RU2744453C2 - Targeted non-invasive transplantation into brain of functionally active mitochondria for treating neurodegenerative diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to treating a neurodegenerative disease. Disclosed is the use of targeted non-invasive transplantation of live mitochondria into the brain by intranasal administration for treating a neurodegenerative disease characterized by mitochondrial dysfunction.

EFFECT: invention provides effective delivery of mitochondria into the brain.

1 cl, 22 dwg, 3 tbl, 1 ex

Description

Technology area

The invention relates to a method for treating Alzheimer's disease (AD) and other neurodegenerative diseases characterized by mitochondrial dysfunction by non-invasive transplantation of functionally active mitochondria into the brain using them for intranasal administration.

State of the art

It is known that mitochondria (MX), being represented in all cell types with the exception of mature erythrocytes, are involved primarily in the production of energy in the form of ATP, fatty acid metabolism, ion transport, and other processes necessary for the normal functioning and survival of the cell. Dysfunction of mitochondria is accompanied by the development of severe cellular pathology. Disorders in mitochondrial activity are associated with such neurodegenerative diseases as Parkinson's disease [1, 2], Alzheimer's disease [3, 4], muscular dystrophy [5], macular degeneration [6], and even age-related neurodegeneration [7]. A whole area of intensive research is currently being devoted to the development of "mitochondrial medicine", where the MX itself is the target of therapeutic interventions. For this purpose, various compounds are used that do not bind to the MX membrane (2,4-dinitrophenel, non-binding protein-2, -3); substrates that increase the efficiency of the electron transport chain (ETC) MX (dichloroacetate, thiamine), improve metabolism (Metformin) and various antioxidants to combat reactive oxygen compounds (ROC) (coenzyme Q10, MitoQ, RP103) [8, 9, 10 ]. In 2018, there were already over 400 completed or ongoing clinical trials registered on ClinicalTrials.gov. [eleven]. However, no effective remedy has yet been found for the treatment of pathologies associated with an insufficient level of energy production in the cell or the loss of the physiological functions of POC. Therefore, at present, the direction of research is actively developing, associated with the possibility of replacing defective or destroyed MX with healthy ones, by transplanting the latter directly into cells. It is important to note that endogenous MX can participate in the processes of “self-healing” of the brain in traumatic brain injury, promoting the acceleration of axon regeneration due to activation of MX division and their active movement in the axoplasm to the injury site [12]. Under in vitro co-cultivation of various cells, as well as in vivo under normal conditions and the development of pathologies, mitochondria exchange between cells, in particular, mesenchymal multipotent stromal cells (MMSC) transplanted into the brain, or astrocytes and neurons, was noted [8]. Various approaches are used to ensure the success of transplantation. Table 1 summarizes data on the use of various experimental approaches for mitochondrial transplantation in modeling various pathologies of both the central nervous system and peripheral organs. Either the direct introduction of the isolated mitochondria into the affected organ is carried out or intravenously. Also used is the transplantation of cells enriched with healthy MX, in particular, mesenchymal stromal cells isolated from the bone marrow, which are capable of forming tunnel nanotubes and transport mitochondria to other cells through them [12].

There are currently 4 studies noted on ClinicalTrials.gov (NCT01631578, NCT02586298, NCT03384420, NCT02851758) using mitochondria that have been approved for clinical trials and are mainly related to the introduction of autologous mitochondria into oocytes for the treatment of infertility or the use of mitochondria-enriched stem cells. for the treatment of mitochondrial diseases such as Piarson's syndrome or ischemic myocardium.

It is obvious that the effectiveness of the action of "mitochondrial therapy" depends on many factors, including the specificity of pathogenesis and the efficiency of delivery of mitochondria, where the method of transplantation plays the main role.

To date, the known routes of delivery of mitochondria in in vivo experiments are direct injection into the target organ or their intravenous administration, along with the introduction of cells enriched with MX. However, these methods have their drawbacks and limitations.

Thus, the use of the introduction of mitochondria locally into the brain in Alzheimer's disease, characterized by mosaic damage to the cortical and deep brain structures, may be ineffective, but in another neurodegenerative disease such as Parkinson's disease, which has a precise defect in the substantia nigra, this MX transplantation method can give a good effect.

Systemic intravenous administration of MX is possible due to their small size (~ 1 μm in diameter) even in comparison with erythrocytes having a diameter of ~ 6-8 μm, while it is believed that free MX will not enter the erythrocytes and interfere with oxygen transport. With systemic administration of mitochondria, in particular, through the tail vein, they are found not only in the brain, but also in the heart, liver, kidneys, and muscles [18], which can also have a negative effect, since MX are actively involved not only in cell survival , but also its death both by the type of necrosis and apoptosis [21, 22, 23].

The use of cells, such as mesenchymal stromal cells (MSC), as sources of mitochondria with any local administration is traumatic, and with intravenous administration there is a threat of thrombosis, in some cases, cancerous degeneration cannot be ruled out.

In our study, it is proposed to use the intranasal method for transplanting mitochondria into the brain.

Numerous CNS drug candidates are effective in in vitro studies but not effective in whole body conditions. One of the important reasons for this can be both the wrong choice of the method of administration and the use of an invalid model of the pathological process. The blood-brain barrier (BBB), CSF-blood barrier (BCSFB) and the arachnoid barrier not only protect the central nervous system from invasive pathogens and various endogenous brain toxic substances, but also prevent drugs from entering the brain when taken orally, intravenously, intraperitoneally, intramuscular and subcutaneous injections. Therefore, the intranasal route seems to be a good alternative, since it carries out direct drug delivery from the nose to the brain, bypassing the BBB [24]. There are no data on the possibility of using intranasal administration for transplanting mitochondria into brain structures. Intranasal administration has several advantages, such as direct nose-to-brain delivery, rapid onset of therapeutic action, and minimal invasiveness by reducing peripheral side effects.

The olfactory epithelium is localized on the cranial side of the nasal cavity and consists of three types of cells: olfactory sensory neurons, supporting and basal cells. Olfactory sensory neurons are regenerated (neurogenesis) at a rate of 3-4 weeks to maintain the ability to smell. In this case, a decrease in the barrier function may occur due to a combination of factors such as the incomplete presence or functioning of tight junction proteins, efflux transporters and proteolytic enzymes, which leads to a "leaky barrier". The decrease in the effectiveness of the barrier function can be used to achieve direct drug delivery to the brain. The axons of the olfactory sensory neurons form the olfactory nerve, which passes through the perforated plate and into the olfactory bulb. The respiratory and olfactory regions are innervated by branches of the trigeminal nerve (oculomotor, maxillary and mandibular), coming from the nuclei of the trigeminal nerve in the brain stem. However, the mandibular nerve does not innervate the nasal epithelium. Consequently, the innervation of the olfactory region and the respiratory region is provided only by the oculomotor and maxillary nerves. Finally, the free nerve endings of these branches of the trigeminal nerve extend into the superficial epithelium, from where they transmit chemosensory information to the brain. Intranasally administered drugs can reach the central nervous system through three transport mechanisms: 1) can penetrate the central nervous system by transport along the olfactory nerve to the olfactory bulb; 2) through the trigeminal nerve, passing through the respiratory and olfactory epithelium, and 3) can enter the systemic circulation (blood) by absorption through arteriovenous anastomoses. Interestingly, the distance between the nasal epithelium and the brain stem (trigeminal tract) in rats is estimated at ~ 20-30 mm, while the distance from the olfactory epithelium to the olfactory bulb (olfactory tract) is estimated at ~ 4-5 mm. The first two mechanisms of drug transport bypass the BBB, which leads to their direct transfer to the brain. In the latter case, drugs from the systemic circulation can enter the brain, overcoming the BBB, through intracellular mechanisms such as passive diffusion, adsorptive endocytosis and receptor-mediated endocytosis. However, extracellular transport along the olfactory nerves occurs faster (from minutes to half an hour) than intracellular transport (from hours to days). Upon reaching the olfactory bulb, the drugs can penetrate into other areas of the central nervous system by volumetric flow mechanisms and by mixing with cerebral enteric fluid (ECF). Thus, both the respiratory and olfactory epithelium are the main absorption regions for drug delivery to the brain, but the olfactory epithelium is most important because it provides a direct connection between the nasal cavity and the central nervous system, bypassing the BBB [24]. Many animal studies have shown that intranasally administered protein preparations can enter the brain bypassing the BBB. Thus, in rats in the central nervous system, proteins such as insulin growth factor 1 (7.65 kDa), erythropoietin (30-34 kDa), interferon βlb (18.5 kDa), ovalbumin (45 kDa), leptin (16 kDa) were found. , human nerve growth factor (26.5 kDa), vascular endothelial growth factor (38.2 kDa), human growth factor β1 (25 kDa) and human fibroblast growth factor (17.2 kDa). It is assumed that delivery to the central nervous system occurs through the components associated with the olfactory and trigeminal nerves, with subsequent spread to other structures of the central nervous system within 30-60 minutes. [25, 26]. At the same time, in experiments on animals, intranasal administration led to a higher bioavailability of the drug and a reduced distribution of it to non-target organs, which made it possible to reduce systemic side effects. Previously, it was believed that the size of particles penetrating into the brain after intranasal administration is limited to 1 kDa; however, a number of studies indicate the possibility of delivery to the brain of stem cells [27, 28], the size of which significantly exceeds the size of MX.

Thus, intranasal drug delivery has a number of significant advantages over local or intravenous routes [24].

Advantages of intranasal drug delivery:

• Large surface area of the nasal mucosa for dose absorption

• Rapid absorption of the drug through the highly vascularized mucous membrane

• Fast onset of action

• Ease of administration and non-invasiveness

• Bypasses the BBB

• Avoiding gastrointestinal tract and first-pass metabolism

• Improved bioavailability

• Possible direct transport to the central nervous system

• Dose reduction / reduction of side effects

• Improved usability and compliance

• Self admission

Limitations:

• The volume that can be delivered to the nasal cavity is limited to 25-200 µl.

• Adverse effects of pathological conditions (rhinitis, etc.)

• Permeability may change due to movement of cilia

• Permeability is limited due to enzymatic inhibition

Despite the considerable efforts of scientists and doctors around the world, it has not yet been possible to find an effective drug against most neurodegenerative diseases, including Alzheimer's disease, the increase in the prevalence of which is observed in all developed countries, mainly due to the increase in the life expectancy of the population. A behavioral manifestation of AD is progressive memory loss against the background of mitochondrial dysfunction and neuronal death, mainly in the cortex and hippocampus, brain structures responsible for learning and memory. One of the reasons for the unfavorable situation with AD therapy is the lack of valid animal models. The most widespread models on transgenic animals reproduce a rare hereditary form of AD. The disadvantages of these models include insignificant neuronal death, weak association between the formation of amyloid plaques and the development of memory deficits [29]; in addition, these animals often carry a complex of genetic changes that never occurs simultaneously in AD patients. Therefore, of the greatest interest are models of the sporadic form of AD, which occurs in 95% of cases. The difficulty in modeling the sporadic form of AD is that under natural conditions, even during aging, mice and rats, the experimental animals most often used for modeling, rarely spontaneously form senile plaques - a morphological sign of AD. Nevertheless, to date, a number of experimental approaches have been described that cause the appearance of signs of sporadic AD in animals.

Our studies to test the effectiveness of intranasal transplantation of functionally active mitochondria were performed on mice with removed olfactory bulbs - olfactory bulbectomized (RBE) NMRI mice. The choice of this particular model of the sporadic form of AD was due to its obvious advantages over the existing “injection” models with the introduction of various neurotoxic substances into the brain of native animals or models in animals with accelerated aging (SAMP8 mice [30] or OXYS rats [31], in the latter case it takes a long time (up to 18 months) for the development of neurodegenerative changes of the Alzheimer's type, which significantly increases the cost of research on this model).

Animals with removed olfactory bulbs, first of all, reproduce a characteristic characteristic of AD - the selective death of neurons in certain areas of the brain: in structures directly related to the processes of learning and memory (entorhinal cortex, hippocampus, temporal cortex), as well as in nuclear formations, containing neurons that synthesize the main neurotransmitters - acetylcholine, serotonin, dopamine and norepinephrine [32, 33]. This mosaic pattern of neuronal death is due to the numerous afferent and efferent connections of the olfactory bulb with the above-mentioned brain structures affected in AD. The localization of amyloid plaques in the brain of AD patients and in RBE animals is not random and is mainly confined to the sites of the terminals of cholinergic neurons [34] and the increased density of alpha-7-nicotinic acetylcholine receptors with which beta-amyloid (Abeta) is capable form complexes, initiating their endocytosis [35] with subsequent activation of caspases and neuronal death. Therefore, the places of accumulation of plaques, as a rule, coincide with the foci of neuronal degeneration. As a result of bilateral removal of olfactory bulbs in different species of rodents (mice, guinea pigs and rats), behavioral, morphological, biochemical and immunological changes are observed, characteristic of the development of Alzheimer's type neurodegeneration, including impairment of spatial memory, an increase in the level of brain Abeta, a decrease in the number of immunoreactive x choline acetyl cells (ChAT) and the density of alpha-7-acetylcholine receptors [36-40]. The development of neurodegeneration in the brain after RBE is manifested in an increase in the number of cells with pycnosis, karyolysis, and cytolysis in the temporal cortex and hippocampal fields, as well as in the nuclei that synthesize serotonin and acetylcholine. Classic amyloid plaques were found in the cortex and hippocampus after RBE in guinea pigs, which, unlike mice and rats, have a primary Abeta structure similar to that in humans [41]. Experiencing brain slices from RBE rats showed a significant impairment of long-term potentiation in the hippocampus [42] and a decrease in the level of synaptophysin, indicating a pathology in synaptic transmission, which is also typical for the brains of AD patients [43]. RBE causes immunosuppression and a decrease in the concentration of TNF-α in the peripheral blood plasma, suppression of its production in peritoneal macrophages and splenocytes, inhibition of mitogen-stimulated proliferation of T and B lymphocytes of the spleen, as well as a decrease in NO production in macrophages, as is the case with AD [ 44]. It is important to note that RBE mice develop a deficit of energy metabolism characteristic of AD, which manifests itself in a low oxidation rate of NADH substrate of complex I of the electron transport chain, a decrease in the generated potential on the inner mitochondrial membrane, and a decrease in cytochrome oxidase activity (complex IV) of the mitochondrial respiratory chain against a background of high the content of soluble Abeta in them (1-40). Disruption of energy metabolism was accompanied by the accumulation of lipid peroxidation products in mitochondria, which indicated the development of oxidative stress. Similar changes in the brain mitochondria are observed in the sporadic form of this disease in humans [45]. A significant acceleration of amyloid plaque deposition was noted in the B6C3-Tg (APPswe, PSENldE9) 85Dbo / J transgenic mice subjected to RBE. These data allowed us to draw a conclusion about the possibility of using the RBE of animals as a valid model of the sporadic form of AD. The body of evidence supporting the important role of damage to the olfactory system, including degeneration of the mitral cells of the olfactory bulb, further strengthens the validity of the use of RBE as a model of sporadic neurodegeneration of the Alzheimer's type. The advantage of RBE as a model of AD is the completeness of the reproduction of this symptom complex, the relatively short time required for the development of pathology (1 month after surgery). However, the main advantage of this model is its adequacy to the natural occurrence and development of this disease. RBE, roughly mimicking damage to the peripheral olfactory system, causes behavioral and molecular-cellular disorders characteristic of AD. Thus, a comparative analysis of the existing models of the most common sporadic form of AD from the point of view of the completeness of reproduction of the molecular-cellular mechanisms of AD makes it possible to distinguish two models - rats with accelerated aging and RBE animals. Our study of the effectiveness of using mitochondria as a drug against AD was carried out on RBE mice, since the results can be obtained in a much shorter time.

Prototype

The prototype for the proposed method of non-invasive intranasal mitochondrial transplantation can be the intravenous administration of mitochondria, described in the proceedings of the Emerging Concepts in Mitochondrial Biology conference, which took place on February 4-8, 2018, in Israel at the Weismann Institute (Orekhon). Authors: Eyai.Ekesner, Efrat.Lkesner, Saada-Reich, A .; Rosenmann, H .; Nitzan, K .; Benhamro, S .; Valitsky, M .; Lorberboum-Galski, H. in the theses entitled Mitochondrial Incorpotion - Characteristics and Treatment for Neurodegenerative Diseases [46].

In cell culture, the authors showed that mitochondria can be incorporated into cells by simply co-incubating isolated mitochondria with cells without the need for any intervention. Using the method of confocal microscopy, the authors showed in in vitro experiments that isolated mitochondria can very quickly be incorporated into various types of recipient cells and remain inside them for several days, co-localizing with endogenous mitochondria. In in vivo experiments, the authors also showed that intravenous injection of human mitochondria into mice is safe, and the injected mitochondria penetrate the organs of animals, including the liver, heart and brain. This is sufficient for the introduction of exogenous mitochondria to improve not only memory, but also an increase in neuronal density in the hippocampus against the background of a decrease in microglial and astroglial cells in animals with an injection model of Alzheimer's disease caused by intracerebral administration of amyloid beta. The authors believe that intravenous MX administration may be promising for the treatment of neurodegenerative diseases.

It was shown in native animals - NMRI mice that the transplantation of mitochondria into the brain can be carried out using their intranasal introduction, and ensure the entry of exogenous mitochondria into the brain. Similar to the prototype, but on another model of the sporadic form of AD - RBE mice, it was found that 3-fold intranasal administration of mitochondria for 3 weeks restores memory in these animals, while RBE mice that did not receive mitochondrial administration demonstrate a complete loss spatial memory. However, intranasal mitochondrial transplantation is non-invasive and does not cause suffering. In comparison with intravenous administration, the penetration of mitochondria into other organs and tissues, except for the brain, which is affected in various neurodegenerative diseases, is practically excluded. This also excludes the appearance of blood clots with the possible formation of aggregates of mitochondria in the blood. In addition, the proposed method allows for dose-dependent administration of mitochondria, which is important to ensure the optimal amount of mitochondria required in the treatment of various neurodegenerative diseases.

The invention solves the problem of expanding the possibilities of targeted delivery to the brain of exogenous mitochondria, which have a therapeutic effect in neurodegenerative diseases.

This problem is solved through the use of non-invasive intranasal transplantation of living mitochondria into the brain.

The essence of the invention

The essence of the invention is the targeted transplantation of mitochondria into the brain using their non-invasive intranasal administration. The present invention relates to therapeutic agents for the treatment of neurodegenerative diseases. When administered intranasally, mitochondria pass into the brain and are localized in the cortex, hippocampus and olfactory bulb - structures responsible for learning and memory and are affected in a number of neurodegenerative diseases.

Another aspect of the invention is to develop doses of the mitochondrial preparation that depend on a number of factors such as the patient's body weight, sex and age. Unlike the prototype, it provides the possibility of dosing and minimizes the occurrence of side effects due to the penetration of mitochondria into other organs and tissues, which cannot be avoided when using their intravenous administration.

The technical result of the invention is the targeted entry of mitochondria into the brain and improvement of memory in animals with induced neurodegeneration of the Alzheimer's type.

List and description of figures

Figure 1. Micrographs of a sample solution with unstained mitochondria at a concentration of 1.8 mg / ml.

Figure 2. Micrographs of a sample of buffer solution filtered from MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 3. Micrographs of a sample solution with MTR-stained mitochondria at a concentration of 0.3 mg / ml.

Figure 4. Micrographs of a sample solution with MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 5. Micrographs of a sample solution with MTR-stained mitochondria at a concentration of 3.3 mg / ml.

Figure 6. Typical curves of oxygen consumption by mouse brain mitochondria in the presence of substrates of the complex (5 mM malate + 5 mM potassium glutamate) (A) and the complex (10 mM succinate + 2.5 mM potassium glutamate) (B) of the respiratory chain. The composition of the incubation medium: 100 mM KCl, 75 mM mannitol, 25 mM sucrose, 5 mM KH2PO4, 0.5 mM EGTA, 10 mM Hepes / KOH (pH 7.4). Additives: 5 mM malate + 5 mM potassium glutamate or 10 mM succinate + 2.5 mM potassium glutamate, 200 μM MADP and 50 μM 2,4-dinitrophenol (DNP). The concentration of mitochondrial protein in the cuvette is 0.5 mg / ml (n = 3).

Figure 7. Micrographs of a smear of olfactory bulbs of a mouse injected with a solution with unstained mitochondria at a concentration of 1.8 mg / ml.

Figure 8. Micrographs of a smear of the neocortex of a mouse injected with a solution with unstained mitochondria at a concentration of 1.8 mg / ml.

Figure 9. Micrographs of a smear of the hippocampus of a mouse injected with a solution with unstained mitochondria at a concentration of 1.8 mg / ml.

Figure 10. Micrographs of a smear of olfactory bulbs of a mouse injected with buffer solution filtered from MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 11. Micrographs of a smear of the neocortex of a mouse injected with buffer solution filtered from MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 12. Micrographs of a hippocampal smear of a mouse injected with a buffer solution filtered from MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 13. Micrographs of a smear of olfactory bulbs of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 0.3 mg / ml.

Figure 14. Micrographs of a smear of the neocortex of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 0.3 mg / ml.

Figure 15. Micrographs of a hippocampal smear of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 0.3 mg / ml.

Figure 16. Micrographs of a smear of olfactory bulbs of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 17. Micrographs of a smear of the neocortex of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 18. Micrographs of a hippocampal smear of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Figure 19. Micrographs of a smear of olfactory bulbs of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 3.3 mg / ml.

Figure 20. Micrographs of a smear of the neocortex of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 3.3 mg / ml.

Figure 21. Micrographs of a hippocampal smear of a mouse injected with a solution of MTR-stained mitochondria at a concentration of 3.3 mg / ml.

Figure 22. Influence of intranasal mitochondrial transplantation at a dose of 20 μg / mouse) on the spatial memory of mice tested in the Morris water maze, where: A is the time (sec) of the stay of mice in the sectors of the water maze, B is the number of entries in them in%. The abscissa shows the numbers of the sectors of the Morris maze (1-4) and the group of animals. Learning Sector 3 is colored black. The ordinate shows the time spent in the maze sectors (A) and the number of entries into them (B). Significance of differences between the data on the Tukey's test: * - p <0.05; ** - p <0.01; *** - p <0.001.

Administration of the mitochondria preparation

The mitochondrial suspension is preferably administered intranasally as a spray or as nasal drops. The liquid form can be injected through a tube or catheter, for example with a syringe.

The suspension of mitochondria can be water-based and can contain various additional ingredients that improve the stability of mitochondria or improve their delivery to the brain when administered through the nasal passages. Such additional ingredients are well known in the art [47]. Examples of additional ingredients to improve the functional state of mitochondria and their preservation for a long time: a) respiration substrates - pyruvate, succinate, malate; b) agents that bind fatty acids - albumin, EGTA; c) agents for maintaining the osmotic pressure of mitochondria (mannitol, sucrose); d) agents for inhibiting aggregation (eg EGTA); e) agents that bind iron ions - EDTA; f) pH control agents (Tris and Hepes). Examples of agents that promote membrane penetration and enhance the efficiency of mitochondrial transplantation include, but are not limited to, for example: a) agents that modulate the physiology of the epithelium; b) a chelating agent (eg citric acid); c) amino acids or their salts (for example, monoaminocarboxylic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline, etc .; hydroxyamino acids such as serine; amino acids such as aspartic and glutamic acids, etc., and basic amino acids such as lysine and the like, including their alkali and alkaline earth metal salts); d) an inhibitor of the synthesis of fatty acids; e) inhibitor of cholesterol synthesis. The membrane permeation enhancing agent can also be selected from short hydrophilic molecules in low concentration, including dimethyl sulfoxide (DMSO). Additional membrane permeation enhancers include acids such as nylon, lactic, malic and citric acids and their salts; triglyceride (eg, amylodextrin, Estaram 299, Migliol 810); cyclodextrins and beta-cyclodextrin derivatives (for example, 2-hydroxypropyl-β-cyclodextrin and heptakis (2,6-di-O-methyl-β-cyclodextrin), pinocytosis activators [48], as well as other penetrating agents that are physiologically acceptable for intranasal administration.

Non-limiting examples of bioadhesive agents include, for example, carbopol, cellulosic materials, starch, dextran, and chitosan.

Delivery

Agents that increase the nasal delivery of mitochondria can be mixed, separately or together, with other substances included in the composition together with the mitochondria. For intranasal administration, it is desirable that any significant changes in mucosal permeability be reversible during the period of drug action. In addition, the drug should not cause toxicity or harmful changes in the barrier properties of the nasal mucosa with prolonged use.

Procedures used in prevention and treatment

In the prevention and treatment of AD, mitochondria can be administered intranasally or in combination with other drugs that may be useful in neurodegenerative diseases. For example, mitochondria can be administered intranasally in combination with oral administration of the following agents:

a) acetylcholinesterase blockers such as donepezil (Aricept ™), galantaminhydrobromide (reminil), rivastigmine (Exelon) and ipidacrine (neuromidine);

b) means for improving memory, reducing the excitotoxicity of glutamate (akatinolamemantin ™);

c) antidepressants (paroxetine ™). Serotonin reuptake inhibitors (trazodone, buspirone) and tricyclic antidepressants with minimal M-anticholinergic action (desipramine and nortriptyline) are commonly used;

d) anxiolytics (thioridazine), antipsychotics (haloperidol), or benzodiazepines (lorazepam);

e) drugs for sleep disorders (diphenhydramine);

f) anti-inflammatory drugs (non-steroidal drug ibuprofen, diclofenac, etc.);

g) antioxidant agents (vitamin E or standardized extract of ginkgo biloba leaves (EGb 761) contained in the memoplant preparation);

h) antihypoxic agents (activators of the mitochondrial ATP-dependent potassium channel, uridine, etc.)

i) cholesterol modulating agents, for example, cholesterol lowering agents, statins (atorvastatin, pravastatin, simvastatin, lovastatin and fluvastatin) and histamine (H2) receptor antagonists (dimebon) (see WO 2013006076).

Injection devices

The present invention encompasses any device that is suitable for intranasal administration of mitochondria and compositions therewith. Preferably, the device is capable of delivering a metered dose of the composition. Examples of intranasal devices include, but are not limited to, for example, catheters, droppers, dispensers, aerosol nebulizers, metered-dose inhalers, and other devices.

To administer the composition in a liquid droplet form, the composition may be placed in a container and provided with a conventional dropper, preferably the dropper having a fixed delivery volume.

MX are preparing ex tempero. The delivery device (or its packaging) can be labeled and / or with instructions for use indicating that the composition is to be used intranasally. A barcode can be printed on the label, which encodes the manufacturer, the time of issue and other necessary information.

Operation 1. Isolation of mitochondria from the brain of NMRI mice

Mitochondria were isolated from the brain tissue of male NMRI mice, 3.5 months old, using the generally accepted method of differential centrifugation with some modifications proposed by us earlier [49]. In short, the procedure includes the following steps.

A) After decapitation of the animal, the brain must be quickly removed and placed in refrigerated saline. Then grind it, rinse twice in saline from blood. Then the saline solution is removed with a pipette, the tissue is poured with an isolation medium containing 0.225 M mannitol, 0.075 M sucrose, ODMM EGTA, 0.2% defatted BSA, 10 mM Hepes / KOH, pH-7.4 (medium # 1).

B) The resulting mixture is manually homogenized until smooth. The ratio of tissue and medium during homogenization is 1:10. The resulting suspension is centrifuged at 2000g x 5 min. The supernatant is centrifuged again at 12000 x 10 min. The sediment after the second centrifugation is resuspended in a washing medium containing 0.225 M mannitol, 0.075 M sucrose, 10 mM Hepes / KOH, pH-7.4 (medium # 2), and re-centrifuged at 12000 g x 10 min. Then the resulting sediment is resuspended in medium # 2 in a ratio of 1 g of brain tissue to 0.1 ml of medium. All procedures must be performed at + 4 ° C.

The resulting suspension contained 35-40 mg of mitochondrial protein per ml. Protein concentration was determined by the Lowry method [50]. The calibration curve was constructed using BSA purified from fatty acids (Sigma, cat # A6003). The mitochondrial suspensions used later, after the corresponding concentration dilution in medium # 2, contained 0.3, 1.8, and 3.3 mg / ml, which corresponds to approximately 2, 10, and 20 μg of injected mitochondrial protein per 6 μL of solution (solution volume for one intranasal injection).

Operation 2. Labeling of brain mitochondria from NMRI mice with MitoTracker Red CMXRos (MTR, Invitrogen, cat # M7512) to control their delivery to the brain during intranasal transplantation

Mitochondria isolated from the brains of NMRI mice were labeled with a MitoTracker Red CMXRos (MTR) fluorescent probe (Invitrogen, cat # M7512) for in vitro and in vivo imaging and tracking. The staining was carried out in accordance with the manufacturer's recommendations. Briefly, 5 mM glutamate, 5 mM malate and 10 mM potassium succinate were added to the resulting mitochondria at a concentration of 20 mg / ml as respiration substrates and incubated with 1 μM MTR for 5 minutes at room temperature, avoiding light. In order to remove unbound dye, the mitochondrial suspension was centrifuged at 12000g x 10 min, 4 ° C. The resulting mitochondrial pellet was then resuspended in medium # 2 supplemented with respiration substrates and centrifuged again to wash from the MTR. The resulting suspension of mitochondria stained and washed from unbound dye after concentration dilution to 0.3 mg / ml (2 μg / 6 μl), 1.8 mg / ml (10 μg / 6 μl) and 3.3 mg / ml (20 μg / 6 μl) in medium # 2, supplemented with respiration substrates, was used for further visualization in vitro and in vivo using a fluorescence microscope (Leica DM IL LED Fluo, Germany). In tandem, a test was carried out confirming the absence of a free (not bound to mitochondria) fluorescent dye in a suspension of isolated mitochondria. For this, MTR-stained mitochondria at a concentration of 1.8 mg / ml were taken into a separate sterile 1 ml syringe, and slowly passed through a Nalgene filter with a pore diameter of 0.2 μm (Thermo Scientific cat # 723-2520), placed at the end syringe. Since the filter interferes with the passage of mitochondrial particles, the resulting solution ("buffer solution filtered from MTR-stained mitochondria") was used as evidence for the absence of unbound fluorescent probe in further experiments to visualize MTR-stained mitochondria in vitro and in vivo.

Operation 4. Checking the functional activity of the isolated mitochondria before their intranasal transplantation

The main indicator of the functional activity of mitochondria is their ability to oxidative phosphorylation. This ability, in turn, depends on the integrity of the inner membrane of the organelles and can sharply decrease in case of disturbances in the process of obtaining a pure mitochondrial fraction [51]. The most common method for quantitative analysis of the efficiency of oxidative phosphorylation is the determination of respiratory control parameters, ADP / O, and phosphorylation time [52, 53]. Respiratory control is the ratio of the rate of oxygen consumption by energized mitochondria (in the presence of respiration substrates and ADP - state 3 according to B. Chance) to the rate of oxygen consumption in the presence of substrates without the addition of ADP (state 4 according to B. Chance). After adding ADP, the quality of the mitochondrial preparation is also assessed by calculating the ratio of the amount of ADP to the amount of oxygen consumed (ADP / O) and the time spent on phosphorylation of all added ADP (T _f ). In the work, the indicated indicators of the efficiency of oxidative phosphorylation of mitochondria isolated from the brain tissue of mice were measured. The rate of oxygen consumption by mitochondria was assessed using a high-resolution respirometer (Oroboros-2k, Austria). The respiration rate of mitochondria was measured in 2 ml of incubation medium with the addition of substrates I of complex I (5 mM malate and 5 mM potassium glutamate) or complex II (10 mM potassium succinate) of the respiratory chain (4 state according to Chance), followed by the addition of 200 μM ADP (State 3 according to Chance) and an uncoupler of oxidative phosphorylation of 50 μM 2,4-dinitorophenol (uncoupled respiration).

Operation 5. Intranasal administration of different concentrations of labeled mitochondria to native NMRI mice in a volume of 6 μl per animal.

The experiments were carried out on native 6-month-old male NMRI mice kept in cages of 2-3 individuals in a specially equipped room under natural light at a temperature of 21-23 ° C with free access to water and pelleted food. All work with animals was performed in accordance with the requirements set forth in Resolution 118 of the US National Institute of Health and approved by the Committee for the Care and Use of Animals in Experiment (NIH Publications N 8023, 119 revised 1978), as well as with the provisions of the Order of the USSR Ministry of Health dated 08/12/1977 No. 755 "On measures to further improve organizational forms of work with the use of experimental animals" along with the decision of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Purposes (Strasbourg, 1986) and the principles of the Declaration of Helsinki (2000). All research protocols are approved by the ITEB RAS Commission on Ethics and Humane Treatment of Experimental Animals (Order No. 173 / K dated 03.10.2011, Protocol No. 09 dated 01.03.2019).

The mitochondrial preparation used for intranasal administration was obtained from the brain of a 3.5 month old male NMRI mouse according to the procedure described above. A freshly prepared suspension of labeled mitochondria at concentrations: 0.3 mg / ml, 1.8 mg / ml, and 3.3 mg / ml was injected into mice intranasally once in a volume of 6 μl per animal, 3 μl in each nostril. Control animals received intranasal administration of buffer solution filtered from MTR-stained mitochondria at a concentration of 1.8 mg / ml, or unlabeled mitochondria (1.8 mg / ml) in the same volume. All animals with intranasal administration were under observation during the day. No toxicity of intranasal MX administration was found.

The first series of experiments is devoted to the study of the penetration of mitochondria into the brain after their intranasal administration. The following groups of animals were formed:

Group 1 - control animals receiving a solution with unstained mitochondria at a concentration of 1.8 mg / ml.

Group 2 - control animals injected with a buffer solution filtered from MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Group 3 - animals injected with a solution of MTR-stained mitochondria at a concentration of 0.3 mg / ml.

4th group - animals receiving a solution of MTR-stained mitochondria at a concentration of 1.8 mg / ml.

Group 5 - animals injected with a solution of MTR-stained mitochondria at a concentration of 3.3 mg / ml.

In 48 hours after intranasal administration, the animals were decapitated under sterile conditions and the brain was removed in the cold. The following structures were identified for the study: hippocampus, neocortex, and olfactory bulbs, which were placed in a refrigerator at -20 ° C. On the same day, a homogenate was prepared from each structure in physiological saline, a part of which in the form of a smear was placed on a previously cleaned glass slide and microscopic examination was carried out using a fluorescent microscope (Leica DM IL LED Fluo (Germany)). The presence of mitochondria stained with a fluorescent dye in each field of view (x20, at least 10 fields of view) was assessed visually in the red region of the spectrum and photographs were taken.

Figures 1-5 show micrographs of suspensions of MTR-stained mitochondria (1 μl) at different concentrations, as well as a buffer solution filtered from MTR-stained mitochondria at a concentration of 1.8 mg / ml (control 1) and a suspension of unlabeled mitochondria 1.8 mg / ml (control 2) immediately before their intranasal administration. A - fluorescence in the red region of the spectrum; B - image of the solution in transmitted light. The scale is 25 microns.

Before the transplantation of mitochondria into the brain, the quality of isolated mitochondria isolated from the mouse brain was assessed. Respiratory control, ADP / O, and phosphorylation time were used to assess the efficiency of mitochondrial oxidative phosphorylation. Typical polarographic curves reflecting the change in the oxygen concentration in the cuvette and the rate of oxygen consumption in the suspension of mouse brain mitochondria are shown in Figure 6.

According to the obtained polarographic graphs, the respiratory control indicators (DC) were calculated, which is equal to the ratio of the rates V ₃ to V ₄ , the ratio of the added ADP to the amount of oxygen consumed (ADP / O) and the phosphorylation time of ADP (Tf) (Table 2).

The composition of the incubation medium: 100 mMCO, 75 mM mannitol, 25 mM sucrose, 5 mM KH2PO4, 0.5 mM EGTA, 10 mM Hepes / KOH (pH 7.4). Additives: 5 mM malate + 5 mM potassium glutamate or 10 mM succinate + 2.5 mM potassium glutamate, 200 μM MADP and 50 μM 2,4-dinitrophenol (DNP). The concentration of mitochondrial protein in the cuvette is 0.5 mg / ml (n = 3).

The composition of the incubation medium: 100 mM KCl, 75 mM mannitol, 25 mM sucrose, 5 mM KH2PO4, 0.5 mM EGTA, 10 mM Hepes / KOH (pH 7.4). Additives: 5 mM malate + 5 mM potassium glutamate or 10 mM succinate + 2.5 mM potassium glutamate, 200 μM MADP and 50 μM 2,4-dinitrophenol (DNP).

Mitochondrial respiration rate V, nmol О ₂ / min⋅mg; ADP / O, μM / nmol O ₂ ; t, s; DC - respiratory control, V ₃ / V ₄ . The concentration of mitochondrial protein in the cuvette is 0.5 mg / ml (n = 3).

Based on the results obtained, it can be concluded that mitochondria isolated from the brain of mice have high values of DC (6.17 ± 0.30 and 4.28 ± 0.20) and ADP / O (2.96 ± 0.09 and 2.04 ± 0.08), recorded when using substrates as complex I (succinate dehydrogenase) and complex II (NADH dehydrogenase) of the respiratory chain, respectively. High values of respiratory control and the ADP / O, and briefly T _f suggests that isolated mitochondria can effectively oxidize written substrates exhibit high enzymatic activity of the respiratory complexes and ATPase activity, and also have a low leakage H ⁺ across the inner membrane of mitochondria, i.e. are well connected and functionally active.

Thus, the obtained preparation of isolated mouse brain mitochondria is of high quality and can be used for the treatment of mitochondrial insufficiency.

Next, the solution was intranasally administered to mice in accordance with the experimental group of the animal. Animals that received intranasally a suspension of mx were kept under observation for a day. According to the results of observation of the acute toxicity of mss during their intranasal transplantation, it was not revealed. Of greatest interest was to assess the possibility of penetration of the stained mitochondria preparation into the olfactory bulbs - the first cerebral structure on the way of mitochondria to the brain after intranasal administration, as well as into the neocortex and hippocampus - the brain areas that are directly involved in learning and memory and, mainly, suffering from AD. ...

The following figures (No. 7-21) show representative micrographs of smears of the corresponding structures of the brain of native mice 48 hours after intranasal administration of both control solutions and suspensions of mitochondria stained with the fluorescent dye MTR. For each figure: A - fluorescence observed in the red region of the spectrum; B - image of a smear of the corresponding area in transmitted light. The scale is 50 microns.

From the above results of the first series of experiments on the transplantation of labeled mitochondria into the brain after their intranasal administration, it follows that, indeed, mitochondria are able to penetrate into the studied structures of the brain, as evidenced by the presence of fluorescent particles in the red region of the spectrum, corresponding to the used mitochondrial dye MTR, and by the form having complete resemblance to their image in suspension before their intranasal administration (micrographs in Figures 3A-5A). An important proof of the localization of the injected mitochondria in the brain is the presence of a dose-dependence in the number of fluorescent particles on the concentration of labeled mitochondria in the suspension administered intranasally. On micrographs of the brain structures of control animals, as expected, there was no fluorescence. When comparing smears of olfactory bulbs, in the case of three different concentrations of transplanted stained mitochondria, an increase in their number in the structure, observed with an increase in concentration, is clearly visible. As for the hippocampus and neocortex, it should be noted that, as expected, the penetration of mitochondria into these structures after 48 hours was less than into the olfactory bulbs. If, at a concentration of 0.3 mg / ml of stained transplanted mitochondria in solution, there was no fluorescence in the red region of the spectrum in the neocortex and hippocampus, then at concentrations of 1.8 mg / ml and 3.3 mg / ml in the photographs of smears shown in Figures 17A , 18A, 20A and 21A, fluorescence in these structures is clearly visible, which is very important, since it is these structures of the brain that are affected in AD and are involved in the performance of such higher brain functions as learning and memory. Thus, the data obtained indicate the possibility of targeted delivery of functionally active mitochondria transplanted into the brain by intranasal administration.

The next series of experiments was devoted to the question of the effectiveness of mitochondrial transplantation into the brain in the development of an induced neurodegenerative process of the Alzheimer's type.

Research method:

A) Bulbectomy operation

At the age of three months, the operation of removing the olfactory bulbs (bulbectomy) is performed in mice under sterile conditions under Nembutal anesthesia (40 mg / kg, intraperitoneally), for local anesthesia during scalping, a 0.5% solution of novocaine is subcutaneously injected. Bilateral removal of olfactory bulbs is carried out by aspiration through the burr hole. Sham-operated (LO) animals served as control, which were subjected to a similar procedure, but without removing the olfactory bulbs.

Two weeks after the operation, mice are injected into the nose with a micropipette, 6 μl of a suspension of mitochondria at a concentration of 3.3 μg / μl. The drug was administered three times over three weeks (two times before training and once at the beginning of training).

B) Learning in the Morris water maze and testing spatial memory.

Mice are trained in the navigation reflex of finding a rescue platform in the Morris water maze [54]. The experimental chamber was a plastic round pool 80 cm in diameter, filled to 30 cm with water at a temperature of 23 ° C. The pool area was conditionally divided into four equal sectors, in one of which there was a rescue platform with a diameter of 5 cm submerged in water. The water was whitened with milk so that the animals could not visually detect the rescue platform.

All experimental and control mice were preliminarily tested for their ability to swim and preservation of vision, which is especially important for RBE mice. For this, tests are carried out in a pool with water and in the presence of a visible, not submerged rescue platform. Determine the latency period for the animals to reach the visible platform. In our experiments, animals of all three experimental groups showed statistically indistinguishable values of latency periods. Then, four training sessions are carried out every day for five days in a rescue platform submerged under the surface of the water. The animals are trained to find an invisible rescue platform for 60 seconds. At the end of the training, the mice are tested for spatial memory in the absence of the rescue platform for one minute. The state of spatial memory is assessed by two criteria: the time spent in the sectors of the pool and the number of visits to them, expressed in%.

C) Results on the effectiveness of brain transplantation of mitochondria for RBE memory in mice

The results of three-time intranasal transplantation of mitochondria for three weeks for the spatial memory of RBE in animals representing a model of sporadic AD are analyzed in detail.

The results of one-way analysis (One-Way ANOVA) of memory testing data in different experimental groups are presented in Table 3.

One-way ANOVA analysis of the effect of intranasal mitochondrial transplantation during the stay of mice in the sectors of the Morris water maze revealed a significant preference for learning sector 3 in the LR and RBE + MC groups of animals (p “0.0001 and p <0.001379, respectively). The reliability of the factor for discriminating the maze sectors was also significant for the RBE group of mice (p <0.021923), but as shown by Post Nose analysis, this was due to a significant excess of the RBE residence time of mice in the 4th indifferent sector of the maze compared to the target 3rd sector. ... Post-Hoc analysis using the Tukey'sHSD test revealed that only mice in the LR and RBE + MC groups identified the third target sector in terms of residence time, but not RBE. Similar data were obtained in the study of the ability of RBE + MC mice to isolate the learning sector by the number of entries into it (p <0.0001), while the RBE mice that did not receive mitochondrial administration showed the smallest number of entries into the target sector in comparison with other indifferent compartments. Figure 22 (A and B) shows the effect of intranasal administration of a suspension of mitochondria on the spatial memory of mice tested in the Morris water maze, where A is the time spent by mice in the sectors of the water maze, B is the number of entries into them. Black bars correspond to indicators in the training sector. Significance of data differences according to Tukey's HSD criterion: * - p <0.05; ** - p <0.01; *** - p <0.001.

The data obtained show that the RBE of mice significantly deteriorates spatial memory, while in RBE animals, which are intranasally injected with a suspension of mitochondria at a dose of 20 μg / animal three times for 3 weeks, the memory remained intact and these animals could allocate a sector. in which, during training, there was a rescue platform, like the LO animals. The positive effect of the mitochondrial preparation was observed according to both criteria for assessing the state of memory. The time spent in the training sector was 22 ± 1.5> 11.4 ± 0.8 <19.3 ± 1.4 sec in LR, RBE, and RBE mice receiving intranasally a suspension of functionally active mitochondria, respectively. The percentage of the number of entries into the learning sector of the total number of entries into all sectors was 35.3 ± 2.2> 21.7 ± 1.3 <30.7 ± 1.6% in LO, RBE, and RBE of mice treated with the mitochondrial preparation, respectively. In general, the results of testing spatial memory in RBE animals indicate that intranasal administration of a suspension of mitochondria prevents the deterioration of spatial memory caused by the removal of olfactory bulbs.

Thus, the results obtained indicate the presence of a neuroprotective effect of transplantation of a suspension of mitochondria after intranasal administration, which is manifested in the inhibition of the development of loss of spatial memory in RBE mice, which are a model of the most common sporadic form of AD.

The simplicity of drug administration, the effectiveness of low doses, which minimizes the occurrence of adverse side effects, indicate the advisability of developing a new therapeutic drug against BA on this basis.

Industrial use

A pharmacological composition containing a suspension of functionally active mitochondria can find prophylactic and / or therapeutic use for the treatment of AD. The high efficiency of the composition based on a suspension of mitochondria makes it possible to increase the efficiency and shorten the time of AD treatment.

The pharmacological composition does not possess toxicity and is biocompatible with the mammalian organism, including humans, since it uses a suspension of functionally active mitochondria as the main element. The low toxicity of the composition makes it possible to increase the effectiveness of treatment due to the simultaneous combined effect of the suspension of mitochondria with additional drugs and biologically active agents.

This is especially important in the treatment of concomitant diseases that complicate AD treatment.

The composition has a high solubility and quickly penetrates into the intercellular space of the body, which makes it possible to treat brain diseases. The composition is compatible with any pharmaceutically acceptable carrier that does not impair the biological activity and viability of mitochondria. The results obtained above are experimental evidence of the effectiveness of using a suspension of mitochondria in the development of a neurodegenerative process of the Alzheimer's type. Ease of administration of the drug, the effectiveness of low doses minimizes the occurrence of adverse side effects.

List of used literary sources

1. Mizuno Y, Ohta S, Tanaka M, Takamiya S, Suzuki K, et al. Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease. Biochem Biophys Res Commun. 1989; 163: 1450-1455.

2. Winklhofer KF, Haass C. Mitochondrial dysfunction in Parkinson's disease. BiochimBiophysActa. 2010; 1802: 29-4.

3. Swerdlow RH, Khan SM. A "mitochondrial cascade hypothesis" for sporadic Alzheimer's disease. Med Hypotheses. 2004; 63: 8-20.

4. Tobore TO. On the central role of mitochondria dysfunction and oxidative stress in Alzheimer's disease. Neurol Sci. 2019; 40 (8): 1527-1540. DOI: 10.1007 / s 10072-019-03 863-x

5. Onopiuk M, Brutkowski W, Wierzbicka K, Wojciechowska S, Szczepanowska J, etal. Mutationindystrophin-encodinggeneaffectsenergy metabolism in mouse myoblasts. BiochemBiophysResCommun. 2009; 386: 463-466.

6. Burdon RH. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Freeradicalbiology & medicine. 1995; 18: 775-794.

7. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006; 443: 787-795.

8. Gollihue JL, Rabchevsky AG. Prospects for therapeutic mitochondrial transplantation. Mitochondrion. 2017; 35: 70-79.

9. Wang W, Karamanlidis G, Tian R. Novel targets for mitochondrial medicine. SciTransl Med. 2016; 8: 326rv3.

10. Hattab AW, Zarante AM, Almannai M, Scaglia F. Therapies for mitochondrial diseases and current clinical trials. Mol Genet Metab. 2017; 122: 1-9.

11. Chang Chu-Yuan, Liang Min-Zong and Chen Linyi Current progress of mitochondrial transplantation that promotes neuronal regeneration Translational Neurodegeneration 2019, 8:17; 1-12. https://doi.org/l0.1186/s40035-019-0158-8.

12. Chien L, Liang MZ, Chang CY, Wang C, Chen L. Mitochondrial therapy promotes regeneration of injured hippocampal neurons. BiochimBiophysActa. 2018; 1864: 3001-3012.

13.Islam MN, Das SR, Emin MT, Wei M, Sun L, Westphalen K, Rowlands DJ, QuadriSK, Bhattacharya S, Bhattacharya J. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury Nat Med. 2012 Apr 15; 18 (5): 759-765. doi: 10.1038 / nm.2736.

14. Masuzawa A, Black KM, Pacak CA, Ericsson M, Barnett RJ, Drumm C, Seth P, Bloch DB, Levitsky S, Cowan DB, McCully JD. Transplantation of autologously derived mitochondria protects the heart from ischemiareperfusion injury. Am J PhysiolHeartCircPhysiol. 2013; 304: H966-982.

15. Hayakawa K, Esposito E, Wang X, Terasaki Y, Liu Y, Xing C, Ji X, Lo EH. Transfer of mitochondria from astrocytes to neurons after stroke. Nature. 2016; 535: 551-555.

16. Chang JC, Wu SL, Liu KH, Chen YH, Chuang CS, Cheng FC, Su HL, Wei YH, KuoSJ, Liu CS. Allogeneic / xenogeneic transplantation of peptide-labeled mitochondria in Parkinson's disease: restoration of mitochondria functions and attenuation of 6-hydroxydopamine-induced neurotoxicity. Transl Res. 2016; 170: 40-56.

17. Kaza AK, Wamala I, Friehs I, Kuebler JD, Rathod RH, Berra I, Ericsson M, Yao R, Thedsanamoorthy JK, Zurakowski D, et al. Myocardial rescue with autologous mitochondrial transplantation in a porcine model of ischemia / reperfusion. J ThoracCardiovasc Surg. 2017; 153: 934-943.

18. Shi X, Zhao M, Fu C, Fu A. Intravenous administration of mitochondria for treating experimental Parkinson's disease. Mitochondrion. 2017; 34: 91-100.

19. Gollihue JL, Patel SP, Eldahan KC, Cox DH, Donahue RR, Taylor BK, Sullivan PG, Rabchevsky AG. Effects of Mitochondrial Transplantation on Bioenergetics, Cellular Incorporation, and Functional Recovery after Spinal Cord Injury. J Neurotrauma. 2018; 35 (15): 1800-1818.

20. Fu A, Shi X, Zhang H, Fu B. Mitotherapy for fatty liver by intravenous Administration of Exogenous Mitochondria in male mice. Front Pharmacol. 2017; 8: 241-249.doi: 10.3389 / fphar.2017.00241.

21. Riedl SJ, Salvesen GS. The apoptosome: signaling platform of cell death. Nat Rev Mol Cell Biol. 2007; 8: 405-413.

22. Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, et al. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron. 1995; 15: 961-973.

23. Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 1997; 57: 1835-1840.

24. Ruigrok, M. J. Emerging insights for translational pharmacokinetic and pharmacokinetic-pharmacodynamic studies: towards prediction of nose-to-brain transport in humans [Electronic resource] / M. J. R. Ruigrok, de Lange E. C. M. // The AAPS journal. - 2015. - V. 17. - No. 3. - P. 493-505. https://link.springer.com/article/10.1208/sl2248-015-9724-x

25. Singh A. K. Nasal cavity, a promising transmucosal platform for drug delivery and research approaches from nasal to brain targeting [Electronic resource] / A. K. Singh, Singh A., Madhv N. V. // Journal of drug delivery and therapeutics. 2012.2 (3): 22-33. http://www.jddtonline.info/index.php/jddt/article/view.File/l63/82

26. Ghori, M. U. Nasal Drug Delivery Systems: An Overview [Electronic resource] / M. U. Ghori [et al.] // American Journal of Pharmacological Sciences. 2015 .. 3 (5): 110-119. http://pubs.sciepub.com/aips/3/5/2/.

27. Jiang Yl, Zhu J, Xu G, Liu X. Intranasal delivery of stem cells to the brain. Expert Opin Drug Deliv. 2011; 8 (5): 623-632. doi: 10.1517 / 17425247.2011.566267.

28. Li G, Bonamici N, Dey M, Lesniak MS, Balyasnikova IV Intranasal delivery of stem cell-based therapies for the treatment of brain malignancies. Expert Opin Drug Deliv. 2018; 15 (2): 163-172. doi: 10.1080 / 17425247.2018.1378642.

29. Irizarry, M. C. APPSw transgenic mice develop age-related Aβ deposits and neuropil abnormalities, but no neuronal loss in CA1 [Text] / M. C. Irizarry [et al.] // Journal of Neuropathology & Experimental Neurology. - 1997. -V. 56. - No. 9. - P. 965-973.

30. Morley, J.E. The senescence accelerated mouse (SAMP8) as a model for oxidative stress and Alzheimer's disease [Electronic resource] / J. E. Morley [et al.] // Biochimica et Biophysica Acta (BBA) -Molecular Basis of Disease. - 2012. - T. 1822. - No. 5. - C. 650-656. Access: http://www.sciencedirect.com/science/article/pii/S0925443911002754, free. - Title from screen.

31. Kolosova N.G. Senescence-accelerated OXYS rats: A genetic model of premature aging and age-related diseases [Electronic resource] / N. G. Kolosova [et al.] // Advances in Gerontology. - 2014. - V. 4. - No. 4. - P. 294-298. Access: https://www.researchgate.net/profile/Natalya_Kolosova/publication/278715099_Se nescence-accelerated_OXYS_rats_A_genetic_model_of_premature_aging_and_agerelated_diseases / links / 5587cbcl08ae71f6ba916e56. - Title from screen.

C. Changes in the serotonergic system in the main olfactory bulb of rats unilaterally deprived from birth to adulthood [Electronic resource] / C. Gomez [et al.] // Journal of neurochemistry. - 2007. - V. 100. - №4. - P. 924-938. Access: http://onlinelibrary.wiley.com/doi/10.l 11 l/j.l471-4159.2006.04229.x/full, free. - Title from screen.32.

C. Changes in the serotonergic system in the main olfactory bulb of rats unilaterally deprived from birth to adulthood [Electronic resource] / C. Gomez [et al.] // Journal of neurochemistry. - 2007. - V. 100. - No. 4. - P. 924-938. Access: http://onlinelibrary.wiley.com/doi/10.l 11 l / j.l471-4159.2006.04229.x / full, free. - Title from screen.

C. Differential effects of unilateral olfactory deprivation on noradrenergic and cholinergic systems in the main olfactory bulb of the rat [Electronic resource] / C. Gomez [et al.] // Neuroscience. - 2006. - V. 141. - №4. - P. 2117-2128. Access:33.

C. Differential effects of unilateral olfactory deprivation on noradrenergic and cholinergic systems in the main olfactory bulb of the rat [Electronic resource] / C. Gomez [et al.] // Neuroscience. - 2006. - V. 141. - No. 4. - P. 2117-2128. Access:

https://www.researchgate.net/profile/Laura_Orio2/publication/6975759_Differenti al_effects_of_unilateral_olfactory_deprivation_on_noradrenergic_and_cholinergic _systems_in_the_main_olfactory_bul8_bulb_ linksof_the_bdf06. - Title from screen.

34. Kus, L. Distribution of high affinity choline transporter immunoreactivity in the primate central nervous system [Electronic resource] / L. Kus [et al] // Journal of Comparative Neurology. - 2003. - V. 463. - No. 3. - P. 341-357. Access: https://s3.amazonaws.com/academia.edu.documents/42880344/Distribution_of_hig h_affinity_choline_tr20160220-27776-

ld9uhqh.pdf? AWSAccessKeyId = AKIAIWOWYYGZ2Y53UL3A & Expires = 1506 953533 & Signature = StvoqMqRPtjUlFSPvFmbXjUK8Hyo% 3D & response-content-disposition = inline% 3B% 20finity_affinity_tline% 3DhDistribution_righoline pdf, free. - Title from screen.

35. Wang, H.Y. β-Amyloidl-42 binds to α7 nicotinic acetylcholine receptor with high affinity implications for Alzheimer's disease pathology [Electronic resource] / H. Y. Wang [et al.] // Journal of Biological Chemistry. - 2000. - V. 275. - No. 8. - P. 5626-5632. Access: http://www.jbc.org/content/275/8/5626.long, free. - Title from screen.

36. Alexandrova, I.Yu. Increased level of beta-amyloid in the brain in bulbectomized mice [Text] / I.Yu. Alexandrova [et al.] // Biochemistry. - 2004. - T. 69. - No. 4. - S. 218-224.

37. Nesterova, I.V. Bulbectomy-induced loss of raphe neurons is counteracted bt antidepressant treatment [Text] / I. V. Nesterova [et al.] // Progress in Neuro-Psychopharmacology and Biological Psychiatry. - 1997. - V. 21. - No. 1. - P. 127-140.

38. Bobkova, H.B. The state of cholinergic structures of the forebrain in bulbectomized mice [Text] / NV Bobkova, Nesterova IV, Nesterov VI. // Bull. expert. biol. - 2001. - T. 131. - No. 5. - S.507-511.

39. Kamynina, A.V. Vaccination with peptide 173-193 of acetylcholine receptor α7-subunit prevents memory loss in olfactory bulbectomized mice [Text] / A.V. Kamynina [et al.] // Journal of Alzheimer's Disease. - 2010. - V. 21. - No. 1. - P. 249-261.

40. Bobkova, H.B. The influence of passive immunization with antibodies against the extracellular fragment of a7-ACHR on the neurodegenerative process of the Alzheimer's type [Text] / N. V. Bobkova [et al.] // Bulletin of new medical technologies. - 2009. - T. 16. - No. 1. - S. 214-216.

41. Beck, M., Guinea pigs as a nontransgenic model for APP processing in vitro and in vivo [Text] / M. Beck, Bigl V., Roβner S. // Neurochemical research. - 2003. - V. 28. - No. 3-4. - P. 637-644.

42. Battaglia, F. Cortical plasticity in Alzheimer's disease in humans and rodents [Text] / F. Battaglia [et al.] // Biological psychiatry. - 2007. - V. 62. - No. 12. - P. 1405-1412.

43. Reddy, P.H. Differential loss of synaptic proteins in Alzheimer's disease: implications for synaptic dysfunction [Text] / Reddy P. H. [et al.] // Journal of Alzheimer's Disease. - 2005. -V. 7. - No. 2. - P. 103-117.

44. Novoselova, E.B. The immune status of bulbectomized mice [Text] / E.B. Novoselova [et al.] // Reports of the Russian Academy of Sciences. - 2003. - T. 393. -№6. - S. 824-826.

45. Avetisyan, A.V. Functional impairment of the mitochondria of the neocortex and hippocampus in mice with bulbectomy - a model of Alzheimer's disease [Text] / A.V. Avetisyan [et al.] // Biochemistry. - 2016. - T. 81. - No. 6. - S. 802-812.

46. Eyai. E. kesner, Efrat. Lkesner, Saada-Reich, A .; Rosenmann, H .; Nitzan, K .; Benhamro, S .; Valitsky, M .; Lorberboum-Galski, H. Mitochondrial Incorpotion - Characteristics and Treatment for Neurodegenerative Diseases. EmergingConceptsinMitochondrialBiology 2018 4-8 February, abstract

47. Zhang Z, Ma Z, Yan C, Pu K, Wu M, Bai J, Li Y, Wang Q. Muscle-derived autologous mitochondrial transplantation: A novel strategy for treating cerebral ischemic injury. Behav Brain Res. 2019, 356: 322-331. DOI: 10.1016 / j.bbr.2018.09.005

48. Conner SD., Schmid SL. Regulated portals of entry into the cell. Nature 2003, 422 (6927): 37-44. DOI: 10.1038 / nature01451

49. Venediktova NI, Gorbacheva OS, Belosludtseva NV, Fedotova IB, Surina NM, Poletaeva II, Kolomytkin OV, Mironova GD. Energetic, oxidative and ionic exchange in rat brain and liver mitochondria at experimental audiogenic epilepsy (Krushinsky-Molodkina model). J Bioenerg Biomembr. 2017, 49: 149-158. DOI 10.1007 / s 10863-016-9693-5

50. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951, 193 (1): 265-275.

51. Chinopoulos C, Zhang SF, Thomas B, Ten V, Starkov AA. Isolation and functional assessment of mitochondria from small amounts of mouse brain tissue / // Neurodegeneration. 2011, 793: 311-324.

52. Wen Y, Li W, Poteet EC, Xie L, Tan C, Yan LJ, Ju X, Liu R, Qian H, Marvin MA, Goldberg MS, She H, Mao Z, Simpkins JW, Yang SH. Alternative mitochondrial electron transfer as a novel strategy for neuroprotection / Y. Wen [et. al] // Journal of Biological Chemistry. 2011,286 (18): 16504-16515.

M, Del Hoyo P,

MA, Rubio JC,

A, Castellano G, Colina F, Arenas J, Solis-Herruzo JA. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology. 2003, 38(4):999-1007.53.

M, Del Hoyo P,

MA, Rubio JC,

A, Castellano G, Colina F, Arenas J, Solis-Herruzo JA. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology. 2003, 38 (4): 999-1007.

54. Morris R. Developments of a water-maze procedure for studying spatial learning in the rat // Journal of neuroscience methods. - 1984. - T. 11. - No. l. - S. 47-60.

Claims (1)

The use of targeted non-invasive transplantation of living mitochondria into the brain by intranasal administration for the treatment of neurodegenerative disease characterized by mitochondrial dysfunction

Images