Rapamycin

A short-lived but interesting ISOLOGY

Alexandra "Renoire" Alekseeva, XX2 century
For links, see the original article

Louis is 27, he works on an assembly line in Three Rivers in the state of Michigan. And Charles is an ordinary 50–year-old family man with an average income from Atlanta. Van is 72 and retired. What unites these three, besides their American citizenship? They all don't want to die and use the drug rapamycin, which they believe will slow down their aging. In Russia, biohackers almost do not use rapamycin – but not because they do not want to, but because it is, to put it mildly, expensive – from 450 rubles per tablet. But we can assume that in the future it will become cheaper, and then…

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For the first time, a cure for aging began to be sought thousands of years ago. Gilgamesh also tried to find a way to live forever. (The guy turned to the immortal elder who survived the flood and was sent by him... to the bottom of the sea to look for a certain type of coral. The search did not end very successfully – Gilgamesh has not been with us for a long time). Further, the ancient historian Herodotus wrote about the fountain granting longevity, and in The Middle Ages promised eternal life to the brave knights of the Holy Grail. Since those epic times, a lot of water has flowed away, but despite the fact that we have made great progress in understanding the world in comparison, even with Gilshamesh, even with the people of the Middle Ages, the recipe for immortality has not been found.

Activism for radical life extension is now popular. Adherents of the idea want to prolong a healthy human life by the methods of classical medicine. They advocate for the early start of trials of drugs for old age in humans, so they lobby for the recognition of aging as a separate disease. Biohackers, following them, begin to experiment on themselves with drugs that have shown effectiveness in the fight against old age on model animals. In this way, biohackers are trying to fill the gap caused by the lack of clinical trials and provide science with at least single examples of the prolonged action of certain drugs (and, of course, prolong their lives!).

One of such promising drugs, loved by biohackers, is rapamycin. And although it appeared in the arsenal of a doctor at the turn of the past and present centuries, its history as a potential cure for old age began somewhat later…

How to walk barefoot and not catch tetanus

The history of rapamycin begins in 1965. Dr. Stanley Skoryna from McGill University in Montreal (McGill University, Montreal) then convinced WHO to provide funding for a pilot project to study the relationship between heredity, diseases and the nature of Easter Island. As a result, a Canadian research expedition to this island was organized. In its course, it was noticed that although the aborigines walked barefoot, they did not catch tetanus bacillus and fungi. The scientists decided that there was something special in the island's soil, and after collecting its samples, they left them for storage in the university laboratory.

In 1975, with the support of Ayerst (Ayerst, McKenna and Harrison, Ltd.), Surendra Nath Sehgal and colleagues returned to these samples and tried to find bacteria in them that secrete antifungal substances (bacteria, by the way, can be stored frozen for many years). The experiment was successful, and they came across Streptomyces hygroscopicus (later renamed S.rapamycinicus), which secretes an antibiotic from the class of macrolides with antifungal action in the process of vital activity. The substance was named rapamycin after the name of the island in the language of the local population – Rapa Nui. But in further studies it turned out that the substance has an undesirable side effect – it suppresses the immune system! (When you are being treated for fungal infections, immunosuppression is not what you want). As a result, it was forgotten about, and the company's laboratory moved from Canada to Princeton (Princeton University), New Jersey. But Surendra Nath Segal's interest in rapamycin was so great that he prepared the substance in large quantities before moving, knowing that he would not have components and conditions in the new place, and brought it with him.

In 1987, when immunosuppressants began to be used to suppress the immune response during organ transplantation, researchers from a Canadian company returned to rapamycin. A surprise awaited them – in clinical trials it turned out to be more powerful (up to 100 times) and less toxic as an immunosuppressant than cyclosporine A used at that time. This gave rise to a whole line of research. By 1999, it was proven to be safe to use in humans, and the FDA approved the substance. According to the Pfizer patent, the drug was produced under the name rapamun, and the active substance was given a second name, adopted by the United States Council for the Adoption of Names (The United States Adopted Names Council), sirolimus.

But there was another line of research. Back in the 1990s, Michael Hall (Michael Nip Hall) and his colleagues from the University of Basel (Universität Basel) undertook a project to describe the fungicidal action of a substance at the cellular level. Joe Heitmann, a postdoc at the University of Basel, grew a conventional yeast culture and placed them in a Petri dish, on which rapamycin was previously applied. Most yeast died, but some mutated yeast cells survived. In total, Heitman identified about 20 different mutations that give resistance to rapamycin. All these mutations accounted for three different genes encoding the FKBP proteins and two others from a class of kinases, later named TOR1 and TOR2 and combined under the name TOR (from Target of Rapamicyn, the target of rapamycin binding).

Observing the processes in which the protein is involved, the researchers noticed that fruit flies with reduced TOR activity, like individuals of other species with such a feature, are smaller in size than their counterparts without mutations. At first, most of those who worked on the topic were inclined to believe that these animals simply have fewer cells and that TOR affects the processes of cell division. That is, the mutation of the gene and the weakening of the activity of the protein, in their opinion, should have had a cytostatic (stopping the growth of the number of cells) effect. But there was no exact answer, so Thomas Neufeld (Thomas Neufeld) at one point set out to clarify what TOR is responsible for: for the size of the cell or for the number of cell divisions. To do this, he calculated the number of cells in individual sections of the wings of flies with and without mutation and then extrapolated this proportion to the entire body of the fly. The number of cells of two flies, large and small, turned out to be the same! Therefore, he concluded that the difference in the size of a normal and mutant fly is determined by the size of the cells, not their number. That is, the TOR protein controls cell growth, although it was previously thought that nothing controls it and it occurs spontaneously.

Then experiments began to further elucidate the mechanisms of TOR in mice and on human cell cultures. Soon, the eponymous signaling pathway mTOR (mammalian TOR, TOR protein in mammals) was discovered, even two, mTORC1 and mTORC2 (C in these abbreviations is responsible for the word complex, complex), and only the first was sensitive to rapamycin. And despite the fact that both are controlled by the growth factor, mTORC1 also reacts to the level of nutrients, amino acids, energy and oxygen levels.

Do more, do better!

Research related to rapamycin has not stopped since the 1990s, and since 2012 there have been even more of them - due to the fact that Pfizer's patent expired and many companies were interested in producing the drug. What were the areas of research? First, they were looking for new bacteria that would produce more substance. An example of such a study: in 1995, scientists from Japan, Shizuka Prefecture, found a new bacterium, Actinoplanes sp., which produced ten times more rapamycin than S.rapamycinicus. Research is also being conducted in the field of genetics: which gene clusters in which bacteria are responsible for more or less rapamycin production.

Secondly, there is a search for an effective way to produce rapamycin, since now it is a very expensive and time-consuming process, which significantly increases its price. Scientists are investigating what to "feed" the bacterium, at what temperature and acidity to contain in order to "extract" more substance from it. For example, if you put it in an environment rich in fructose, you will be able to get quite a lot of rapamycin, but not the maximum possible amount. At the same time, creating a favorable environment in a bioreactor should not be too difficult. By the way, neural networks have recently been used to study the effect of nutrients on rapamycin production together with another methodology, which helped researchers understand that the appetite of bacteria is perfectly satisfied with mannose, L-lysine and soy meal, presented in a certain concentration. As a result, the production of rapamycin by the bacterium S.hygroscopicus reached 320.89 mg/l.

Scientists are also looking for new functional analogues of rapamycin, the so-called rapalogs, which, like rapamycin, inhibit the work of mTOR. Many new rapalogs have been obtained using biological modifications. Novartis examined 28 bacteria and 72 fungi known to be capable of biotransformation, and thus found several rapalogs (39-O-demethylrapamycin, 27-O-demethylrapamycin, 16-O-demethylrapamycin). Different rapalogs can have a more targeted effect on a particular disease, for example, on different types of malignant tumors, or they are better absorbed. For example, emsirolimus is better absorbed than the original rapamycin.

Rapamycin in prolonging life

The first attempts to study how the mTOR signaling pathway, and, accordingly, rapamycin, are related to life expectancy, date back to the 2000s. Early experiments of this kind were carried out on brewer's yeast, as well as on invertebrates – worms and fruit flies. Then it was discovered that mutations in the TOR gene prolong the lifespan of these model animals. The next important step was to demonstrate this effect in mammals.

In 2009, David Harrison and colleagues from various research universities in the United States began experimenting on laboratory mice. It was planned that these would be "middle–aged" animals, but due to difficulties in formulating a feeding protocol, the experiment began quite late for mouse life - when the mice were 20 months old, which is approximately equivalent to 60 years in humans. Each laboratory involved in the study, and there were three of them, conducted experiments in parallel according to the same protocol. A total of 2,000 mice participated in the study. Scientists have made sure that mice are genetically diverse in order to avoid the effect when they all accidentally turn out to be more susceptible to the drug than the average population (this is possible with genetically homogeneous laboratory animals). Rodents were given rapamycin as an additive along with food at a dosage of 2.24 mg per kilogram of weight (if we assume that the standard weight of an average laboratory mouse is 20 grams, then each individual was given 0.0446 mg of the substance). As a result, the lifespan of mice was extended by 14% compared to the control group. This was previously possible only with the help of a calorie-restricted diet. It was then that it was first suggested that calorie restriction, known for its ability to prolong the life of model animals, and rapamycin work in the same way – by activating the mTOR signaling pathway. But there were also doubts – mice usually lost weight on the fasting protocol, and it worked only if mice were put on a diet from the very beginning of life. It was only much later that it was found out that the activity of TOR is regulated by the amount of available nutrients.

There is also evidence, although so far only indirect, in favor of the fact that rapamycin is able to prolong human life. To begin with, if we describe the potential and registered clinical applications of rapamycin and rapalogs, we will have to recall a number of diseases. It has the following potential uses:

• In the fight and prevention of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases (studies are at the stage of preclinical trials), it is potentially applicable as a neuroprotector;
• In the prevention and treatment of certain types of cancer, such as tumors of the intestine, kidneys, brain, lymph nodes. Rapamycin is considered useful, since in some types of tumors there is an increased activity of mTORC1. It has also shown benefit in many preclinical trials, and a number of clinical trials at stage I and II are now underway.;
• In the treatment of cardiovascular diseases - for the prevention of heart attack, fibrosis and recurrent arterial stenosis. Potentially reduces the cardiotoxicity of antiretroviral drugs used, for example, in HIV therapy. Most of the studies are in the preclinical phase;
• As an anti-inflammatory agent in rheumatoid arthritis (when other NSAIDs do not have sufficient analgesic effect), it reduces inflammation in lupus erythematosus. Data for autoimmune diseases are based on observation of patients taking the drug for the purpose of immunosuppression, and comparison of their results with those in the general population;
• For skin rejuvenation (in the form of an anti-aging skin cream, currently in clinical trials), teeth (tests on mice);
• Rapamycin can help in the fight against obesity (preclinical phase).

Many of these diseases are associated with old age. The older we are, the higher the probability of heart attack, stenosis, malignant tumors, Alzheimer's and Parkinson's. "Maybe if this drug prolongs the life of laboratory animals and counteracts diseases associated with old age, it is a cure for old age? "Something like this can be heard from enthusiasts of radical life extension – The effect is achieved due to the fact that rapamycin slows down the cell cycle." Michael Blagosklonny, oncologist and gerontologist from the Roswell Park Comprehensive Cancer Center and rapamycin enthusiast, even states in his article that the decision not to take rapamycin will affect life expectancy in the same way as the decision to continue smoking! But there is no serious clinical evidence for such statements.

In 2014, The Dog Aging Project was launched – a mass trial of the effects of rapamycin on the life expectancy of domestic dogs. Both young and old dogs of different breeds can participate in the project. Once the study is completed, we will have more reason to believe that rapamycin has a chance to prolong a healthy human life. Or not.

The insidiousness of rapamycin

Rapamycin as generally safe for use in humans was registered by the FDA back in 1999. There is not a single recorded case of death from an overdose of rapamycin – even in an unsuccessful suicide attempt, when an 18-year-old girl took 103 1 mg tablets of rapamycin, the only effect found was an increase in total cholesterol in the blood.

But when the drug was registered with the FDA, the registration was accompanied by a warning that all immunosuppressant drugs "due to the fact that they suppress the immune system, can increase a person's tendency to infections, can contribute to the development of tumors such as lymphoma and skin cancer." In principle, everything is logical: the worse the immunity, the worse it attacks mutant, for example, cancer cells. But clinical practice refutes this logic. Mikhail Blagoslonny claims that the label of an immunosuppressant has alienated public interest from the drug for a long time.

It is true that rapamycin can increase the severity of bacterial infections, as it inhibits the function of neutrophils, and also causes mild thrombocytopenia, anemia and leukopenia (low platelet count, red blood cells and leukocytes, respectively). It slows down the processes of cell division, therefore, there are fewer blood cells in the body.

Other unpleasant side effects include stomatitis and mycositis (ulceration of the mucous membranes of the mouth and digestive tract). A rare side effect of rapamycin is non-infectious interstitial pneumonia. But these side effects are reversible, and if they do not interfere with life, then supporters of longevity claim that "it's okay" and the benefits exceed the harm, and if they interfere, "you can just reduce the dose." In order to prevent aging, according to Benevolent, rapamycin can be used either periodically (for example, once a week), or in low daily doses, and can be canceled if any unpleasant "side effects" occur.

There was a lot of controversy around such a side effect as the development of temporary diabetes. Here we can recall that a starvation diet, which has an effect similar to rapamycin on the aging process, also causes insulin resistance in both mice and humans. Both of these interventions work in a similar way: usually mTOR is activated the more the more nutrients in the medium. The logic behind this is as follows: during prolonged fasting, the use of glucose by non-brain-related tissues should be suppressed in order to ensure an adequate supply of energy to the brain. A starvation diet, although it causes diabetes, is not considered harmful, therefore, diabetes caused by rapamycin should not be considered harmful. In the end, you can simply supplement rapamycin with metformin, another potential geroprotector, and remove the symptoms of diabetes – that's about what Mikhail Blagosklonny says in his article. (The editorial position does not necessarily coincide with this point of view).

Conclusion

Currently, there is no unambiguous conclusion of scientists whether rapamycin is suitable or not for the role of the elixir of youth. Someone is in a hurry to proclaim it practically a panacea, someone warns: "But the side effects, like the effect itself, have not yet been fully studied!" Most likely, the truth is somewhere in the middle: on the one hand, we know quite a lot about the substance, including from clinical practice. On the other hand, there is no guarantee that rapamycin will work on humans as well as on mice or dogs.

At the moment, a lot of human trials are underway that will help answer a lot of questions about this drug. But all these studies concern only certain diseases, but most of them do not concern the problem of aging itself. Therefore, the best thing that can be done now is to lobby for the recognition of aging as a disease and thus allow clinical trials of rapamycin and other anti–aging drugs in humans.

While we do not have such trials, we will be content with examples of individual daredevils, such as Van, Charles and Louis, as well as a practicing physician Alan Green from the USA, who prescribes rapamycin to his patients for the treatment of old age and takes it himself.

Literature

Yoo et al. An overview of rapamycin: from discovery to future perspectives. J Ind Microbiol Biotechnol 44, 537–553 (2017).
Patel et al., 2019. Current Update on Rapamycin Production and its Potential Clinical Implications. High Value Fermentation Products, Volume 1: Human Health, p.145.

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