by Ivan SENIN, Dr. Sc. (Chem.), Head of the Ophthalmology Group, Institute of Mitoengineering, Lomonosov Moscow State University, Valery ERICHEV, Dr. Sc. (Med.), Deputy Director of the Institute of Eye Diseases, RAMS, Academician Vladimir SKULACHEV, Director of Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University

Traditionally high interest of ophthalmologists to glaucoma is explained by its leading position in the sad statistics of irreversible blindness and poor eyesight. An increase of intraocular pressure forms a base for its development, eventually leading to death of ocular nerve fibers and loss of visual functions. It is therefore essential to create drugs for neuron support in patients and prevention of death of these cells. A group of Russian scientists from the Moscow State University and Institute of Biophysics of the USSR Academy of Sciences (today the Russian Academy of Sciences) on the brink of the 1960s and 1970s made a discovery, which served as a basis for creation of a new class of drugs. We hope that the use of these drugs will promote a significant reduction of blindness among glaucoma patients in the near future.

Glaucoma is a large group of diseases characterized by periodical or constant elevation of intraocular pressure.

HOW DOES

THE PATHOLOGY ARISE?

Glaucoma-a group of diseases characterized by periodical or permanent elevation of intraocular pressure. The interest to this group of diseases from medical and scientific viewpoints is explained by the fact that up to the present time all causes of the disease are unknown. The social significance of the problem is explained by the leading position of glaucoma among the causes of irreversible blindness and poor eyesight. About 100 mln people on our planet suffer from it, and their number can double by 2030, as a result of which sooner or later 30 mln patients may become blind in one eye and about 10 mln may become completely blind.

It seems that the disease develops as a result of a successive chain of risk factors. The effects of these factors are summed up, and the mechanism leading to the disease is eventually triggered. However, it remains insufficiently studied. One thing is obvious: the main aftereffect of elevated intraocular pressure is impairment of blood circulation in the eye, resulting in a lesser delivery of nutrients and oxygen to the eyes. However, different structures of this organ differently react to these changes. The majority of ocular tissues are capable of realizing their vital activity without oxygen during several minutes, while insufficient oxygen delivery to the neurons in the ocular structures arrests metabolic processes in them within just few seconds. Hence, no

Living cell (diagram).

wonder that the neurons are the first that suffer in glaucoma, and an appreciable part of the neurons constitute the optic nerve (consisting of retinal ganglion cell* axons).

Rapid progress of the disease in the majority of patients is not caused by merely elevation of intraocular pressure, but by its constant fluctuations throughout day and night. Even a slight (5-10 mm Hg) shift of this parameter can reduce the blood flow in the optic nerve. The alternation of periods of limited delivery of oxygen (because of ischemia) and subsequent recovery of its delivery (as a result of reperfusion)** to the neurons decrease their defense properties and lead to irreversible destructive processes.

The particular danger of glaucoma is its asymptomatic course, impeding timely diagnostics. As a result, the clinical picture becomes obvious when more than 20 percent neurons of the optic nerve are dead, and hence, the disease cannot be completely arrested. And then the aim of the treatment is to preserve visual functions for as long as possible. Therefore, as soon as the diagnosis is made, drug treatment, aimed at normalization of intraocular pressure, should be started directly. But as the drugs used for this purpose do not directly protect the neurons from consequences of ischemia and reperfusion, their use is essential, but not sufficient for effective treatment. That is why the important constituent of this treatment is neuroprotective therapy, that is, prevention of concomitant abnormalities in the optic nerve, specifically, in retinal ganglion cells. Let us emphasize that normalization of metabolism in these cells is a key factor of effective rehabilitation of patients.

Today ophthalmologists can use such drugs as nifedipine, memantine, brimonidine, etc. modulating many components in the pathogenesis of neurodegenerative damage in glaucoma. However, their application and efficiency are very often limited. Therefore, search for drugs with more effective protective characteristics and, the last but not the least, with the minimum side-effects is in progress.

WHY DO NEURONS DIE?

First, let us make a digression to one of the sections of cellular biology.

All living organisms provide energy for themselves by using some external resources. Energy production in the cells is realized by special organelles--mitochondria. They are characterized by an intricate enzymatic system, due to which they realize the most important function for cells: a synthesis of energy "currency" of the organism--adenosine triphosphate (ATP)--by oxidizing (with oxygen) the organic compounds. During ATP synthesis, oxygen participates in certain chemical reactions and eventually reduces to water (H₂0) by binding four electron and four hydrogen ions to 0₂ molecule (O₂+4e-+4H⁺=2H₂O). This complex reaction is catalyzed by special enzymes-proteins. A small amount of O₂ (from 0.1 to 2 percent) transforms into reactive oxygen species (ROS) from precursors--superoxide O^2- ions (with unpaired electron). It is demonstrated that the ROS cause neuronal damage. Normally, a complex system of antioxidant defense works in the mitochondria, which prevents such destructive processes.

* Ganglion cells are neurons, whose axons, released from the eye, conduct stimulation from the retina to the central nervous system.--Ed.

** Reperfusion is recovery of the blood flow.--Ed.

Mitochondrion (x 25,000).

As has been mentioned above the development of glaucoma is linked not only with intraocular pressure elevation, but also with frequency of fluctuations, the blood flow is restored after a period of oxygen starvation, which is seemingly to reduce the pathological processes. But in reality oxygen inflow after ischemia augments negative processes in the cells, as in reperfusion the concentration of oxygen reducers in the tissues drastically increases, thus promoting additional potent generation of active oxygen forms in the mitochondria. This explains rapid progress of glaucoma in patients with pronounced frequent fluctuations of intraocular pressure.

NEUROPROTECTION: NEW APPROACHES

Modern concepts on the pathophysiological processes in the optic nerve structures during the disease in consideration help to determine approaches to protection and maintenance of neuron life. That is why modern studies are mainly focused on search for methods and means to inhibit all stages of the pathogenetic cascade, including search for antioxidants preventing the development of oxidation stress, regulators of ionic channels* and neurotransmitter** release. Of these three listed ones, the latter are best studied. However, the clinical trials did not confirm the preclinical exper-imental data on the pronounced neuroprotective effects of this group of drugs. Calcium, sodium, and potassium channel blockers also proved to be unfit for glaucoma control.

Antioxidants are the least studied in this respect. Good perspectives of vitamins E and C, emoxipin, tocopherol, lipoic acid, cytochrome c have been demonstrated. Unfortunately, the studies have also revealed their certain drawbacks in combined therapy for the disease.

Why do common antioxidants exhibit limited efficiency in glaucoma? It is very simple to answer this question if we recollect where the major part of free radicals are forming when glaucoma develops. In mitochondria. All drugs used today in the treatment of glaucoma do not penetrate into these organelles and do not protect them from the destructive effects of ROS. Therefore, this therapy does not protect mitochondria of the optic nerve neurons from destruction. The technological approach that we suggest is free from these flaws. It implies addressed modulation of the mitochondria--regulation of the amounts of ROS produced by them. This is a kind of mitochondria engineering, or mitoengineering. One of its main trends is addressed delivery of highly effective antioxidants to these organelles.

What are the presumable characteristics of a substance protecting the optic nerve neurons in glaucoma patients? First, it should be addressed directly into the mitochondria. Secondly, it should be safe, as reacting with ROS, the "protector" molecules transform into radicals and hence, the cell should possess a reliable

* Ionic channels are special transport proteins supporting the difference of potentials between the outer and inner sides of the cell membrane. Due to them, sodium, potassium, chlorine, and calcium proteins pass through the membrane in accordance with their electrochemical gradients.--Ed.

** Neurotransmitters (neuromediators) are bioactive chemical substances through which an electric pulse is transmitted between neurons.--Ed.

Lipophilic cations-molecules-electric transport delivering the "load" to mitochondria.

SkQ1 and a model of its penetration into mitochondria.

mechanism for their immediate neutralization, better with restoration in the initial form. Thirdly, all antioxidants in high doses are characterized by a pro-oxidant (stimulating the lipid peroxidation processes) effect, which limits their use; that is, their efficiency should be high, while their dose as low as possible. It must be pointed out that the traditional substances of this series, even if they reach the mitochondrial membrane, are natural compounds whose excess can be cleaved by cellular enzymes. In other words, the body has actually systems for self-defense not only from oxygen, but from antioxidants as well. The matter is that ROS are involved in certain biological functions essential for full-value life (for example, they are directly involved in bacterial and viral control). Hence, the "protector" should eliminate not all ROS, but just their excess forming in the mitochondria.

Unfortunately, none of the antioxidants known by the end of the 20th century meets all these requirements. A real candidate appeared only at the beginning of the new century.

The main distinctive feature of the mitochondria from other cell organelles is a negative charge on the inner membrane. This factor can be used for addressed accumulation of substances. On the brink of the 1960s and 1970s, one of the authors of this paper, Vladimir Skulachev together with Yevgeny Liberman, Dr. Sc. (Phys. & Math.), from the Institute of Biophysics, found out that some compounds--lipophilic* cations (for example, phosphonium ion)--can penetrate into living cell mitochondria. The positively charged atom in these compounds is surrounded by hydrophobic residues. The charge of these ions is evenly distributed in a large volume around the central atom. This construction prevents the ion hydration--the main cause of impermeability of membranes for charged molecules. It is justified that in 1974 Douglas Green, an American biochemist, called these compounds "Skulachev ions" (SkQ). And at the beginning of the 1970s, a team of scientists from the Moscow State University suggested

* Lipophilia is chemical affinity for organic substances, in fact, a synonym of hydrophobia.--Ed.

Selective accumulation of SkQ1 in mitochondria of man's fibroblasts: a-order of SkQ1 (red); b-order of mitochondria (green) in fibroblast cells; c-laying of photos a and b on each other.

that the penetrating cations can be used by the mitochondrion as "molecules-electric transport" for accumulation of uncharged substances bound to these cations.

At the end of the 1990s, Michael Murphy, a British biochemist, attempted to create a mitochondrion-addressed antioxidant based on these ideas. He bound vitamin E to the lipophilic ion. Unfortunately, this substance, as its somewhat improved variant with ubiquinone (also an antioxidant) instead of vitamin E, is still not used in medicine, presumably because of a strong pro-oxidant effect and poor efficiency in low doses. As a result, the perspectiveness of the approach turned out to be doubtful. However, in 2003-2005, a new mitochondrion-addressed antioxidant has been created at the Moscow State University. Plastoquinone (a substance derived from plant chloroplasts) was used to improve its efficiency. As to their saturation with oxygen, chloroplasts are unique in living nature. The SkQl compound has been created and synthesized, and its efficiency proved to be hundreds of times higher than of the previous compounds.

MITOENGINEERING TODAY AND TOMORROW

As a result, the efficiency of mitoengineering as a means to fight against mitochondrial ROS was proved in 2005, the possibilities to realize the new approach in practice were confirmed. It has turned out that SkQl easily penetrates through membranes of cells and mitochondria, selectively accumulates in the latter. According to our estimates, concentration of this substance in them can be higher than in the extracellular medium 2 •10⁸ times, which will help achieve required therapeutic effect using minimal doses. It is important that SkQl is an antioxidant of multiple action, as its oxidized form is reduced by mitochondria into initial one.

Experimental treatment of glaucoma by SkQl in model systems on animals was carried out in 2005-2009 and the results were promising. A new preparation form-Visomitin eye drops-has been created. Clinical trials of these eye drops were carried out in 2010 at several ophthalmological institutes in Russia. In addition, official preclinical trials are starting within the framework of the project in the USA in order to get permission for clinical trials in other countries.

All this means that SkQl can be widely used in clinical medicine. In addition, studies of the role and significance of free oxygen radicals in metabolic disorders has shown that they are involved in the pathogenesis of almost 100 diseases. Each of these diseases is associated with high levels of free radicals and their oxidation products in the cell mitochondria of various tissues.

At present experiments are in progress within the framework of the investment project "Practical Use of Skulachev Ions". About 300 scientists working in 40 groups in more than 20 centers of Russia, USA, and Sweden participate in the project. The results indicate that SkQl inhibits the development of ocular and cardiovascular diseases, aging of the reproductive system, emergence of malignant tumors, prolongs the life span of animals of various species.

The work was supported by a grant of the President of the Russian Federation No. MD-4423.2010.4.