RU2846338C1 - Heterocomplex based on telomeric sequence for recovery of muscular tissue - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, particularly to a method for preparing a preparation for muscular tissue tonus, firmness and elasticity recovery. Said method includes: (i) recovering nuclear DNA from a patient cell sample; (ii) obtaining a telomeric DNA sequence of 600 bp using the isolated nuclear DNA according to step (i); (iii) formation of complex due to non-covalent bonding of peptide-carrier with SEQ ID NO: 3 with the telomeric DNA sequence obtained at step (ii). Present invention also relates to a preparation containing such a complex, as well as the use of such a preparation to restore the tone, elasticity and elasticity of muscular tissue by intramuscular administration.

EFFECT: present invention provides muscular tissue tonus, elasticity and resilience recovery.

7 cl, 3 dwg, 7 tbl, 5 ex

Description

The invention relates to the field of genetic engineering, medicine and cosmetology, and can be used to improve muscle condition by preventing cellular aging of muscle tissue, stabilizing telomeres and normalizing its functions.

State of the art

Atrophic degenerative changes in skeletal muscles result in loss of their strength and volume. This process is associated with aging; long-term observational studies have shown that the mass of adipose tissue replacing muscle increases with age. Fatty infiltration of muscles is associated with a decrease in strength and contractility of muscles. Muscle mass and strength begin to gradually decrease after 30 years, and after 60 years this decrease progressively accelerates. From 20 to 80 years, a decrease in muscle mass by 30% is observed, and a decrease in the cross-sectional area of muscles by about 20%. This dynamics is due to a decrease in the size and number of muscle fibers.

Skeletal muscles are particularly vulnerable to oxidative stress because their work requires a large amount of oxygen, and therefore myocytes accumulate significant amounts of peroxidation products. Reactive oxygen species lead to damage to many cellular components, including DNA, membrane lipids, and proteins. Physical exercise as an integral component of sarcopenia prevention programs causes aerobic bioenergetic reactions in mitochondria and cytosol, which result in increased production of reactive oxygen species (Misnikova I.V. et al., Sarcopenic obesity, RMJ, 2017, 1:24-29).

Physiological regeneration – renewal of muscle fibers – constantly occurs in skeletal muscle. Mature skeletal muscle fibers are not capable of mitosis, but resting myosatellite cells can divide. During muscle fiber renewal, myosatellite cells enter proliferation cycles with subsequent differentiation into myoblasts and their inclusion in the composition of the preceding muscle fibers. During reparative regeneration (in case of damage), myosatellite cells are activated, differentiate into myoblasts, and fuse to form muscle tubes with their characteristic central arrangement of nuclei. The assembly of myofibrils, the formation of sarcomeres, and the migration of nuclei to the periphery complete the formation of muscle fibers.

It is known that the key mechanism of cellular aging induction is telomere shortening (Shay J.W., Telomeres and aging, Current Opinion in Cell Biology, 2018, 52: 1-7). Telomeres are ten-thousandth hexameric tandem (TTAGGG)n repeats associated with the Shelterin protein complex, located at the ends of chromosomes, and are necessary for maintaining genome stability (preventing activation of DNA repair pathways and maintaining the linear shape of chromosomes). The signal for the onset of replicative aging is a critical shortening of the telomeric ends of chromosomes (the Hayflick limit) due to the sequential loss of part of the telomeric sequence during cell division (due to DNA underreplication).

Telomere stabilization plays a key role in cellular maintenance of functions, including tissue repair, cellular response, and adaptation to stress conditions. Endogenous factors that cause telomere shortening include aging, inflammation, and oxidative stress. Currently, both large differences between tissues in susceptibility to initiation of cellular aging processes and unique individual profiles of telomere attrition have been shown (Tarry-Adkins J.L. et al., Exploring Telomere Dynamics in Aging Male Rat Tissues: Can Tissue-Specific Differences Contribute to Age-Associated Pathologies? Gerontology, 2021, 67(2):233-242).

In skeletal muscle, genomic stabilization and telomere regulation play an important role in tissue health, influencing the repair of muscle damage. (Trajano L.A.D.S.N. et al., Genomic stability and telomere regulation in skeletal muscle tissue, Biomedicine & Pharmacotherapy, 2018, 98: 907-915; Arsenis N.C., et al., Physical activity and telomere length: impact of aging and potential mechanisms of action, Oncotarget., 2017, 8(27): 45008-45019).

Facial muscles deserve special attention in aesthetic medicine, hypotrophic processes in which are the most important component in the loss of aesthetic appeal. It is age-related hypotrophy of facial muscles, which form the muscular framework, that largely determines individual facial features. The condition of the facial and chewing muscles determines the contours of the face, the presence of atrophic muscle areas changes the biomechanics of the facial muscles, its replacement with fatty tissue leads to the formation of deformational changes in the face and contributes to skin atrophy.

Unfortunately, up to now there have been no means in the arsenal of modern cosmetology that affect this process. The main procedures are aimed at improving the properties of the skin, but the impact on the muscles at the level of regulating plastic processes in the hypotrophic muscle is an unresolved issue. There are methods of hardware effects on muscles for the purpose of their biostimulation and improving blood supply using various hardware methods.

A well-known method for increasing the tone and elasticity of muscle tissue and skin is the use of methods aimed at non-surgical hardware lifting of the muscles and skin of the face (Wong A. et al., Ultrasound Therapy for the Skin, Facial Plast Surg Clin North Am., 2023, 31(4):503-510. doi: 10.1016/j.fsc.2023.05.008, PMID: 37806683).

The essence of the method is the application of microfocused ultrasound to various layers - from the superficial to SMAS (muscular-aponeurotic system) - their point heating occurs, while the skin surface is not damaged. Microfocused ultrasound penetrates into different layers of the skin to a depth of 1.5 to 4.5 mm, stimulating the synthesis of new, young collagen in the dermis, and promotes lifting at the SMAS level, without damaging the skin surface. After such exposure, new young collagen and elastin are actively produced, which allows you to tighten and tighten the skin and muscles of the face, neck and décolleté, as well as correct wrinkles.

The disadvantages of this method include a low safety profile: too much energy supply can cause a burn in the area of impact. Insufficient and/or too weak energy supply will not bring the expected result. Another disadvantage is the temporary effect.

Another well-known method used for muscle tightening is the so-called facebuilding or facial exercises (Benz R., Facebuilding: The Daily 5-Minute Program for a Beautiful Wrinkle-Free Face/ John Walter, Reinhold Benz. — Sterling Publishing Co., Inc, 2008, 100 p.).

The essence of the method is to perform a set of exercises, which, according to adherents, allows you to improve your appearance and smooth out traces of age-related changes without invasive interventions. Facial gymnastics allows you to tone some muscles, while others, on the contrary, relax. As for expression wrinkles, during gymnastics, the muscle temperature increases, which activates fibroblasts, provokes collagen compression, ultimately stimulating oxygen exchange and vital processes occurring in the skin. In fact, there is a stimulation of metabolism in muscle tissue, which is the same as with hardware procedures.

The disadvantages of the method include a short-term effect, the risk of additional wrinkles if the technique of performing the exercises is violated. However, all these methods provide only a temporary change in homeostasis due to the stimulation of tissue trophism at the tissue and cellular levels.

Modern methods of increasing the tone, firmness and elasticity of muscle tissue at the cellular and subcellular level include muscle rejuvenation methods aimed at increasing the proliferation and differentiation of muscle cells with growth factors NGF, IGF-1 or bFGF (Guthridge M. et al., The Role of Basic Fibroblast Growth Factor in Skeletal Muscle Regeneration, Growth Factors, 1992, 6:1, 53-63, DOI: 10.3109/08977199209008871; Papadakis M.A. et al., Growth hormone replacement in healthy older men improves body composition but not functional ability. Ann Intern Med., 1996, 124(8):708-16. doi: 10.7326/0003-4819-124-8-199604150-00002).

A well-known method for stopping age-related loss of muscle mass and function is the method of transferring the IGF-1 gene by an adeno-associated viral (AAV) vector into mouse skeletal muscles (Barton-Davis E.R. et al., Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function, BIOLOGICAL SCIENCES, 1998, 95, (26), 15603-15607, https://doi.org/10.1073/pnas.95.26.15603).

In a mouse model, direct injections of insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), and to a lesser extent nerve growth factor (NGF), result in accelerated muscle healing in injured, contused, and damaged muscles at 5 and 7 days post-injury.

The disadvantages of this method are:

- the effectiveness of direct injection of recombinant growth factors is limited by their high concentration required to obtain the desired effect, which is often fraught with side effects at the injection site;

- uncontrolled mitotic activity of the IGF-1 gene carried by the AAV vector, which is a potent mitogen for fibroblasts located in the interstitial space between myofibrils in adult skeletal muscles, which can lead to oncotransformation of cells;

- increased synthesis of matrix components such as collagen due to the action of IGF-1, which can lead to the formation of fibrous tissue and scarring at the injection site.

From the Eurasian patent EA 26251 B1, published on 31.03.2017, a method is known for the restoration and regeneration of damaged muscle tissue, for example, after injury, inflammation or loss of muscle mass in age-related atrophic involution, which involves the administration of a therapeutically effective amount of an active therapeutic agent - a retinoic acid receptor gamma agonist. The retinoic acid receptor γ (RAR γ) is a member of the subfamily of nuclear receptors RARα, β and γ, which are transcription factors that regulate various endocrine metabolic pathways. RARs are capable of transmitting a signal and activating gene transcription, which is initiated by the binding of a ligand to a specific retinoic acid receptor, i.e. RARα, RARβ or RARγ. The use of the proposed therapeutic agent helps to increase the area of muscle fibers in damaged tissue and completely restore the normal muscle structure.

The disadvantages of the specified method, which can be considered as the closest analogue of the proposed invention, are:

- risks of cell oncotransformation. Thus, targeting the RARγ signaling axis may lead to redundancy and random changes in gene activity. Much effort has been made to study the mechanism of RARγ regulation in cancer, but there are conflicting opinions regarding its role in cancer development. Recent studies have shown that RARγ is involved in the development and progression of cancer. The exact role of RARγ has not been determined: it may function as an oncogene stimulating cancer cell growth and metastasis, and, conversely, RARγ may act as a tumor suppressor factor that suppresses cancer cell proliferation and invasion. Thus, stimulation of RARy can also lead to oncotransformation of cells (see Gan WJ. et al., RARγ-induced E-cadherin downregulation promotes hepatocellular carcinoma invasion and metastasis. J Exp Clin Cancer Res., 2016, 35, p.164, https://doi.org/10.1186/s13046-016-0441-9).

Thus, the main disadvantage of known methods of therapy that increase the replication potential of cells by transferring various biologically active substances into muscle tissue cells is the selective focus on isolated links in muscle tissue reparation, which creates the trigger for uncontrolled processes of cell growth and oncotransformation.

Methods have also been proposed that are aimed at restoring telomeric chromosome regions shortened due to terminal underreplication and affecting the cell's own protein-synthetic function and restarting cellular homeostasis. Currently, the main focus of researchers on cell immortalization by lengthening telomeric DNA sequences is associated with finding ways to regulate the activity of telomerase, a ribonucleoprotein enzyme whose main function is to build up the G-chain of telomeric DNA.

The effect of telomeric region elongation due to the action of an exogenous telomeric sequence was first described in the article by Runge K.W. et al., Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation. Mol Cell Biol. 1989:1488-97, where it was suggested that a possible mechanism of action of the introduced telomeric DNA consists in competitive binding of proteins interacting with chromosomal telomeres, which increases the availability of telomeres for binding to telomerase or for the process of gene conversion – mechanisms of telomeric region elongation.

Later, it was proven that the processes of alternative telomere lengthening are based on recombination and retrotransposition (Nabetani A. et al., Alternative lengthening of telomeres pathway: Recombination-mediated telomere maintenance mechanism in human cells, J. Biochem., 2011, 149 (Is. 1): 5–14. https://doi.org/10.1093/jb/mvq119; Cesare A.J. et al., Alternative lengthening of telomeres: models, mechanisms and implications, Nat Rev Genet., 2011, 11, 319–330; Lee J.J. et al., Alternative paths to telomere elongation, Semin Cell Dev Biol., 2021, 113:88-96. doi: 10.1016/j.semcdb.2020.11.003; Wang F. et al., Inhibition of LINE-1 retrotransposition represses telomere reprogramming during mouse 2-cell embryo development, J Assist Reprod Genet., 2021, 38(12): 3145-3153. doi:10.1007/s10815-021-02331-w; Kordyukova M. et al., Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis, PLoS One, 2018, 13(8): 0201787. doi: 10.1371/journal.pone.0201787; Casacuberta E., Drosophila: Retrotransposons Making up Telomeres, Viruses, 2017, 9 (7):192. doi:10.3390/v9070192).

From the Russian Federation patent No. 2766120, published on 08.02.2022, a method is known for lengthening telomeres by short-term increase in telomerase activity in the cell, which is achieved by intracellular introduction of synthetic ribonucleic acid encoding telomerase reverse transcriptase. The proposed method ensures preservation of elasticity, thickness, smoothness and appearance of the skin.

The disadvantages of the known method include:

- the absence of targeted intranuclear delivery of the active compound in target cells, since the use of an exosome, lipid nanoparticle or polymer nanoparticle as a delivery carrier ensures delivery only to the cell cytosol;

- not a direct, but an indirect effect on increasing the proliferative potential of the cell through the processes of translation and subsequent assembly of the protein (the enzyme telomerase reverse transcriptase) into a functional unit that initiates the elongation of telomeric sequences, which makes the degree of manifestation of the therapeutic effect dependent on the potential of the protein-synthesizing system of the patient's body;

- the mechanism of action due to the activation of telomerase is often associated with the oncotransformation of the cell.

The technical problem of the inventionthere is a need to increase the efficiency of recovery and normalization of the condition of muscles and their functions both by improving the efficiency of transfection of the compound initiating telomere elongation and by minimizing insertional mutagenesis by activating processes alternative to the action of telomerase.

The technical problem of the invention consists in developing a method for increasing the proliferative function of muscle cells based on a genetically engineered therapeutic approach through an alternative to the action of telomerase, telomere lengthening (ALT) by introducing an exogenous telomeric sequence.

Disclosure of the essence of the invention

Unless otherwise defined, all terms and concepts used in the disclosure of the present invention, including technical and scientific terms, have the meaning that is commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

As a rule, the classification used and the methods of cellular and molecular biology, immunology, analytical, preparative, medicinal and pharmaceutical chemistry described in this document are well known to those skilled in the art and are widely used in this field.

The methods for isolating nuclear DNA from a patient cell sample, amplifying DNA by polymerase chain reaction, and complexing telomeric DNA with a transfer peptide are performed according to the manufacturer's instructions, as is commonly performed in the art, or as described in detail herein.

All documents cited or incorporated by reference in this specification, and all documents cited or incorporated by reference in documents cited in this specification, together with any manufacturer's instructions, descriptions, and modes of use of a method or products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in practicing the invention.

The technical result of the invention is an increase in muscle tone, elasticity and resilience by at least 1.5 times, as well as an increase in the diameter of skeletal muscle fibers by an average of more than 15% compared to the control due to an increase in the reparative potential of cells by introducing the proposed composition of the drug based on a synthetically created telomeric sequence as part of a heterocomplex with a peptide carrier, wherein the therapeutic effectiveness when using the proposed composition of the drug is accompanied by low risks of oncotransformation of cells due to the absence of mutagenic potential.

The technical result is achieved by implementing the proposed method for obtaining a preparation for restoring tone, elasticity and flexibility of muscle tissue by intramuscular administration.

The proposed method includes the following steps:

(i) isolation of nuclear DNA from a cellular sample from the patient;

(ii) obtaining, using the isolated nuclear DNA according to step (i), the active ingredient of the drug - a 600 bp telomeric DNA sequence by amplification during the polymerase chain reaction;

(iii) formation of a complex by non-covalent binding of the carrier peptide with SEQ ID NO: 3 in an amount of 8-10 μg with 200-800 ng of the telomeric DNA sequence obtained in step (ii) in 1 ml of 0.05 M phosphate buffered saline solution with pH 7.0, supplemented with glucose to a final concentration of 1%.

The said technical result is also achieved by the preparation for intramuscular administration obtained by the proposed method. The preparation is intended to restore the tone, elasticity and flexibility of muscle tissue and contains an effective amount of a complex formed by non-covalently binding 8-10 μg of a carrier peptide with SEQ ID NO: 3 with 200-800 ng of a 600 bp telomeric DNA sequence (SEQ ID NO: 1) in 1 ml of a 0.05 M phosphate buffered saline solution with a pH of 7.0, supplemented with glucose to a final concentration of 1%.

In this case, according to the proposed invention, the telomeric DNA sequence can be characterized by telomeric repeats (TTAGGG)n, where n=100, or the nucleotide sequence SEQ ID NO:1. The carrier peptide is a fusion protein "NLS-peptide carrier" of the structure GRKKRRQRRRPPQPKKKRKV (SEQ ID NO:3), containing a protein transduction domain that imparts the ability of the peptide to penetrate the plasma membrane of cells and is a 13-amino acid sequence of TAT (48-60) of the human immunodeficiency virus (HIV), as well as a nuclear localization signal, which is a sequence of 7 amino acids of T-ag of the SV-40 virus, which transfers the peptide containing it from the cytoplasm into the cell nucleus.

Preferably, the heterocomplex preparation is characterized by the following composition: telomeric DNA sequence with SEQ ID NO: 1 - 0.2-0.8 μg/ml, carrier peptide with SEQ ID NO: 3 - 8-10 μg/ml, 100 μl of 10% glucose solution and 0.05 M phosphate buffered saline (PBS) at pH 7.0 - the rest up to 1 ml.

Also, the specified technical result is achieved through the use of the proposed drug for restoring tone, elasticity and flexibility of muscle tissue by intramuscular administration.

In a preferred embodiment, the drug is administered in an effective amount to restore muscle tissue tone, firmness and elasticity.

The term "effective amount", including "pharmacologically effective amount", "prophylactically effective amount" or "therapeutically effective amount" refers to an amount that provides a desired response or desired effect, alone or in combination with additional doses, in particular that will restore tone, firmness and/or elasticity of muscle tissue.

The desired response is preferably associated with the restoration of muscle tone, firmness and/or elasticity. The desired effect may be a delay in the onset or prevention of the loss of muscle tone, firmness and/or elasticity.

The effective amount may depend on the condition being treated, the severity of the condition, individual patient parameters including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy if any, the specific mode of administration and other factors. Accordingly, the administered doses of the drug described herein may depend on a variety of such parameters. In the event that the patient's response or the effect achieved is insufficient at the initial dose, higher doses may be used or effectively higher doses achieved, for example, by another more precisely localized route of administration.

Preferably, the effective amount, according to the present document, is an amount selected depending on the muscle size from the range of 0.1-0.5 ml per injection.

The administration can be carried out either in the form of a single injection or in the form of multiple injections, preferably into the muscle tissue of the patient, once or repeatedly at intervals of one to several days, months and/or years, preferably once a year, most preferably once every six months.

In this case, the intramuscular injection is carried out on a subject, preferably a mammal, in an area selected as being subject to restoration by means of the introduction of the proposed preparation for restoration of tone, elasticity and flexibility of muscle tissue.

The terms "subject", "patient", "individual" and the like are used interchangeably herein and refer to any animal susceptible to the proposed means and methods. Preferably, the subject, patient or individual is a mammal, most preferably a human of either sex and any age.

The term “repair” in relation to muscle tissue as used herein refers to the regeneration of damaged skeletal muscle tissue with restoration of function, tone, firmness and/or elasticity of muscle tissue lost as a result of aging, injury or disease.

Indications for the muscle restoration procedure are:

- age-related flabbiness of skin and muscles;

- damage to the skin and musculoskeletal system.

The effect of using the drug is an increase in the proliferative function of cells due to an increase in the number of divisions that a cell is capable of performing, as well as maintaining genomic and telomeric stability.

When using the proposed drug, there is an increase in muscle density and elasticity, an increase in skeletal muscle tone and an increase in the adaptive potential of cells to oxidative stress and inflammation.

At the same time, the general reparative capacity of tissues increases due to an increase in the possible number of divisions that a cell can perform by lengthening chromosomal telomeres, and the acceleration of muscle fiber regeneration due to an increase in the mitotic potential of myoblasts, which significantly increases the ability of muscles to recover from injuries. Intramuscular injection of the drug for flabbiness of facial muscles allows for an increase in the tone of facial muscles, elimination of gravitational ptosis of the face. Visible effect - the face becomes more compact, the volume of some areas is restored (for example, in the cheekbones) to create a more youthful appearance; deep age folds and wrinkles are eliminated.

The advantage of the proposed invention also lies in the ability to improve the regenerative potential of tissue at both the cellular and subcellular levels, which allows not only to stimulate the regeneration of cells that are labile in terms of regenerative potential, but also to increase the homeostasis of cells that are stable in division by restoring the functions of mitochondria and the endoplasmic reticulum.

The principle underlying this technology can be used to influence the cells of all human tissues and organs, so the scope of application is very wide. The drug can be used in all areas of medicine to accelerate tissue reparation (not only muscles), as well as in tissue engineering to grow the necessary tissues and organs. In addition, the principles of the proposed invention can be used to extend the life of organs and tissues, which in turn is of decisive importance in traumatology, burn therapy and transplantology.

Brief description of the drawings

The invention is illustrated by the following graphic materials.

Fig. 1 shows a schematic representation of the amplification of a specific region of DNA during the polymerase chain reaction using forward and reverse primers (highlighted in color) and template DNA.

Fig. 2 shows a schematic representation of the formation of a heterocomplex of the NLS carrier peptide with telomeric DNA.

Fig. 3 (A-C) shows the results of the evaluation of the efficiency of transporting the heterocomplex into the cell nucleus (in vitro in cell culture), where Fig. 3 (A-B) shows the detection of labeled telomeric DNA in the nuclei of target cells, with Fig. 3 (A) showing an image with the DNA label fluorescein, and Fig. 3 (B) showing an image with the DNA label rhodomin; Fig. 3 (C) shows the efficiency of transferring the innovative heterocomplex into the cell nuclei.

Implementation of the invention

Example 1. Stages of the method implementation

The method for obtaining a drug for restoring tone, elasticity and flexibility of muscle tissue by intramuscular injection includes the following steps:

Stage I. Isolation of DNA from biological material (blood, skin epithelium, muscles, and other tissues);

Stage II. Obtaining a telomeric sequence (amplification of a specific DNA region using polymerase chain reaction);

Stage III. Formation of a molecular heterocomplex: PCR of the synthesized telomere sequence with a cationic carrier peptide (NLS peptide).

I. Isolation of DNA from biological material

Genomic DNA was isolated from biological material, such as the patient's peripheral blood, using the phenol-chloroform extraction method (Sambrook J. et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Woodbury, NY, 1989). When isolating DNA from peripheral blood, erythrocyte lysis was performed using the Kunkel method (Lahiri DK et al., A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested, J Biochem Biophys Methods, 1992, Dec; 25(4):193-205, doi: 10.1016/0165-022x(92)90014-2). To 500 μl of blood were added 500 μl of erythrocyte lysis solution (29 mM Tris-HCl pH 7.4, 10 mM NaCl, ₃ mM MgCl2, 5% sucrose, 1% Triton X100), incubated for 5 min, gently mixing, then centrifuged for 10 min. at +9°C, 4000 rpm. The supernatant was decanted and the leukocyte pellet was resuspended in membrane lysis solution (29 mM Tris-HCl pH 7.4, 10 mM NaCl, 1 mM _MgCl2 , 0.25 M sucrose). The mixture was centrifuged under the same conditions, the supernatant was decanted. 300 μl of proteinase K solution (0.01 M TrisHCl, 0.01 M NaCl, 0.01 M EDTA pH 8/0), 30 μl of 30% SDS and proteinase K to a final concentration of 100 μg/ml were added to the sediment. The mixture was incubated for 2-3 hours at +50°C for proteolysis. The resulting homogeneous solution was successively treated with 300 μl of phenol, 300 μl of a phenol-chloroform mixture in a ratio of 1:1, 300 μl of chloroform. After each extraction for 10 min with gentle stirring, the mixture was centrifuged for 10 min at +20°C 4000 rpm and the aqueous phase was collected. After extraction, 1/10 volume of 3 M sodium acetate and 2 volumes of 96% ethanol were added to the aqueous phase. The precipitate was washed twice with 70% ethanol and, after drying in a thermostat, dissolved in 100 μl of sterile water. The DNA yield as a result of such purification was 40-50 μg of DNA from 500 μl of whole blood.

II. Obtaining the telomere sequence

The active ingredient of the drug contains a 600 bp telomeric sequence. - 100 repeats of the classical vertebrate sequence TTAGGG, created in vitro during the polymerase chain reaction by amplification with the following primers: Forvard - (TTAGGG)x5 and Revers - (TAACCC)x5 in 30 µl of the reaction mixture containing 67 mM Tris-HCL (pH 8.8), 16.6 mM (NH4) ₂ SO ₄ , 0.1% Triton X-100, 2.0 mM MgCl ₂ , 0.5% DMSO, 4% Formamide, 3 µl of 25 mM dNTP, 30 pmol of Forvard primer, 20 pmol of Revers primer, 3 units of Taq DNA polymerase (Fermentas, Lithuania). The matrix used is human genomic DNA isolated from peripheral blood leukocytes using the phenol-chloroform extraction method in stage I. The scheme for amplifying a specific DNA region during the polymerase chain reaction using forward and reverse primers and matrix DNA is shown in Fig. 1.

Both primers were obtained according to the method proposed in the work of Lorite et al., Conservation of (TTAGG)(n) telomeric sequences among ants (Hymenoptera, Formicidae), J. Hered., 2002, Jul-Aug; 93(4):282-285, doi: 10.1093/jhered/93.4.282). PCR was carried out on a thermal cycler using hot-start. After the initial denaturation at 94°C for 3 min with the Forward primer, Taq polymerase was added and incubated for an additional 7 min at the same temperature. Then 10 amplification cycles were carried out in the following mode: melting 94°C for 1 min, annealing 70°C for 1 min, synthesis 72°C for 1 min. After completion of 10 cycles, a pause of 90°C was set for 10 min, and the Reverse primer was added. Then 35 amplification cycles were carried out in the same temperature-time mode as for the previous 10 cycles. After completion of 35 amplification cycles, the final synthesis was carried out at 72°C for 7 min.

1 μl of the obtained PCR product was taken, diluted with water in a ratio of 1/100 and 1 μl of the resulting solution was added to the reaction mixture for a new PCR, which was carried out under the conditions and mode described above.

20 μg of the PCR product obtained after reamplification of the reaction mixture were subjected to electrophoresis in a 1% agarose gel containing TAE (Tris-acetate buffer, 40 mM Tris-base, 20 mM acetic acid, 1 mM EDTA) at a gradient of 8 V/cm. After electrophoresis, the gel was stained with ethidium bromide and photographed under UV light. Stratagen kits (USA) were used as molecular weight markers.

Agarose gel strips corresponding to 600 bp telomeric DNA fragments were excised from the gel and DNA was isolated from them using a Qiagen kit (Qiagen, Germany) according to the manufacturer's instructions. 20 μl of solution containing 100 ng of telomeric DNA sequences were obtained.

In this way, the telomeric DNA sequence included in the active ingredient is obtained with SEQ ID NO: 1.

III. Formation of a molecular heterocomplex: PCR of the synthesized telomere sequence with a cationic carrier peptide

The NLS-peptide carrier was used as a carrier for delivering the gene construct into the cell nuclei - the chemically synthesized peptide GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 3), which has the maximum efficiency of transporting the active ingredient into the cell nucleus and is a fusion peptide - a hybrid of two natural peptides PKKKRKV - the T-antigen sequence of the SV40 virus containing a nuclear localization signal (NLS), capable of initiating the transfer of DNA into the nuclei of eukaryotic cells (Jans D.A. et al., Signals mediating nuclear targeting and their regulation: Application in drug delivery, Med Res Rev, 1998, 18:189-223) and GRKKRRQRRRPPQ HIV TAT(48-60) - a 12-amino acid peptide of the human immunodeficiency virus HIV, which has the ability to penetrate cells (including the nucleus) in cell cultures (Green, M. et al., Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein, Cell, 1988, 5, 1179–1188). The heterocomplex is formed by non-covalent binding of the cationic NLS carrier peptide to the telomeric DNA with SEQ ID NO:1, based on the electrostatic interaction of the negatively charged DNA with the positively infected NLS carrier peptide (the scheme is shown in Fig. 2). To form the heterocomplex, 8–10 μg of the cationic NLS carrier peptide with SEQ ID NO:3 were mixed with 200–800 ng of the telomeric DNA with SEQ ID NO:1 in 1 ml of 0.05 M phosphate buffered saline solution with the addition of 100 μl of 10% glucose solution to a final glucose concentration of 1% for 20 min at room temperature and pH 7.0.

In this way, a heterocomplex preparation is obtained, which is in a 0.05 M phosphate-buffered saline solution with 1% glucose (pH 7.0), wherein the heterocomplex includes the telomeric DNA sequence with SEQ ID NO: 1, obtained during PCR, and a chemically synthesized NLS carrier peptide with SEQ ID NO: 3 (hereinafter referred to as the heterocomplex preparation).

The use of 0.05 M phosphate buffered saline with 1% glucose at pH 7.0 provides the most optimal conditions for the formation of the heterocomplex and simultaneously allows the creation of a finished dosage form suitable for direct administration of the drug to the patient without additional manipulations to bring the composition of the drug to a physiologically acceptable level.

Examples of compositions of the heterocomplex preparation are given in Tables 2-4. If necessary, the preparation is lyophilized; the concentration of the components in Tables 2-4 is indicated before drying.

Example 2. Evaluation of the efficiency of transporting an innovative heterocomplex drug into cell nuclei (in vitro on cell culture)

On a non-immortalized primary cell culture of human dermal fibroblasts (HDF) isolated from the skin of adult donors (provided by the staff of the Institute of Cytology of the Russian Academy of Sciences, St. Petersburg), an in vitro assessment was carried out of the efficiency of transporting the innovative heterocomplex drug into the nuclei of cultured cells.

DFC (1x10 ⁶ cells/ml) were cultured in 6-well plates (Nunc, Denmark) in RPMI-1640 medium (Sigma, USA) containing 10% fetal calf serum (Gibco, USA), 1 mM L-arginine (Sigma, USA), 1% Hepes (Sigma, USA), 0.2% NaHCO ₃ , 50 units/ml penicillin and 50 μg/ml streptomycin (Gibco, USA), in a CO ₂ incubator at 37°C, 5% CO ₂ and 95% humidity.

A heterocomplex preparation with fluorescently labeled telomeric DNA in its composition was added to a 24-hour DFC culture at a rate of 4 μg of the heterocomplex preparation per well of the plate in 100 μl of 0.05 M phosphate-buffered saline solution (pH 7.0) with 1% glucose. When adding to the cells, CaCl ₂ was introduced into the medium to a concentration of 20 mM. After incubating the cells with the heterocomplex preparation for 2 h (in a CO ₂ incubator at 37°C, 5% CO ₂ and 95% humidity), the cells were washed from non-internalized heterocomplexes with a heparin solution and incubated in RPMI-1640 medium with 10% serum for 48 h.

Telomere DNA labeling was performed in two ways: using fluorescent PCR primers (rhodamine 6G) or by enzymatic incorporation of an amine-modified nucleotide (aminoallyl-dUTP) followed by chemical labeling with Alexa Fluor® dyes (ARESTM Alexa Fluor® 488 DNA Labeling Kit, Molecular Probes) during the polymerase chain reaction.

After 48 hours, the efficiency of transfer of the innovative heterocomplex into cell nuclei was assessed using an AxioScope1 fluorescence microscope (Carl Zeiss, Germany). About 80% of the cells had intranuclear fluorescent staining (Fig. 3(A-C)).

Example 3. Evaluation of functional activity (elasticity and muscle rigidity) in rabbits after intramuscular administration of a heterocomplex preparation

The aim of the study was to examine the state of muscle tone, elasticity and flexibility of muscles after a single administration of a heterocomplex preparation obtained using the proposed technology.

Materials and methods:

The study was conducted on 50 female rabbits aged 3-5 years (two groups were formed by random sampling: experimental and control groups - 25 rabbits each). The heterocomplex preparation was administered under short-term ether anesthesia into the buccal muscle on both sides - into the superficial layer of m. buccinator 0.5 ml per injection (experimental group at a dose of 0.5 ml, in the control group 0.9% physiological solution 0.5 ml was injected into m. buccinator).

The result was assessed 30 days after drug administration.

The assessment of muscle tone, firmness and elasticity of muscles was performed using a digital palpation device, such as MyotonPRO (MYOTON AS, Estonia) for non-invasive assessment of tone, firmness and elasticity of superficial skeletal muscles and tendons.

Myoton technology has been successfully used to study age-related muscle changes (see Agyapong-Badu S. et al., Measurement of ageing effects on muscle tone and mechanical properties of rectus femoris and biceps brachii in healthy males and females using a novel hand-held myometric device. Arch Gerontol Geriatr. 2016, Jan-Feb; 62:59-67. Doi: 10.1016/j.archger.2015.09.011. Epub 2015 Oct 23. PMID: 26476868).

The measurement technique is as follows: the probe of the device is placed over the muscle being examined, measurements are taken. To improve the accuracy of diagnostics, the measurement is taken three times, the device automatically selects the average value. The measurement is non-invasive, painless for both animals and humans, and lasts only a few seconds. The measurement results were compared with reference values developed by the manufacturer of the digital palpation device.

Quantitative parameters corresponding to five characteristics of muscle condition were recorded:

the value F is the oscillation frequency that characterizes the muscle at rest, reference values 12-18 Hz,

value S - resistance to contraction or external impact, reference values 220-380 N/m,

D value - elasticity, reference values 1.0-1.6,

R value is the relaxation time of the muscle after contraction, reference values are 14-30 ms,

value C - rheology similarity criterion - Deborah number, reference values 1.0-2.0.

The program "Statistica 10", IBM SPSS Statistics 17 and the program Microsoft Excel 2010 were used for data storage and statistical processing. When determining the correlation relationship, the Spearman correlation coefficient (r) was calculated. For all listed statistical criteria, the value p<0.05 was accepted as statistically significant.

Research results

The dynamics of observations of muscle tone parameters in rabbits demonstrated a reliable increase in muscle tone to normal physiological values, elasticity and flexibility of muscles after the introduction of the heterocomplex preparation into the superficial layer of m. buccinator in the experimental group (Table 5). Lower values of elasticity parameters (D) and relaxation time (R) in m. Buccinator were characteristic of animals in the control group, which were administered a physiological solution, indicating pronounced structural age-related changes in muscles.

Example 4. Tightening of skeletal muscles by intramuscular administration of a heterocomplex preparation

The aim of the study was to investigate the morphological changes in muscles after the application of the heterocomplex drug to the femoral muscle.

Materials and methods:

The study was conducted on 30 non-inbred male laboratory rats aged 1.5 years, which were in a state of immobilization (the animals were suspended in such a way as to “unload” the hind limbs for 10 days, achieving a decrease in the mass of the femoral muscle by 30%, the diameter of muscle fibers by 45%).

The heterocomplex preparation was administered into the anterior surface of the thigh on both sides into the superficial layer of the quadriceps muscles m. quadriceps femoris. In the experimental group, the heterocomplex preparation was administered at a dose of 0.1 - 0.2 ml per 1 injection, in the control group, 0.9% physiological solution was administered at 0.1-0.2 ml. The volume was distributed by point intramuscular injections of 0.1-0.2 ml. The result was assessed 30 days after the administration of the preparation.

Pathomorphological examination was performed on the 1st, 14th, 30th day after exposure, for this purpose 5 rats were euthanized by ether for anesthesia. The obtained material of the quadriceps muscle of the thigh was fixed in 10% formalin solution with subsequent embedding in paraffin.

The sections were stained in 10% Mayer's hematoxylin solution (cell nuclei) and 1% eosin solution (cell cytoplasm, stroma). The histological preparation was examined under a light microscope.

Rated by:

The study material was tissue fragments of m. quadriceps femoris.

The following morphometric parameters were recorded: the proportion of connective tissue in the field of view at standard magnification (×800), the average diameter of muscle fibers (μm), the ratio of the number of blood vessels to the number of muscle fibers in the field of view at a magnification of ×200.

When using the heterocomplex preparation in the form of intramuscular injections, histological examination showed functional restoration and revascularization in muscles with minimal fibrosis (Table 6).

Proliferation of myogenic cells in damaged or atrophic muscles was observed. This effect was manifested by improvement of muscle tone and strength, body contours.

Thus, the proposed method of muscle recovery in laboratory rats using the heterocomplex preparation is highly effective, namely: it provides a pronounced mitogenic effect on muscles, which is demonstrated by an increase in the diameter of the muscle fiber. It does not cause side effects, including myopathies, neoplasms in muscle tissue, pathological forms of hypertrophy, leads to an increase in the elasticity and resilience of facial muscles by 1.5 times, an increase in the diameter of the fiber of skeletal muscles from 40.4 to 46.5 microns.

Example 5. Evaluation of the mutagenic potential of a heterocomplex drug

Overcoming cellular senescence caused by telomere shortening is of crucial importance in oncogenesis. The vast majority of cases of cellular oncotransformation associated with impaired regulation of telomeric complex components occurs due to an increase in the level of telomeric DNA transcription induced by telomerase expression. However, a small percentage of carcinogenesis mechanisms are associated with alternative elongation of the telomeric sequence based on recombination processes in critical violations of genome integrity. The introduction of an innovative drug of a heterocomplex with telomeric DNA of a certain size (600 bp), on the one hand, allows increasing the proliferative potential of the cell, and on the other hand, avoiding the development of insertional mutagenesis.

In the course of the work, the mutagenic potential of the heterocomplex preparation was assessed in vitro by taking into account chromosome aberrations in the M-FetMSC cell line (human mesenchymal stem cells from the limb muscle of a 5-6 week old embryo). This model for assessing the mutagenic effect of the heterocomplex preparation was chosen as the most suitable for extrapolating the results to humans. The widely used Ames test for assessing the mutagenic potential of chemical compounds was found to be unsuitable for studying the heterocomplex preparation due to differences in the structures of the genomes of prokaryotes and eukaryotic cells.

The experimental sample was M-FetMSC cells (1×10 ⁶ cells/ml) treated with the heterocomplex preparation (as described above for DPF), which were cultured in 6-well plates (Nunc, Denmark) in DMEM/F12 medium (Sigma, USA) containing 10% fetal bovine serum (Gibco, USA) in a CO ₂ incubator at 37°C, 5% CO ₂ and 95% humidity for 48 hours. At the end of the incubation time, metaphase chromosome preparations were prepared. M-FetMSC cells treated with cyclophosphamide were used as a positive control; M-FetMSC cells incubated with 0.05 M phosphate-buffered saline (pH 7.0) with 1% glucose were used as a negative control. Immunocytochemical staining was performed using monoclonal antibodies.

The results of cytogenetic analysis were assessed by comparing the proportions of cells with chromosomal aberrations in the control and experimental series of the experiment. Comparison of the proportions of cells with gaps and breaks along the centromere was considered as additional criteria for the cytogenetic activity of the heterocomplex preparation under study.

The absence of a mutagenic effect of the heterocomplex preparation was demonstrated by statistically equal proportions of aberrant cells in the experiment compared to the negative control. The data are presented in Table 7.

Table 1 - Nucleotide and amino acid sequences

Name Nucleotide sequence (5`-3`) Direct telomeric sequence TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG SEQ ID NO1 Complementary CCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCСTAACCCTAACCCTAACCCTAACCCTAA CCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA CCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA CCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA SEQ ID NO2 Amino acid sequence cationic carrier peptide GRKKRRQRRRPPQPKKKRKV SEQ ID NO3

Table 2. Composition of the heterocomplex preparation (option 1)

Components Concentration, µg/ml Telomeric DNA sequence with SEQ ID NO:1 0.2 Carrier peptide with SEQ ID NO:3 8 10% glucose solution 100 Phosphate buffered saline (PBS) at pH 7.0, 0.05M the rest (up to 1 ml)

Table 3. Composition of the heterocomplex preparation (option 2)

Components Concentration, µg/ml Telomeric DNA sequence with SEQ ID NO:1 0.55 Carrier peptide with SEQ ID NO:3 9 10% glucose solution 100 Phosphate buffered saline (PBS) at pH 7.0, 0.05M the rest (up to 1 ml)

Table 4. Composition of the heterocomplex preparation (option 3)

Components Concentration, µg/ml Telomeric DNA sequence with SEQ ID NO:1 0.8 Carrier peptide with SEQ ID NO:3 10 10% glucose solution 100 Phosphate buffered saline (PBS) at pH 7.0, 0.05M the rest (up to 1 ml)

Table 5. Indicators of the properties of masticatory muscles with and without the introduction of the heterocomplex preparation

Group Elasticity, D Relaxation time, R ms Oscillation frequency, F Hz Resistance, S N/m Elasticity, C Experimental group (administration of the drug) 1.5<0.05 25<0.05 16<0.05 330<0.05 1.5<0.05 Control group (administration of saline solution) 1.1<0.05 12<0.05 11<0.05 200<0.05 0.8<0.05

Table 6. Dynamic changes in morphometric parameters of the quadriceps femoris muscle m. quadriceps femoris

Indicator 1st day 14th day 30th day Proportion of connective tissue, % experimental group 7.6±1.5 6.4±1.8 1.8±0.7 control group 8.1±1.6 7.9±1.9 8.0±0.1 Average diameter of muscle fibers, µm experimental group 40.4±1.9 41.6±2.0 46.5±1.0 control group 39.1±1.2 40.5±0.1 39.5±1.1 Number of vessels per muscle fiber, pcs. experimental group 0–4 0–4 2–8 control group 0–3 0–4 0-5

Table 7. Results of cytogenetic analysis of cells in the control and experimental series

Experimental conditions Number of cells examined Aberrations (quantity) Cells with multiple aberrations (%) Proportion of damaged cells (%) Single fragments Paired fragments Exchanges Prototype 100 3 1 4 26 0.06 Positive control 100 33 12 47 69 0.41 Negative control 100 2 1 5 25 0.06

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Claims (10)

1. A method for obtaining a drug for restoring tone, elasticity and flexibility of muscle tissue by intramuscular administration, including the following steps: (i) isolation of nuclear DNA from a cellular sample from the patient; (ii) obtaining, using the isolated nuclear DNA according to step (i), the active ingredient of the drug - a 600 bp telomeric DNA sequence by amplification during the polymerase chain reaction; (iii) formation of a complex by non-covalent binding of the carrier peptide with SEQ ID NO: 3 in an amount of 8-10 μg with 200-800 ng of the telomeric DNA sequence obtained in step (ii) in 1 ml of 0.05 M phosphate buffered saline solution with pH 7.0, supplemented with glucose to a final concentration of 1%. 2. A method for obtaining a preparation according to claim 1, characterized in that the telomeric DNA sequence is characterized by the nucleotide sequence SEQ ID NO: 1. 3. A preparation for intramuscular administration intended to restore the tone, firmness and elasticity of muscle tissue, containing an effective amount of a complex formed by non-covalently binding 8-10 μg of a carrier peptide with SEQ ID NO: 3 with 200-800 ng of a 600 bp telomeric DNA sequence in 1 ml of 0.05 M phosphate buffered saline solution with pH 7.0, supplemented with glucose to a final concentration of 1%. 4. A preparation for intramuscular administration according to paragraph 3, characterized in that it is obtained by the method according to any of paragraphs 1 or 2. 5. A preparation for intramuscular administration according to any one of claims 3 or 4, characterized in that the telomeric DNA sequence is characterized by the nucleotide sequence SEQ ID NO: 1. 6. Use of the drug according to any of paragraphs 3-5 to restore tone, elasticity and flexibility of muscle tissue by intramuscular administration. 7. Use of the drug according to paragraph 6, characterized in that to restore tone, elasticity and flexibility of muscle tissue, the drug is administered in an amount of 0.1-0.5 ml per intramuscular injection.