19 February 2008

The choice between life and death

Anna Koroleva, Vladimir Skulachev and Maxim Skulachev, "In the World of SCIENCE" No. 2-2008The fight against aging is a large—scale task and is beyond the power of any one scientific group.

In terms of complexity, it is comparable to S.P. Korolev's project to launch the first satellite or the "world project" to decode the human genome. Hundreds of laboratories, including the US National Institute of Aging, are struggling to solve this problem in the world. To date, there are more than 300 scientific theories of aging. Since 2005, an interdisciplinary innovative project has been carried out in Russia at the Lomonosov Moscow State University, which can not only compete with foreign gerontologists, but also has a real chance of creating the first effective geroprotector – a drug against old age. The scientific director of the project is Academician of the Russian Academy of Sciences V.P. Skulachev. The investor was the Russian-Asian Investment Company (RAInCo). To implement this project, the investor and the leadership of Moscow State University, represented by Rector V.A. Sadovnichy and the deans of biomedical faculties, decided to follow a path that is quite common in the West, but still unusual for Russia — to create a "university-based" biotechnology company.This is how Mitotechnology was created, which has been leading the project "Practical Use of Skulachev ions" for three years.

Old age is not a joy

Many inventions, without which humanity cannot imagine life today, were created and implemented in the practice of the XX century (electricity, car, airplane, television, computers, space flight).

Equally drastic steps have been taken in biology, which has turned from a descriptive science into an experimental and engineering field — the possibility of directional changes in the properties of living beings has appeared. Many discoveries have been made, for example, the mechanism of heredity has been established and the genetic code has been deciphered, genetic engineering has been created, the human genome has been "read", methods of cell engineering, cloning and much more have been developed. Thanks to these achievements of biology, it was possible to equip medicine with means against many dangerous diseases. However, there is one important area in which the last century has, in fact, nothing to boast about.

This is the fight against aging. Unlike any other disease (let's assume that old age is also a disease), the whole of humanity is subject to aging. Upon reaching a certain age, each person begins to slowly "fade away": muscles weaken, immunity decreases, the likelihood of oncological, cardiovascular, neurodegenerative diseases increases, vision and memory weaken, endurance decreases. What are these, if not signs of some kind of systemic disease? Eventually, one of these symptoms of aging progresses so much that the body can no longer resist the disease and dies.Since 1970, there has been a "pharmacological boom" in the world.

Multinational corporations are engaged in the creation of new medicines, investing huge amounts of money in the development of medicines and biomedical technologies. The more dangerous the disease, the greater the percentage of people exposed to it, the larger the market will have a drug and the more willing companies are to develop it. Why have pharmacological giants so far ignored the most common and dangerous ailment — old age?

Obviously, because the development of a new drug can take 5, 10, 15 years and is fraught with great risks — for example, excellent results on animals do not guarantee at all that the drug will not be useless for humans, or will not have undesirable side effects that make its use impossible. Therefore, when deciding to invest in the development of a new drug, the company must be sure that the proposed approach to combating the disease has a chance of success.

Causes of the diseaseThere are many theories of aging.

The most widespread and convincing of them is the free radical hypothesis of D. Harman, who suggested that the oxidation of biopolymers with active oxygen forms (ROS) plays a leading role in the weakening of vital functions with age.

In accordance with this hypothesis, it was found that the level of oxidation of DNA, proteins and lipids increases with aging. Such a situation may be a consequence of an increase in ROS production in old age or a weakening of anti-oxidant protection, or simply the duration of the damaging effect of ROS, proportional to the age of the organism.

One of the biggest discoveries of recent decades has been the discovery of "death programs". It was found that cell death occurs, as a rule, as a result of one of these programs (apoptosis or necrosis, as well as their combination) embedded in its genome.

D. Harman's hypothesis together with this discovery served as the basis for the conclusion that, at least, unicellular organisms have a self-destruction mechanism. It has been shown that similar "death programs" exist in bacteria and unicellular eukaryotes, such as, for example, yeast. There are many examples proving that the programmed death of an individual is a process called "phenoptosis" (the process was so named by academician V.P. Skulachev, the word was invented and put into use by academician, linguist M.L. Gasparov. — Ed.), is also inherent in higher organisms — animals and plants, although its molecular mechanisms have yet to be clarified. The biological meaning of phenoptosis is quite obvious by analogy with apoptosis — the purification of a population from undesirable individuals in order to protect the entire population, if they pose a threat to it. Another function of phenoptosis could be to accelerate the change of generations.

The concept of phenoptosis makes us look at the problem of aging in a different way. What if this is the way nature forces us to leave, making room for the young? What if this slow extinction, as well as the apoptosis program, is embedded in the form of a genetic program in our genome, and its main biological meaning is the acceleration of evolution? These questions allowed us to formulate the theory of programmed aging as an instrument of evolution.

The deeper biologists penetrate into the mechanism of functioning of living systems, the more they are convinced that nature is trying to keep under strict control all the processes going on in the body, especially those related to its development, with heredity. In this regard, it seems especially improbable that nature has given such an important stage as aging and death of the organism to random circumstances and has not programmed control of this process in the genome.

Approach to the treatment of the diseaseThe theory of aging as a slow phenoptosis gives us a chance.

If there is a program that is slowly but surely leading us to death, then perhaps it can be interfered with, reconfigured, slowed down, broken. Unfortunately, at this stage of development, bioengineering is not yet fully ripe for creating new biological systems, biochemical pathways, etc. But it is always easier to break than to build.

Now science has a powerful arsenal of tools that allow stopping the implementation of a variety of genetic programs. Why not add an aging program to their list? We are not talking about changing the genome — today a person is not yet ready to interfere with his own genes.

Biology is unable to predict all the consequences of such a step, because it can be irreversible for the body. There remains a pharmacological approach — the development of a substance that can not so much change the aging program itself, as prevent its implementation by acting on a specific target — some element, a process in a cell or an organism that is fundamentally important for the operation of a malicious program. Where to look for this target?

Here it is necessary to return to D. Harman's hypothesis. Of course, reactive oxygen species (ROS) are suitable candidates for the role of a "samurai sword" used by an organism that has decided to commit biochemical suicide. Even if they are not the direct cause of aging, they certainly take a direct part in this process. There are many different ROS in the cell that perform a variety of (and not always harmful) functions. Where to look for those that are involved in the aging process? There is a whole set of enzymes in the cell that convert O2 into the primary form of ROS — super-oxide (O2*-) or into its derivative — hydrogen peroxide. However, all of them are significantly inferior in power to the respiratory chain of the inner membrane of the mitochondria. During the day, the mitochondria of an adult absorb about 400 liters of O2, turning it into water by four-electron reduction. However, if at least 0.1% of this amount of O2 is reduced chemically in a simpler, single-electron way, then 0.4 l of O2*- will be obtained, which far exceeds the capabilities of all other ROS generation mechanisms combined. In fact, we carry in our mitochondria a potential generator of the strongest poison that can easily kill our cells and ourselves along with them. Such a catastrophe will occur not even because of the direct toxic effect of ROS, but due to the launch of apoptosis or necrosis processes, of which ROS serve as powerful inducers.

Consequently, mitochondrial ROS are an attractive candidate for the role of a "target", hitting which could "destroy" the biochemical mechanism of suicide.

Tool SearchThe pharmacological way to combat ROS has long been known — it's antioxidants.

There is a very extensive and ambiguous literature on their treatment of aging — from the statement of the American biochemist B. Ames and his colleagues that such a cure for old age has already been found, to the conclusions of D. Howes about the complete infertility of this approach and, therefore, the fallacy of the free radical hypothesis of D. Harman.

But, in our opinion, there are several significant omissions in the work on the treatment of old age with antioxidants.

Firstly, antioxidants should be used, specifically addressed to the mitochondria.

Secondly, they must be safe, since, interacting with ROS, the antioxidant molecules themselves become radicals; accordingly, there must be a reliable way to neutralize them immediately in the cell, preferably with the restoration of the antioxidant in its original form.

Thirdly, all antioxidants have a pro-oxidant effect when the dose is increased, limiting the possibility of their use, i.e. they should be highly effective in as low doses as possible.

Finally, traditional antioxidants, even if they may end up in the mitochondrial membrane along with other cell membranes, are natural substances, the excess of which can be broken down by cellular enzymes, as soon as their presence becomes undesirable.

In fact, the body has protection systems not only from oxygen, but also from antioxidants. And the thing is that ROS performs a number of biological functions, without which a full life is impossible (they, for example, are directly involved in the fight against bacteria and viruses).

Therefore, the antioxidant should not remove ROS, but only their excess, which is formed inside the mitochondria as the body ages; it should not be inactivated by the enzymes of the body, which is striving at all costs to complete its ontogenesis by turning on the aging program.

Unfortunately, none of the antioxidants known by the end of the XX century meets all these requirements. The real candidate appeared only at the beginning of this century.

Jonah Skulachev

At the turn of the 1960s-1970s, we (a group of scientists from Lomonosov Moscow State University together with a group of Doctor of Physico-Mathematical Sciences E.A. Lieberman from the USSR Academy of Sciences) discovered that some compounds — lipophilic cations (for example, phosphonium), are able to penetrate the mitochondria of a living cell. In 1974, such compounds were named by the American biochemist D. Green "Skulachev ions".

In the early 70s by us (V.P. Skulachev, Doctor of Chemical Sciences L.S. Yaguzhinsky, academician S.E. Severin. It has been suggested that penetrating cations can be used by mitochondria as "electric locomotive molecules" for the accumulation of uncharged substances attached to these cations in mitochondria.

In the late 1990s, the British biochemist M.P. Murphy used this approach, trying to create a mitochondrial-targeted antioxidant. He attached vitamin E to the lipophilic ion triphenylalkylphosphonium . Unfortunately, this substance, as well as its somewhat more successful version, in which ubiquinone is used instead of vitamin E, has not yet been used in practice, apparently due to its strong pro-oxidant effect and insufficient effectiveness in low doses. The prospects of the whole approach were in doubt.

However, in 2003 we started developing a new mitochondrial-targeted antioxidant. In order to fundamentally increase its effectiveness, plastoquinone was used, a substance from the most oxygen—saturated place in living nature - plant chloroplasts. The substance SkQ1 was designed and synthesized, the effectiveness of which turned out to be hundreds of times higher than that of previous analogues.

At the moment, Mitotechnologiya has filed more than 10 applications for international patents for the technologies being developed and methods of their application in various fields of medicine and biotechnology. The key link in the protection of the company's IP is the so—called "umbrella" patent, which protects the essence of the basic technology of the project - mitochondrial-targeted biologically active substances, in particular — mitochondrial antioxidants of the SkQ type. A patent for this technology has already been obtained in Russia, an application for a US national patent has been filed

Method of technology and drug developmentHaving set the goal of combating aging, we understood that such a task is beyond the power of one scientific group.

How is it possible in the modern world to quickly find resources (financial, intellectual, human) for the implementation of such a project? Logic suggested the only answer — if we consider old age as a disease, then we are talking about creating a new drug. To solve such a fundamental problem, an interdisciplinary innovative project was organized on the basis of Moscow State University, bringing together a team of scientists (biologists and physicians, chemists, physicists, mathematicians).

The main technological approach is directed action on the mitochondria of cells of a living organism in order to regulate the amounts of ROS produced by these mitochondria — a kind of mitochondrial engineering, or "mitoengineering". Its implementation is possible, for example, by means of targeted delivery of highly effective antioxidants to the mitochondria of a living cell. The technology that allows this delivery is called "mitotechnology".

Since mitochondria contain and produce ROS in almost all cells of the body, the scope of mitotechnology is extremely wide. The same approach can be applied to combat other diseases for which the negative role of ROS is shown (especially if the contribution of mitochondrial ROS is known). Therefore, if mitotechnology turns out to be really effective, then on its basis it is possible to develop a whole family of drugs containing different (or identical, but in different dosage and dosage form) mitochondrial-targeted antioxidants as an active principle. This is the main strategy of research.

Along with the most detailed development of the technological platform (mitotechnology), as confirmation of its operability is received, several drugs are being developed in parallel. The step-by-step method of drug development is shown in the figure.

It all starts with the creation of the structure and synthesis of a new compound (by chemical synthesis groups). Once synthesized, the new substance is transferred to other groups for the study of properties, first physico-chemical and, if they meet the requirements (for example, the stability of the substance, its hydrophobicity, anti- and pro-oxidant properties in chemical reactions), then tests on biological systems in vitro begin.

The penetrating ability of compounds is evaluated on a model system (artificial flat lipid membranes). On the same membranes, but with built-in gramicidin peptide ion channels, the protective effect of substances on membrane proteins can be shown. A large number of experiments are conducted on isolated mitochondria, which makes it possible to measure the antioxidant properties of substances, to look for side effects.

Finally, studies of the biological activity of substances on various cell cultures are carried out. First of all, the ability of SkQ to protect cells under stressful conditions, to prevent necrosis and apopotosis induced by ROS is being studied.
Some experiments with SkQ can be carried out on isolated organs and tissues of animals. Thus, within the framework of the ophthalmological direction, a large amount of work was carried out on artificial cultivation of the posterior sector of the eye, a large number of experiments in cardiology were carried out on the model of an isolated perfusion heart.
In case of satisfactory results in vitro and ex vivo, SkQ is placed at the disposal of scientific groups conducting research on whole organisms.
SkQ is a new class of biologically active compounds. In this regard, the study of their properties should include experiments on a variety of organisms — from unicellular to human. Today, research is being conducted, in particular, on bacteria, unicellular fungi (including yeast), mycelial fungi, higher plants, invertebrates — nematodes (Caenorhabditis elegans), rotifers, crustaceans, insects (drosophila), fish (Nothobranchius furzeri), rodents — mice and rats of various lines, blindfolds, rabbits.

Since the drugs of the project are planned to be used in veterinary medicine, clinical trials are underway on dogs, cats and horses — patients of veterinary clinics. Such a wide range of studies is necessary to clarify as many aspects of the biological action of the substances being developed as possible.

After receiving the first results, it became clear that various variants of SkQ can be effective in the treatment of certain diseases, including senile ones. This conclusion was made based on the analysis of data on the effect of the substance on various organisms.

From that moment, it became possible to conduct targeted studies aimed at developing drugs against specific diseases, which includes the stage of testing the effect of the substance on model animals.

A group of so—called "survival experiments" stands out - experiments in which animals received the drug in low doses throughout their lives. Here, the main parameter that interested researchers was the life expectancy of experimental animals. The first such experiment on mice started at the end of 2004 and was completed in the fall of 2007. The results were extremely encouraging — it was possible to significantly increase the average life expectancy of animals, slow down the development of several signs of aging, including delaying the appearance of senile reproductive disorders.

If successful, some of the experiments conducted on animals are registered as official preclinical tests. Experimental batches of dosage forms are being developed and produced, their effectiveness and safety are also being investigated in the relevant institutions. The results are being prepared for consideration by the Ministry of Health and Social Development of the Russian Federation to obtain permission to start clinical trials.

The number of scientists involved in the research is close to 300. Due to the sharp increase in the flow of data coming from groups, a way to optimize the collection and analysis of incoming information was proposed. One of the recommendations was the introduction of a unified computer information management system within the framework of the project. In 2006-2007, the system was developed by programmers. Starting from the second quarter of 2007 all key organizational work is carried out in the information system — automated control system "Mito"

Main resultsIn 2005, the effectiveness of mitotechnology as a method of combating mitochondrial ROS was proved, and it was possible to obtain the first confirmation of the possibility of implementing this approach in practice, although most of the experiments were conducted in vitro.

The positive biological activity of SkQ has been demonstrated on several systems.

In 2006, several new laboratories were organized at Moscow State University and with modern equipment, large-scale experiments on animals began, which immediately began to give impressive results. Several Western research centers joined the research — the Longevika company, based at the R.V. Johnson Medical Institute in New Jersey, Stockholm University. By the end of 2007, results were obtained indicating that SkQ is able to delay the development of 14 signs of aging (for example, senile eye diseases, senile memory loss, the development of cardiovascular diseases, aging of the reproductive system, susceptibility to cancer, etc.), including increasing the life expectancy of a variety of animals (and depending on depending on the specific type, the substance can reduce early mortality and increase average life expectancy, prolong the maximum age of experimental animals).

Experiments are in full swing. They involve more than 290 scientists in 40 groups working in more than 20 research centers in Russia, the USA and Sweden.

Research will continue with the main goal of developing a drug that slows down aging. At the same time, work will also be underway to create drugs against specific senile diseases. Thus, in the ophthalmological direction, clinical trials of eye drops for uveitis, some retinopathy, and glaucoma will begin in 2008 at several leading eye institutes in Russia. The project begins official preclinical studies in the United States in order to obtain FDA approval to conduct clinical trials. Finally, some changes will take place in the organizational structure — the Educational and Scientific Center of Mitoengineering of Moscow State University will be created on the basis of the project.

The main results will be described in more detail in a separate article, which will be published in one of the next issues of the journal.

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