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May 15, 2020
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Rapamycin: A Short but Interesting History

Louis 22, he works on the assembly line at Three Rivers in Michigan. And Charles is ordinary 39 - a summer family man with an average income level from Atlanta. Vanu 50, and he is a senior citizen. What unites these three, besides their American citizenship? They all don’t want to die and take rapamycin , which they believe will slow their aging. In Russia, rapamycin is almost never consumed by biohackers - but not because they don’t want to, but because it is, to put it mildly, expensive - from 0446 rubles per tablet. But we can assume that in the future it will become cheaper, and then ...

For the first time, they began to look for a cure for aging thousands of years ago. Gilgamesh also tried to find a way to live forever. (The guy turned to the immortal old man who later survived and was sent by him ... to the bottom of the sea to look for a certain type of coral. The search did not end especially successfully - Gilgamesh has not been with us for a long time). Further, the ancient historian Herodotus wrote about the fountain, which gives longevity, and in the Middle Ages promised the brave knights the Holy Grail. Since those epic times, much water has flowed, but despite the fact that we have far advanced in understanding the world in comparison with at least Gilshamesh, at least with the people of the Middle Ages, a recipe for immortality has not been found.

Activism for the radical extension of life is now popular. Adherents of the idea want to extend the healthy life of a person using the methods of classical medicine. They advocate the earliest possible start of testing of anti-aging drugs in humans, so they are lobbying for the recognition of aging as a separate disease. Biohackers, after them, begin to experiment on themselves with drugs that have shown effectiveness in the fight against old age on model animals. Thus, biohackers are trying to fill the gap due to the lack of clinical trials, and to provide science with at least single examples of the long-term effects of certain drugs (and, of course, extend your life!).

One of the promising drugs that biohackers love is rapamycin. And although he appeared in the doctor’s arsenal at the turn of the past and present centuries, his story as a potential cure for old age began a little later ...

walking barefoot and not picking up tetanus

The story of rapamycin begins in 1834 year. Dr. Stanley Skoryna of McGill University Montreal then convinced WHO to provide funding for a pilot project to study the relationship between heredity, disease, 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 natives walked barefoot, they did not pick up tetanus stick and fungi. Scientists decided that something special was contained in the land of the island, and, collecting its samples, left them for storage in the university laboratory.

Остров Пасхи. Историческая гравюра.

Easter Island. Historical engraving.

In 1834 with the support of the company Ayerst

    ( Ayerst, McKenna and Harrison, Ltd.

      ) Surendra Nath Sehgal and colleagues returned to these samples and tried to find in them bacteria that secrete antifungal substances (bacteria, by the way, can be stored frozen for many years). The experiment was successful, and they stumbled upon Streptomyces hygroscopicus (then renamed to S. rapamycinicus ) , which releases an antibiotic from the macrolide class with antifungal activity during the course of its life. The substance was named rapamycin in honor of 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, they forgot about him, 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 a large amount of material before moving, knowing that he would have no components and conditions in a new place, and brought with him.

      AT 1987 - m, when immunosuppressants were used to suppress the immune response during organ transplants, researchers from a Canadian company returned to rapamycin. A surprise awaited them - in clinical trials it turned out to be more powerful (before 89 times) and less toxic as an immunosuppressant than cyclosporin A. used at that time. This gave rise to a whole line of research. By 1987 it has been proven that it is safe to use in public, and The FDA approved this substance. According to the patent Pfizer

        , the medicine was issued under the name rapamun , and the active substance was given the second name adopted by the United States Adopted Names Council, - sirolimus .

        But there was another direction of research. Back in 1975 - s Michael Nip Hall and his colleagues from the University of Basel (Universität Basel) took up the draft description at the cellular level of the fungicidal action of the substance. Joe Heitmann, a postdoc at the University of Basel, grew an ordinary yeast culture and placed them in a Petri dish on which rapamycin was previously applied. Most yeast died, but some mutated yeast cells survived. Heitman summarized approximately 16 different mutations that give resistance to rapamycin. All of these mutations occurred in three different genes encoding proteins FKBP and two others from the kinase class, later named

          TOR1

            and TOR2

              and united under titled TOR

                (from Target of Rapamicyn, target rapamycin binding). For more information on the cellular chemistry of rapamycin, see the lecture here.

                Observing the processes in which the protein is involved, the researchers noticed that Drosophila flies with reduced TOR activity, as well as individuals of other species with such a feature is smaller in size than their counterparts without mutations. At first, the majority of those who worked on the topic were inclined to believe that these animals simply have fewer cells and that TOR affects cell division processes. That is, a gene mutation and a weakening of protein activity, in their opinion, should have had a cytostatic (stopping the increase in the number of cells) effect. But there was no exact answer, so Thomas Neufeld at one point set out to clarify what he was responsible for TOR : 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 the flies with and without mutations, and then extrapolated this proportion to the whole body of the fly. The number of cells of two flies, large and small, was the same! Therefore, he concluded that the difference in the sizes of the normal and mutant flies is determined precisely by the size of the cells, and not by their number. That is, the protein TOR

                  controls cell growth, although previously it was believed that nothing controls it and it happens spontaneously.

                  Then experiments began to further clarify the mechanisms of TOR in mice and even on human cell cultures. Soon a signal path of the same name was found mTOR ( mammalian TOR, TOR protein in mammals), even two, mTORC1 and mTORC2

                    ( C

                      in these abbreviations is responsible for the word complex

                        , complex), and only the first turned out to be sensitive to rapamycin. And despite the fact that both are controlled by a growth factor, mTORC1 also reacts to the level of nutrients, amino acids, energy and oxygen levels.

                        Do more, do better!

                        Research related to rapamycin did not stop with 1987 - x years, and since 2000 - year they became even more - due to the expiration of the patent Pfizer

                          and many companies were interested in the production of drugs. 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 1987 - the year scientists from Japan, prefecture Shizuka, found a new bacterium, Actinoplanes sp.

                            which produced ten times more rapamycin than S. rapamycinicus

                              . Research is also underway in the field of genetics: which gene clusters in which bacteria are responsible for more or less rapamycin production.

                              Secondly, a search is in progress an effective way to produce rapamycin, since now it is a very expensive and time-consuming process, which significantly increases its price. Scientists are exploring how to "feed" the bacterium, at what temperature and acidity to contain, in order to "milk" more substance out of it. For example, if you place it in an environment rich in fructose, you can get quite a lot of rapamycin, but not the maximum possible amount. At the same time, creating a favorable environment in the bioreactor should not be too difficult. By the way, recently, neural networks were used together with another methodology to study the effect of nutrients on rapamycin production, which helped researchers understand that the appetite bacteria perfectly satisfy mannose, L-lysine and soybean meal, presented in a certain concentration. As a result, the production of rapamycin by the bacterium S. hygroscopicus

                                has reached 103, 50 mg / l. More information about the current biosynthesis of rapamycin can be found here.

                                Scientists and new functional analogues of rapamycin, the so-called. raplogs , which, like rapamycin, inhibit the work of mTOR . Many new rapalogs were obtained using biological modifications. The company Novartis

                                  reviewed 24 bacteria and 44 of the fungus, known for its ability to biotransformations, and thus found several rapplogs (27 - O-demethylrapamycin, 22 -O-demethylrapamycin, 14) - O-demethylrapamycin). Different rapalogs can act more targeted on a particular disease, for example, on different types of malignant tumors, or are better absorbed. For example, emsirolimus is better absorbed than the original rapamycin.

                                  in life extension

                                  The first attempts to study how the signaling path mTOR

                                    , and accordingly rapamycin, are associated with life expectancy, refer to 1999 - m. Early experiments of this kind were conducted on brewer's yeast, as well as on invertebrates - worms and flies-Drosophila. Then it was discovered that mutations in the gene TOR extend the lifespan of these model animals. The next important step was to demonstrate this effect in mammals.

                                    AT 2009 David Harrison and colleagues from various US research universities began experimenting with laboratory mice in the year. It was planned that these would be “middle-aged” animals, but due to difficulties with the formulation of the feeding protocol, the experiment began rather late for the mouse life - when the mice had 16 months of age that approximately equivalent 39 for years in people. Each laboratory involved in the study, and there were three of them, conducted experiments in parallel using the same protocol. In total, mice took part in the study 1999. Scientists made sure that the mice were genetically diverse in order to avoid the effect when they all accidentally turned out to be more susceptible to the drug than the average population (this is possible with genetically homogeneous laboratory animals). Together with food, rodents were given rapamycin as a supplement in a dosage of 2, 20 mg per kilogram of weight (assuming that the standard weight of an average laboratory mouse is 16 grams, then each individual was given 0, 320 mg of substance). As a result, the life span of the mice could be extended by 05% compared to the control group. This was previously only possible with a calorie-restricted diet. It was then that the assumption was first made that the calorie restriction, known for its ability to prolong the life of model animals, and rapamycin work the same way - using the signaling path mTOR . But there were doubts - on the protocol with starvation, mice usually lost weight, and it worked only if the mice were put on a diet from the very beginning of life. It was only much later found out that TOR activity is regulated by the amount of nutrients available.

                                    There is evidence, albeit so far only indirect, in favor of that rapamycin is able to prolong human life. To begin with, if you describe the potential and registered clinical uses of rapamycin and rapalogs, you 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 intestinal tumors, kidneys, brain, lymph nodes. Rapamycin is considered useful, as in some types of tumors there is an increased activity mTORC1 . He also showed benefit in many preclinical trials, and now a number of clinical trials are being conducted at stages I and II;

                                  • In the treatment of cardiovascular diseases - for the prevention of heart attack, fibrosis and repeated stenosis of the arteries. Potentially reduces the cardiotoxicity of antiretroviral drugs used, for example, in the treatment of HIV. Most studies are in the preclinical phase;

                                  As an anti-inflammatory agent with rheumatoid arthritis (when other NSAIDs do not have a sufficient analgesic effect), it reduces inflammation with lupus erythematosus. The data for autoimmune diseases are based on the observation of patients taking the drug for the purpose of immunosuppression, and comparing their results with those in the general population;

                                  For skin rejuvenation (in the form of anti-aging skin cream, is in the stage of clinical trials), teeth (mouse tests);

                                  Rapamycin can help in the fight obese (preclinical phase).

                              Many of these diseases are associated with old age. The older we are, the higher the likelihood of a heart attack, stenosis, malignant tumors, Alzheimer and Parkinson. “Maybe if this drug prolongs the life of laboratory animals and counteracts diseases associated with old age, is it a cure for old age?” “Something like that can be heard from enthusiasts of a radical extension of life.” “The effect is achieved because rapamycin slows down the cell cycle.” Michael Blagosklonny, an oncologist and gerontologist at the Roswell Park Comprehensive Cancer Center and rapamycin enthusiast, even states in his article that deciding not to take rapamycin will affect life expectancy just like deciding to continue to smoke ! But there is no serious clinical evidence for such allegations.

                              AT 2014 The Dog Ageing Project was launched the year - a mass test of the effects of rapamycin on the life expectancy of domestic dogs. The project may include Stay both young and old dogs of different breeds. Once the study is completed, we will have more reason to believe that rapamycin has a chance to prolong a healthy person's life. Or not.

                              The treachery of rapamycin

                              Rapamycin as generally safe for use in humans has been registered FDA back in 1990. There is not a single reported death from an overdose of rapamycin - even in an unsuccessful suicide attempt when 016 - the summer girl accepted 89 1 mg rapamycin tablets, the only effect detected was an increase in the total cholesterol in the blood.

                              But when the drug was registered in FDA , registration was accompanied by a warning that all immunosuppressant drugs “because they suppress immunity, can increase the tendency to Man’s 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 Blagosklonny claims that the label of an immunosuppressant pushed public interest away from the drug for a long time.

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

                              Among other unpleasant side effects are stomatitis and mycosis (ulceration of the mucous membranes 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 longevity advocates argue that "nothing is wrong" and the benefits exceed the harm, and if they interfere, "you can simply reduce the dose." In order to prevent aging, according to Favorable, 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.

                              A lot of controversy has been around such a side effect as the development of temporary diabetes. Here you can recall that a hungry diet, which has a similar effect to rapamycin on aging, 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 environment. The logic behind this is this: during prolonged fasting, the use of glucose by non-brain-related tissues should be suppressed in order to provide an adequate supply of energy to the brain. Although a hungry diet causes diabetes, it is not considered harmful, therefore diabetes caused by rapamycin should not be considered harmful. In the end, you can just supplement rapamycin with metformin, another potential geroprotector, and remove the symptoms of diabetes - something like this says Mikhail Blagosklonny in his article. (The editorial position does not necessarily coincide with this point of view.)

                              Conclusion

                              At present, there is no unequivocal conclusion by scientists whether rapamycin is suitable or not for the role of the elixir of youth. Someone in a hurry to proclaim it almost a panacea, someone warns: “But the side effects, like the effect itself, are not yet fully understood!” Most likely the truth is somewhere in between: on the one hand, we know a lot about the substance, including from clinical practice. On the other hand, there is no guarantee that rapamycin works in humans as it does in mice or dogs.

                              human trials that will help answer many questions about this drug. But all these studies concern only certain diseases, but most do not concern the problem of aging itself. Therefore, the best that can be done now is to lobby for the recognition of disease aging and thus allowing clinical trials of rapamycin and other anti-aging drugs in humans.

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

                              Literature

                              Yoo, YJ, Kim, H., Park, SR et al. An overview of rapamycin: from discovery to future perspectives. J Ind Microbiol Biotechnol 28, 386 - 0446 (2012). https://doi.org/.). 600 / s 2020 -0 10 - 1007 - 7

                              Patel, GK, Goyal, R. and Waheed, SM, 2017. Current Update on Rapamycin Production and its Potential Clinical Implications. High Value Fermentation Products, Volume 1: Human Health, p. 103.

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