E., et al., Low protein intake is associated with a major reduction in IGF-1, cancer, and overall mortality in the 65 and younger but not older population. Cell Metab, 2014. 19(3): p. 407–17.
90. Cannata, D., et al., The GH/IGF-1 axis in growth and development: new insights derived from animal models. Adv Pediatr, 2010. 57(1): p. 331–51.
91. Guevara-Aguirre, J., et al., Growth hormone receptor deficiency is associated with a major reduction in pro-aging signaling, cancer, and diabetes in humans. Sci Transl Med, 2011. 3(70): p. 70ra13.
92. Reagan-Shaw, S., M. Nihal, and N. Ahmad, Dose translation from animal to human studies revisited. FASEB J, 2008. 22(3): p. 659–61.
93. Lee, S.J., A.B. Hwang, and C. Kenyon, Inhibition of respiration extends C. elegans life span via reactive oxygen species that increase HIF-1 activity. Curr Biol, 2010. 20(23): p. 2131–6.