RU2711111C1 - Hybrid protein igf-1-long for treating stroke, nucleic acid, vector, cell, pharmaceutical composition, method of treatment - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, specifically to recombinant production of neuroprotective proteins, and can be used in medicine for treating stroke. Hybrid protein comprising a sequence of insulin-like growth factor 1 (IGF-1) or an isoform thereof, having neuroprotective activity, to which from the C-terminal is attached directly or through a linker, the sequence SEQ ID NO: 3. Invention also relates to a nucleic acid, a vector, a host cell, a pharmaceutical composition, a method of treating stroke and use based on said protein.EFFECT: invention enables to obtain a neuroprotective protein named by the authors "IGF-1-long", characterized by a longer therapeutic effect compared to IGF-1, not fused with an additional domain.16 cl, 3 tbl, 3 ex

Description

FIELD OF THE INVENTION

The present invention relates to medicine, in particular to a new therapeutic agent for the treatment of the effects of stroke - the recombinant protein IGF-1-long.

BACKGROUND OF THE INVENTION

Vascular diseases of the brain are an urgent medical and social problem. The main place among them is occupied by strokes, and more than 10 million people are annually diagnosed with this disease in the world. According to WHO, strokes are invariably the second leading cause of death, for example, in 2015, 6.24 million people died from strokes in the world. No less acute is the problem of the disability of survivors after a stroke, most of which lose their ability to work, and often need constant care. Age is one of the most significant risk factors for stroke, and after 30 years, this risk begins to increase steadily. Most strokes (95%) occur at the age of 45 years and above. Given the process of demographic aging, which recently covers not only developed, but also developing countries, there is an obvious need to concentrate efforts in the field of health on improving existing and developing new preventive and therapeutic approaches to the fight against stroke.

Strokes, depending on their causes, are divided into two types: ischemic and hemorrhagic. Ischemic stroke - the most common type (70-75% of all cases of strokes) - is a violation of cerebral circulation with damage to brain tissue and impaired function due to a decrease or cessation of cerebral blood flow, thrombosis or embolism associated with diseases of blood vessels, heart or blood . Hemorrhagic stroke is understood to mean non-traumatic intracerebral hemorrhage; subarachnoid hemorrhage, as a rule, is also referred to this species.

Treatment of stroke includes a set of emergency care and subsequent rehabilitation. Emergency care includes resuscitation, including maintaining adequate hemodynamics and oxygenation, as well as eliminating the causes and / or consequences of a stroke, such as thrombectomy. In general, early diagnosis and quick start of treatment are important for the most effective restoration of brain functions. Drug treatment of stroke usually prevails in treatment regimens, and there are a large number of drugs that need to be carefully selected by the attending physician depending on the type of stroke and other circumstances. The complexity of the selection of drug treatment necessitates the search for new improved drugs that are effective in the treatment and prevention of stroke. At the same time, the need for more universal medicines that are effective in both ischemic and hemorrhagic stroke is obvious. In addition, there is a need for medicines that can be quickly applied after an attack without the help of medical personnel, for example by intranasal administration.

One of the promising drugs with neuroprotective activity that can be used to treat stroke is insulin-like growth factor 1 (IGF-1 or IGF-1, also known as somatomedin C).

From a publication by Xin-Feng Liu et al. Intranasal administration of insulin-like growth factor-I bypasses the blood – brain barrier and protects against focal cerebral ischemic damage. J Neurol Sci. 2001 Jun 15; 187 (1-2): 91-7, the use of IGF-1 in a model of ischemic stroke induced in rats by intranasal administration of 150 μg IGF-1 is known.

In the publication of Lioutas V.A. et al. Intranasal Insulin and Insulin-Like Growth Factor 1 as Neuroprotectants in Acute Ischemic Stroke. Transl Stroke Res. 2015 Aug; 6 (4): 264-75, describes the beneficial effects of intranasal administration of insulin and IGF-1 in animal models of acute ischemic stroke.

WO 93/08828 discloses the use of pharmaceutical compositions containing neurotrophic factors, including IGF-1, for intravenous administration to a subject suffering from damage to the neurons in the central nervous system.

WO 90/14838 discloses various approaches to the modification of IGF-I and IGF-II for their use in the treatment of diseases associated with neuronal damage, which include chemical alteration of the amino acid and carboxyl groups of IGF, replacement, removal or inclusion of new amino acid residues, addition of additional chemical groups, etc. to increase the possibility of passing through the blood-brain barrier (BBB).

From the publication Dłużniewska J. et al. A strong neuroprotective effect of the autonomous C-terminal peptide of IGF-1 Ec (MGF) in brain ischemia. The FASEB Journal. 2005 Vol. 19, No. 13, pp. 1896-1898, it is known that the C-terminal domain of the MGF protein, which is an isoform of the IGF-1 protein, exhibits a pronounced neuroprotective activity in the ischemic stroke model.

Known is the synthetic protein IGF-1 LR3 (long arginine 3-IGF-1, Lifetech Labs, Hong Kong), which differs from the native mature IGF-1 in that it has the substitution E → R at position 3 and 13 amino acids (MFPAMPLLSLFVN) with N - end, having a length of 83 amino acids. IGF-1 LR3 has been shown to have enhanced IGF-1 activity and improved metabolic stability; its half-life is about 20-30 hours compared to 12-15 hours in native mature IGF-1. Protein IGF-1 LR3 can be considered as the closest analogue of the present invention, which differs from IGF-1 LR3 in that it has an additional domain attached at the C-terminus of IGF-1.

Despite the promising potential of IGF-1 and its active isoforms, there is a problem of an insufficiently long half-life of a protein, which limits the effectiveness of therapy using these proteins. In addition, there is a need to expand the range of modified proteins with IGF-1 activity, which would have increased metabolic stability, but at the same time, acceptable therapeutic properties, in particular for the treatment of stroke. The present invention is directed, inter alia, to solving this problem.

SUMMARY OF THE INVENTION

The inventor of the present invention unexpectedly discovered that the half-life of IGF-1 in the body of a subject who has been given the indicated protein can be increased by attaching to the indicated protein from the C-terminus of a domain having the sequence of SEQ ID NO: 3. Thus, the present invention relates to a hybrid protein having neuroprotective activity containing the IGF-1 sequence, to which the sequence SEQ ID NO: 2 is attached directly or via a linker from the C-terminal. Various isomorphs can be used as IGF-1 Having activity inherent IGF-1, in particular neuroprotective activity.

In addition, the present invention has the following further embodiments:

- a nucleic acid encoding the aforementioned fusion protein;

- a vector containing the above nucleic acid, which preferably may be an expression vector, but not limited to;

- a host cell that contains the above vector; the host cell is preferably a producer of the fusion protein of the invention;

- a pharmaceutical composition for the treatment of stroke, comprising a fusion protein according to claim 1 in a therapeutically effective amount and a pharmaceutically acceptable carrier; preferably the composition is adapted for intranasal administration;

- a method of treating a stroke, comprising intranasally administering to a patient a fusion protein according to claim 1 in an amount of 0.1-3 mg in each nasal passage;

- the use of a hybrid protein according to claim 1 for the manufacture of a medicament for the treatment of stroke.

A stroke includes both ischemic stroke and hemorrhagic stroke.

The introduction of the fusion protein according to the invention is preferably carried out as soon as possible after the onset of a stroke, preferably the first introduction of the protein should be carried out within 120 minutes from the specified time. A typical window for introducing the fusion protein of the invention is 10-120 minutes.

The fusion protein of the invention is preferably administered in the form of a course, for example, daily 1-3 times a day for 7-30 days.

The introduction of the fusion protein of the invention may preferably be accompanied by co-administration of an amino acid selected from arginine, lysine, glutamine, or any mixture thereof. Accordingly, the pharmaceutical composition of the invention may contain at least one of these amino acids in addition to the fusion protein and carrier, for example, in an amount of 0.1-50 mg (each amino acid) per ml of composition.

The present invention is not limited to the use of a fusion protein as a medicament for the treatment of stroke, but also for the treatment or prophylaxis of other diseases or conditions for the treatment of which neuroprotective activity or other activity inherent in IGF-1 is necessary or favorable. Moreover, the fusion protein according to the invention can be used as a means to stimulate human muscle growth due to the anabolic effect, which is useful for athletes, particularly bodybuilders. The proposed fusion protein will also be useful in pediatrics to treat physical developmental delay and weight loss.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise specified in this application, the scientific and technical terms used in connection with the present invention have meanings that are commonly understood by those skilled in the art. In general, the nomenclature and methods used in cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry of proteins and nucleic acids, hybridization, analytical chemistry, synthetic organic chemistry and medical and pharmaceutical chemistry described in this application are nomenclature and methods well known and commonly used in the art.

The terminology used here is intended to describe specific embodiments of the invention and is not limiting. When used in the present description and claims, the singular forms include the plural, unless the context clearly indicates otherwise.

The bioengineering methods and methods of the present invention are usually carried out in accordance with traditional methods well known in the art, and as described in various general and more specific sources, which are cited and discussed in the present description, unless otherwise indicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

Methods for introducing a mutation into the amino acid sequences of a protein are well known to those skilled in the art (see, for example, Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T. Maniatis, EF Fritsch and J Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989)).

References to amino acid residues used in this application are indicated by a generally accepted single or three letter code (see, for example, Lehninger, Biochemistry , 2 ^nd edition, Worth Publishers, New York, 1975, p. 72).

The term "peptide" or "polypeptide" refers to a polymer of amino acids, regardless of the length of the polymer; thus, protein fragments, oligopeptides and proteins are included in the definition of a peptide or polypeptide. The term also encompasses post-expression modifications of polypeptides, for example, polypeptides, which include covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups, and the like. The definition also includes polypeptides that contain one or more amino acid analogs (including, for example, non-naturally occurring amino acids, amino acids that are found in nature only outside the unrelated biological system, modified amino acids from mammalian systems, etc.), polypeptides with substituted bonds, as well as other modifications known in the art, both natural and non-natural.

The term "identical sequence" together with the percent identity means that in a sequence identical, for example, to 80% of any other sequence, 80% of identical amino acids or nucleotides are present in the same position when aligning sequences that can be carried out known in this areas of technology in ways.

The amino acid or nucleotide sequence identity can be determined conventionally using known computer programs such as Bestfit, FASTA or BLAST (see, for example, Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132 : 185-219 (2000); Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucelic Acids Res. 25: 3389-3402 (1997)).

The term “about” and similar ones, such as “about” and “about”, when used here together with a specific value, indicate that this value can vary within a typical measurement error. For example, the use of the indicated terms with a certain value here indicates this value plus / minus 10%.

The structure of the hybrid protein according to the invention

The present invention is based on the use of the known neuroprotective activity of insulin-like growth factor 1 in the treatment and prevention of stroke.

The igf1 gene encoding an IGF-1 protein is translated into a pre-pro-IGF-I precursor protein that includes a signal peptide, a signal peptide cleavage site, IGF-I protein and an E-peptide (C-terminal peptide). During translation, cleavage of the signal peptide produces Pro-IGF-I. Further protease cleavage separates mature IGF-I from the E-peptide.

Mature IGF-1 consists of 70 amino acid residues forming four domains (BCAD), has a molecular weight of 7.6 kDa and enters the bloodstream mainly from the liver, and its secretion is stimulated by growth hormone. IGF-1 is known to have a direct regulatory effect on hematopoiesis, in particular lymphopoiesis and the functioning of the immune system (Clark P., 1997), and it stimulates the differentiation and transition of pro-B cells into pre-B cells (Landgreth K. et al, 1992 ) and in synergy with IL-7, activates B cell proliferation (Gibson LF et al, 1993), functions in the metabolism of oligodendrocytes and myelin in vivo (Komoly, S. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1894-1898). The secretion of IGF-1 is influenced by substances such as glutamine, arginine, lysine, angiotensin-II, baclofen, ghrelin, thyroid hormones, growth hormone, sterilglucosides (SG), such as acylated sterile-β-glucosides (ASG) and others.

While mature IGF-1 produces the effects generally attributed to it with respect to a wide variety of cells and tissues, other isoforms of IGF-1 can also be used in the invention insofar as they contain the sequence of mature IGF-1 and / or produce the indicated effects. Such isoforms include, for example, isoform 1 (IGF-1B), commonly referred to as “canonical”, as it is a full-sized pre-pro-IGF-I precursor protein; isoform 2 (IGF-1A) with a length of 153 amino acid residues, isoform 3 with a length of 137 amino acid residues expressed in the liver, isoform 4 with a length of 158 amino acid residues, isoform IGF-1E, including variants predominantly expressed in muscle (in humans - IGF-1Ec) and in the liver (IGF-1Ea).

The term “IGF-1 isoforms” also includes functional fragments for which it is known that they have desired biological activity, for example, neuroprotective activity. These include, for example, des (1-3) IGF-1, which is a shortened version (67 amino acids) in which the first three amino acids of GPE are absent; as well as, for example, the E-peptide of the isoform IGF-1Ec, and others. The term also covers mutant forms of IGF-1 having amino acid substitutions, for example, naturally occurring variants with substitutions R → W at position 98, A → T at position 115, A → D at position 187, and others, including not naturally occurring options.

For brevity purposes, the term “IGF-1” as used herein means any of the above forms, unless expressly indicated otherwise. Moreover, the invention encompasses a hybrid protein in which the IGF-1 sequence is flanked at the N- and / or C-terminus with additional amino acid sequences, preferably from 1 to 50 amino acids in length, in particular from 1, 2, 3, 4, 5, 10, and up to 45, 40, 35, 30, 25, 20, 15 amino acids, including the indicated values. Such flanking sequences should not alter the activity inherent in IGF-1.

When referring to “IGF-1”, preferably human IGF-1 is meant, but the invention is not limited thereto, and one skilled in the art can use the IGF-1 ortholog to carry out the invention, for example, in veterinary practice.

In one preferred, but not limiting embodiment, the following are used as the IGF-1 sequence in the fusion protein of the invention:

GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGMVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 1) or

MGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGMVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 2; Met ^-1 -rhIGF-1; GenBank: CAA01954.1).

The second important element of the invention is an additional domain developed by the inventor, which provides unexpected benefits of the fusion protein of the invention. The specified 3 kDa additional domain has the sequence of SEQ ID NO: 3 and is attached to the IGF-1 protein from the C-terminus. Not limited to a specific theory, the author believes that since the structure of this additional domain includes certain repetitions of hydrophobic amino acids, they provide the molecule with high thermodynamic stability, which leads to greater stability of the protein in the blood and other body fluids, and, therefore, prolongs the effect exerted by IGF- 1. The additional domain does not affect the interaction of IGF-1 with a specific receptor.

As an alternative to SEQ ID NO: 3, sequences having at least 80% identity with the sequence of SEQ ID NO: 3 can be used, in particular a sequence identical to at least 81, 82 identical to the sequence of SEQ ID NO: 3 can be used, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

The specified additional domain is attached to the C-terminus of IGF-1 either directly (for example, SEQ ID NO: 4 or 5) or via a linker (spacer), usually used to create hybrid proteins (see, for example, Chen X. et al. Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews Volume 65, Issue 10, 15 October 2013, PP. 1357-1369). Typically, the linker is from 3 to 35, for example 3-16, amino acid residues in length, may be of synthetic origin or may be fragments of naturally occurring proteins. For the purposes of the invention, it is advisable not to use linkers that are designed to cleave the binding domains. It is preferable to use a linker that is acceptable from a pharmacological point of view. Examples of linkers include, but are not limited to (GGGS) n, (GGGGS) n, GSAGSAAGSGEF.

Preferably, said additional domain is attached to the C-terminal amino acid of domain D of IGF-1 or the corresponding amino acid of a sequence comprising the sequence of IGF-1, either directly or via a linker.

The present invention also encompasses variants of a protein with sequences having equivalent chemical functions and properties that are obtained by any method known in the art, for example, by site-directed mutagenesis or by chemical synthesis. Such variants are the result of conservative amino acid substitutions, insertions and / or deletions in the sequence of IGF-1 and / or an additional domain (SEQ ID NO: 3).

Thus, the fusion protein of the invention comprising the above-described additional domain and also referred to herein as “IGF-1-long” has an increased half-life compared to the same protein not fused to the specified additional domain. In addition to this, the author found that this domain does not affect the binding of the protein and the corresponding receptor, i.e., maintains the activity of IGF-1, and also reduces the possible toxicity of the hybrid protein and increases patient tolerance to therapy with such a protein. Like the non-extended variant, IGF-1-long protein has a beneficial effect, particularly protective, on brain neurons, preventing their damage and / or death, and / or reducing the level of damage due to a stroke.

Obtaining a fusion protein according to the invention

The fusion protein of the invention, as well as parts thereof, can be obtained using various expression systems and using recombinant nucleic acid molecules. Suitable expression systems include prokaryotes and eukaryotes, for example, yeast, such as S. cerevisiae , bacteria, such as E. coli , a baculovirus-based system (using insect cells), animal cells, for example mammalian cells, in particular human cells.

In one embodiment of the invention, the fusion protein may be conjugated to another molecule, for example, to increase its half-life, solubility, or bioavailability. Molecules that can be conjugated to the fusion protein of the invention include, without limitation, biotin, polyethylene glycol, nucleic acids, polysaccharides and proteins of other organisms. Conjugation can be direct or indirect (for example, through a linker).

In some embodiments, the fusion protein may be coupled to a label, secretion signal, or other signal sequence. Examples of such labels and sequences are described in WO 2013078299.

Nucleic acid

The invention also provides a nucleic acid molecule encoding the fusion protein of the invention. These molecules can be inserted into the expression vector to clone the sequence and / or expression of the protein in a suitable expression system. The nucleic acid may be DNA or RNA, preferably DNA. The nucleic acid may be isolated or be a segment of a longer sequence.

The invention also encompasses any modifications of the nucleic acid sequence of the invention that are obvious to a person skilled in the art, i.e., those in which the nucleotide sequence retains its biological properties, i.e., is capable of expressing a protein possessing the properties provided for in this invention, providing adequate transcription and translation. For a specialist in the field of genetic engineering who is familiar with the concepts of conservative substitutions and the degeneracy of the genetic code, it is not difficult to choose modifications to the sequences of the invention that satisfy the above criteria.

A nucleic acid encoding a fusion protein of the present invention is capable of being expressed in a host cell. The nucleotide sequence encoding the fusion protein of the invention can be optimized to provide a stable and high expression level of the fusion protein.

The nucleic acid molecule in the present invention also refers to a hybrid gene containing in functional linking with each other at least one promoter that is functional in the host organism, a sequence encoding a hybrid protein in accordance with the invention, and a terminator element that is functional in the same host organism. The various elements that the fusion gene may contain are, firstly, elements that regulate the transcription, translation and maturation of proteins, such as a promoter, a sequence encoding a signal peptide or a transit peptide, or a terminator element constituting a polyadenylation signal, and, secondly, a polynucleotide encoding a protein. The choice of regulatory elements depends on the host cell in which they must function, and those skilled in the art are able to select the necessary regulatory elements without further experimentation.

The expression "in functional linkage with each other" means that these elements of the hybrid gene are linked to each other so that the function of one of these elements is affected by the function of the other. For example, a promoter is operably linked to a coding sequence when it is capable of influencing the expression of said coding sequence. The construction of a hybrid gene in accordance with the invention and the assembly of its various elements can be carried out using methods well known to specialists in this field of technology, in particular, described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press). The choice of regulatory elements that make up the hybrid gene substantially depends on the host organism in which they must function, and those skilled in the art are able to select regulatory elements that are functional in the host organism, which is an object of interest. The term "functional" indicates the ability to function in a given host organism.

Promoters that may contain a hybrid gene in accordance with the invention are either constitutive or inducible. For example, the universally effective promoter used for expression in mammalian cells is pCMV (cytomegalovirus promoter).

In accordance with the invention, the hybrid gene may also contain other regulatory sequences that are located between the promoter and the coding sequence, such as transcriptional activators (enhancers).

Vector

The present invention also relates to a vector comprising the nucleic acid of the invention. The vector may be circular or linear, single-stranded or double-stranded.

In one embodiment of the present invention, there is provided a vector for expressing a hybrid gene in a host cell. The vector of the invention is capable of expressing said hybrid gene in a mammalian cell, in particular in a human cell. In one preferred embodiment, said vector is a nucleic acid structure capable of replication in a bacterial host cell.

Suitable vectors are known to those skilled in the art and include, for example, plasmids, cosmids, artificial chromosomes (e.g. bacterial or yeast), bacteriophages, viral vectors (e.g. retroviruses, lentiviruses, adenoviruses, adeno-associated viruses), in particular baculovirus vectors. Preferably, the vector in accordance with the invention is a plasmid. As a rule, the main qualities of this vector should be the ability to maintain itself and self-replicate in the cells of the host organism, in particular, due to the presence of the origin of replication, and to express the ligand in them.

In order to stable transformation of the host organism, the vector can also be integrated into the genome. The selection of a suitable vector, as well as methods for inserting a gene into a vector, are known to those skilled in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press) and Maniatis et al. (1984. Molecular Cloning).

In one preferred embodiment, the vector may be selected from pcDNA3 and pcDNA3.1 (Invitrogen), which contain a cytomegalovirus promoter and a polyadenylation signal of the bovine growth hormone gene. In another preferred embodiment, the vector may be selected from pVAX (Invitrogen), pCI, VR1012 DNA (Vical Inc.), pJW4303, pVAC1-mcs, pVAC2-mcs (InvivoGen). In yet another preferred embodiment, the vector may be a viral vector, for example, based on adenoviruses (e.g. AD5), orthopoxviruses, alphaviruses or any other suitable vector of the invention that is commercially available and / or known to one skilled in the art.

The regulatory elements necessary for expression are described above with respect to the nucleic acid of the invention, and their selection lies within the competence of a person skilled in the art.

The initiating codon may be located above the coding sequence of the antigen. The stop codon may be located below the coding sequence of the antigen. The initiating and stop codons can be located in the same reading frame with the coding sequence of the antigen.

Examples of polyadenylation signals suitable in the framework of the present invention, in particular for the production of a human genetic vaccine, are, without limitation, SV40 and LTR polyadenylation signals (long terminal repeats).

The promoter may be the promoter of monkey virus 40 (SV40), the promoter of the mouse breast tumor virus (MMTV), the promoter of cytomegalovirus (CMV), the Epstein-Barr virus (EBV) promoter. The promoter may also be a promoter of a human gene, for example, a human actin gene, a human myosin gene, a human hemoglobin gene. The promoter may also be a tissue-specific promoter, for example, a muscle and skin specific promoter, natural or synthetic. In particular, the desmin gene promoter is suitable for expression in hepatocytes, and the promoter of the vitamin D3 hydroxylase gene for expression in keratinocytes. Examples of such promoters are described in US20040175727.

The vector may include genes for transformation markers indicative of the transformation of the bacterial cell by the vector. The transformation marker may be a selective marker gene used to distinguish and select cells that are effectively transformed, that is, those in which the vector is present, from cells without the vector. Such markers are well known to those skilled in the art. Commonly used selective markers include genes that confer resistance to specific antibiotics such as ampicillin, neomycin, etc. Some vectors may also include marker genes that only indicate which cells have been transformed by the vector. Such genes may be genes encoding pigments or enzymes that control the formation of pigments in transformed cells. Such selective marker genes are, in particular, described in WO 91/02071, WO 95/06128, WO 96/38567 and WO 97/04103. The indicator marker gene may be a lacZ gene sequence encoding a β-galactosidase enzyme. Other transformation markers used include, for example, luciferase and GFP genes.

A preferred vector of the invention is a plasmid. A preferred plasmid is a plasmid designed to allow expression of transgenic genetic sequences. A large number of vectors are known and characterized (for example, the base http://genome-www.stanford.edu/vectordb/vector_pages/plasmid_pages/Plasmid.html). Examples of plasmids that allow relatively easy insertion of expression cassettes are well known in the art and are commercially available (WO1990002189, US 20140065699, US 2001/0016351).

In one embodiment of the present invention, the vector may be suitable for transfection of cells with a nucleic acid encoding a fusion protein of the invention, wherein the transformed host cell is cultured and maintained under the conditions under which the expression of the fusion protein occurs. Transformation methods for introducing vectors into various cells and organisms are well known. For example, a vector construct can be introduced by the method of chemically competent cells (calcium chloride / heat shock), electroporation, by plasmid conjugation, particle bombardment, using a gene gun, liposome carriers, etc.

Cell

The invention also provides a host cell comprising a nucleic acid molecule or vector according to the invention and preferably designed to produce copies of a nucleic acid or vector or to produce a fusion protein according to the invention. Any cell commonly used in combination with the expression vectors described above can be used. A cell for producing the fusion protein of the invention may be a eukaryotic or prokaryotic cell, as described above. Examples of eukaryotic cells include animal or plant cells, for example, yeast cells, CHO (Chinese hamster ovary cells), Vero. A prokaryotic cell can be, for example, an E. coli cell and its strains BL21 (DE3), BLR (DE3), origami 2 (DE3), Bacillus, or other gram-positive bacteria such as Lactococcus lactis . Preferred cellular expression systems include E. coli, such as E. coli DH10B.

Pharmaceutical composition and methods of treatment

The term "treatment" as applied to a person, animal or other subject refers to therapeutic treatment, prophylactic or preventive measures for research and diagnostic applications. The term “treating” refers to treating, alleviating, inhibiting the progression of, or preventing a disorder or condition in relation to which the term is used, or one or more symptoms of such a disorder or condition. The term covers the full range of treatment methods for a particular condition from which the subject suffers, for example, administering an active substance or composition containing it in order to alleviate or alleviate symptoms or complications; slowing the progression of the condition, partial relief of the clinical manifestations of the disease or disorder; cure or eliminate the condition, disease or disorder and / or prevent or reduce the risk of developing the condition, disease or disorder. The terms "prevention" or "prophylaxis" mean a system of measures and patient care to prevent the development of a condition, disease or disorder and mean the introduction of active compounds to prevent or reduce the risk of symptoms or complications.

The term "introduction" when applied to biological objects, such as an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contacting of an exogenous pharmaceutical, therapeutic agent, diagnostic agent or composition with a biological object comprising an animal, human, subject, cell, tissue, organ, or body fluid. The term “administration” may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. The treatment of cells includes bringing the reagent into contact with the cell, as well as bringing the reagent into contact with the fluid, where the fluid is in contact with the cell. The term "administration" also means methods of processing in vitro and ex vivo , for example, a cell, a reagent, a diagnostic agent, a binding compound, or another cell.

The term “subject,” as used herein, refers to any mammal, including rodents, such as a mouse or rat, guinea pigs and primates, such as Javanese macaques ( Masasa fascicularis ), rhesus macaques ( Masasa mulatta ) or humans ( Homo sapiens ). Preferably, the subject is a primate, most preferably a human.

As used herein, the terms “therapeutically effective dose” or “therapeutically effective amount” or “pharmaceutically effective dose” or “pharmaceutically effective amount” means a dose having effects for which it is administered. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more diseases or conditions of a patient or population to be treated (either by inducing regressions of these diseases or conditions, or by inhibiting the progression of such diseases or conditions to any clinically measurable degree.

The term "pharmaceutical composition" refers to a mixture containing one or more active substances in accordance with the present invention together with other components, in particular physiologically / pharmaceutically acceptable excipients (excipients). The pharmaceutical composition should have a composition optimized for the desired route of administration of the active ingredient into the body and, ultimately, the manifestation of the biological effect.

"Pharmaceutical" or "pharmaceutically acceptable" refers to chemical compounds and compositions that do not lead to an adverse, allergic or other undesirable reaction when administered to a mammal, in particular humans, or such reactions are minimized. A pharmaceutically acceptable excipient refers to a non-toxic solid, soft, or liquid excipient, diluent, capsule material, or other excipient of any type.

"Pharmaceutically acceptable carrier" means one or more compatible solid, semi-solid or liquid excipients that are suitable for administration to a subject and must be of sufficient purity and sufficiently low toxicity. “Compatibility” herein means that the components of the composition can be mixed with the compounds of the invention or with each other, and they will not significantly reduce the effectiveness of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), moisturizer (such as sodium dodecyl sulfate), color whether, flavors, stabilizers, antioxidants, preservatives, water (sterile), etc.

Methods for administering the fusion protein of the invention

The introduction of the fusion protein or pharmaceutical composition of the invention can be accomplished in a variety of ways, including, without limitation, the oral, subcutaneous, intravenous, parenteral, intranasal, intraorthical, intraocular, rectal, vaginal, transdermal, intraperitoneal, intramuscular, intrapulmonary or intrathecal routes of administration. Preferably, the introduction of the fusion protein or pharmaceutical composition of the invention is carried out intranasally, with the introduction being carried out in each nasal passage.

The composition of the invention can be administered together with other therapeutic agents, i.e., the therapeutic agents described herein can be administered in parallel, simultaneously or sequentially with other pharmacotherapeutic agents or against other therapeutic effects on the body, for example radiation therapy, physiotherapy , surgery, etc. It is understood that the fusion protein or pharmaceutical composition of the invention may be part of a comprehensive treatment for stroke, including yuchayuschego other recommended for the treatment of stroke, drugs and / or other therapeutic effects. The pharmaceutical composition according to the invention may contain, in addition to the fusion protein according to the invention, other active substances, provided that they do not lead to an undesirable from the pharmaceutical and therapeutic points of view, for example, inhibition of each other. The principles and practical approaches to the creation of combined medicines are part of the competence of a specialist.

Any of the active substances included in the compositions of the invention can be introduced indirectly by introducing an exogenous nucleic acid, for example, DNA or RNA encoding the corresponding active substance, resulting in the expression of the introduced nucleic acid. For example, arginine, lysine, or glutamine, or any mixture thereof, may be used as an additional therapeutic agent. These agents may be included in the same composition as the fusion protein of the invention, or in a separate pharmaceutical composition that makes up a kit with the fusion protein composition.

The pharmaceutical compositions of the invention are formulated into a dosage form suitable for the desired route of administration.

Liquid dosage forms can, for example, be a solution or suspension of one or more of the present compounds and optional pharmaceutical adjuvants in a carrier, such as, for example, water, physiological saline, aqueous dextrose, glycerin, ethanol and the like. If necessary, the pharmaceutical composition for administration may also contain other non-toxic adjuvants, such as wetting or emulsifying agents, pH buffering agents, etc. Typical examples of such adjuvants are sodium acetate, sorbitan monolaurate, triethanolamine, triethanolamine oleate, etc. Practical methods for preparing such dosage forms are known or will be apparent to those skilled in the art, for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. , 16th Edition, 1980. In one embodiment, aerosols or sprays are used for intranasal administration. In one embodiment, solutions of the fusion protein of the present invention, in a sterile aqueous solution, in aqueous propylene glycol, or in sesame or peanut oil are used for parenteral administration. Aqueous solutions should be appropriately buffered, when appropriate, and the liquid diluent made isotonic, for example, using appropriate saline or glucose. Aqueous solutions are particularly suitable for intranasal, intravenous, intramuscular, subcutaneous and intraperitoneal administration. All sterile aqueous media used can easily be obtained by standard methods known to those skilled in the art.

Suitable pharmaceutical carriers include inert solid diluents or excipients, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, magnesia, sucrose, cyclodextrin, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid and lower alkyl cellulose ethers. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. In addition, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or in admixture with a wax.

The pharmaceutical compositions of the invention may be in the form of a lyophilisate, suitable for the manufacture of a solution by adding sterile water or another suitable solvent immediately prior to administration of the composition.

Under the pharmaceutical compositions in the present invention are understood as pharmaceutical substances, which are raw materials for the production of drugs, and drugs themselves, having a specific dosage form, and, in general, finished pharmaceutical products, for example, nasal drops or nasal spray dosage, including Packed in packaging, including instructions for use. Preferably, the composition of the invention is presented in unit dosage form.

Doses and duration of treatment

When selecting a dose of a fusion protein according to the invention, one skilled in the art can be guided by the commonly used dose of the corresponding IGF-1 without the additional domain of the invention, which are known in the art. The amounts of active substances to be administered according to the invention are usually determined based on the body weight of the subject.

For example, the approximate range of a single dose of IGF-1-long may be from 1 μg / kg to 15 mg / kg of body weight of a subject, including subranges from 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 μg / kg of body weight of the subject, from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 , 7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 , 5, 5.5 or 6 mg / kg of subject body weight and up to 15, 14, 13, 12, 11, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5 mg / kg of body weight of the subject, including, in particular, the specified boundary values.

When calculating a single dose of IGF-1-long for intranasal administration to humans, a specialist can focus on a range from 0.1 to 3 mg per nasal passage or from 0.2 to 6 mg on both nasal passages. The specified range includes subranges from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3 , 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 mg per nasal passage and up to 3.0, 2.9, 2.8, 2.7, 2, 6, 2.5, 2.4, 2.3, 2.2, 2.1 or 2.0 mg per nasal passage, including, in particular, the indicated boundary values.

In one aspect of the present invention, the fusion protein of the invention is administered in conjunction with at least one of the following amino acids: arginine, lysine, or glutamine. Medicines containing these amino acids are well known in the art and are available on the market, and they can be used in finished form; alternatively, these amino acids can be included in the pharmaceutical composition of the invention in amounts in which these amino acids are commonly used, i.e., one skilled in the art can be guided by known dosages and concentrations of amino acids. Exemplary doses of arginine, lysine and glutamine are from 1, 5, 10, 15, 20 or 25 mg / kg of body weight of the subject and up to 50, 45, 40, 35 or 30 mg / kg of body weight of the subject, including, in particular, values. For example, amino acids are administered in an amount of from 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 40 and up to 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, or 6 g / kg of subject body weight, including, in particular, the indicated boundary values. The daily dose of these amino acids when taken by a person may, in particular, be from 0.15 g to 1.5 g of each amino acid. When using amino acids in the pharmaceutical composition of the invention, in particular in a liquid dosage form, for example, spray or drops, the concentration of each amino acid may be 0.1-50 mg / ml of the composition. This range includes sub-ranges from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 25, 30 mg / ml of the composition and / or up to 45, 40, 35, 30, 25, 20 mg / ml, including, in particular, the indicated boundary values.

All of these doses are single and are preferably administered 1, 2 or 3 times a day. In this case, in the case of intranasal administration, the indicated doses are administered in each nasal passage. However, depending on the need determined by the doctor, the frequency of administration can be either reduced or increased. For example, if a dosage form with a long release is used, the frequency of administration can be reduced to 1 time in 2 days, 1 time in 3 days, 1 time in 4 days, 1 time in 5 days, 1 time in 6 days or 1 time per week . On the other hand, the frequency can be increased to more than two times a day, for example 3, 4 or 5 times a day.

The duration of administration of the active substances or compositions according to the invention is preferably from 7 to 30 days, for example from 7, 8, 9, 10, 14, 21 days and / or up to 30, 28, 24, 23 days. Of course, depending on the severity of the therapeutic effect and possible side effects, the duration of treatment can be either reduced or increased relative to the indicated values. Treatment may include several courses of administration of the fusion protein or composition of the invention, or their substantially continuous administration.

The subject is administered the composition of the invention as soon as possible after a stroke, not later than 120 minutes. In this context, it is preferable to understand the first signs of a stroke, which can be determined using the FAST test (Face-Hand-Speech) or USP. As a rule, the therapeutic window is 5-120 minutes after the onset of a stroke. Preferably, a composition of the invention is administered to a subject within 10-60 minutes after a stroke. Most preferably, the composition is administered within 10-15 minutes after a stroke.

The exact dosage will depend on the purpose of the treatment and should be determined by the attending physician using well-known recommendations. The amount of therapeutic agent that is effective in alleviating a particular disease or condition (also referred to as a “therapeutically effective amount”) may vary according to several factors, such as disease status, age and body weight of the patient, and others. The dosage can, if necessary, be adjusted directly during treatment, depending on the severity of the therapeutic effect and side effects.

The amount of fusion protein in the compositions for use according to the invention can vary widely depending on the desired dosage form. In any case, the content of active substances in the compositions according to the invention can be selected based on the approximate doses indicated above, so that the finished dosage form provides the easiest way for the patient to follow the prescribed treatment regimen.

The present invention is not limited by the scope of exemplary embodiments that are intended only as illustrations or specific aspects of the present invention. Various modifications of the present invention, in addition to those disclosed herein, will be apparent to those skilled in the art, taking into account existing knowledge of the art. It is assumed that all such modifications are covered by the attached claims.

Experimental models

In connection with the relevance of this disease, it is important to adequately test drugs at the preclinical stage. From this point of view, it is important to choose a suitable model of the disease in animals.

Such models as the model of ischemic stroke caused by ligation of two carotid arteries (global cerebral ischemia), the model of hemorrhagic stroke caused by craniotomy, the model of global cerebral ischemia, focal cerebral ischemia caused by intraluminal implantation, are most often used to study the neuroprotective effect of the drug. hematomas without craniotomy and others.

Different types of animals can act as biological test systems, but rats are the most suitable and often used.

Based on practical and ethical considerations, experimental modeling of stroke should be reproduced with adequate anesthesia. In the experimental practice of stroke modeling, the choice of anesthesia is one of the most important components, since anesthesia can affect the main indicators of stroke: survival and area of brain damage. Many anesthetics have a fairly high cerebroprotective potential and are able to change the adaptation of the brain to hypoxia, and therefore affect the outcome of a stroke.

Global cerebral ischemia model by occlusion of two vessels in combination with arterial hypotension (GBI)

GBI is an acute violation of cerebral circulation with damage to brain tissue, a violation of its functions due to difficulty or cessation of blood flow to a particular department. According to ICD 10, this condition refers to ischemic strokes. According to statistics, ischemic stroke is the most common and common cerebrovascular disease. GBI was first described in 1972 by Eklof and Siesjo. This model is characterized by relative simplicity in execution and fairly good reproducibility, as a result of which it is widely used in laboratory practice to reproduce the state of global cerebral ischemia in rats.

To reproduce this model, the use of various drugs for anesthesia is described. For example, the use of Zoletil 100 in combination with xylazine at a dose of 2.6 mg / kg intravenously allows for deep, prolonged anesthesia and to prevent the effect of the anesthetic on the development of pathology.

After preparation of the surgical field, a midline incision of the neck and layer-by-layer dissociation of the muscles and soft tissues of the neck is performed in a blunt manner until the neurovascular bundle of the neck is left and right. During this manipulation, it is important not to damage n. vagus. After isolation of the common carotid artery, ligation is performed on both sides. On the left, one ligature is superimposed on the common carotid artery. First, a ligature is applied distally to the right, closer to the bifurcation of the common carotid artery, after which, under the control of blood pressure (BP), bloodletting is performed by piercing the common carotid artery with a catheter. According to the recommendations, it is necessary to lower the pressure to 30 mm Hg. Art. At the end of blood pressure reduction by bloodletting, the common carotid artery is ligated proximal to the injection site of the catheter needle. Then the wound is sutured in layers and treated with an antiseptic.

When using this model, species differences in animals can also be taken into account. So, for example, the gerbil has no Wilizian circle, and anastomoses between the anterior cerebral arteries are limited. This anatomical feature allows the induction of unilateral forebrain ischemia by occlusion of one carotid artery, with a success rate of approximately 40%, bilateral occlusion of the common carotid arteries - up to 90%.

To approximate the method to the natural conditions for the development of ischemic stroke, a modified model was developed.

In a rat, soft tissues of the anterior surface of the neck are dissected with a midline incision. Allocate common carotid and left external carotid arteries. Ligatures are brought under the vessels, while under the left common carotid artery there are two ligatures (upper and lower). Bandage the left external carotid artery to prevent the occluder from being carried into this vessel and the development of massive bleeding with the introduction of the occluder. The left common carotid artery is ligated with a lower ligature to prevent massive blood loss with the introduction of the occluder. Immediately before bifurcation, a clamp is applied to the left common carotid artery. A surgical needle with a circular cross section produces perforation of the wall of the left common carotid artery just above the ligation site. An occluder is introduced through an injection into the arteries, after which the clamp is removed and the occluder is slowly, to prevent damage to the vessel at the bifurcation site, carried out to a depth of 17-20 mm along the general and internal carotid arteries to the middle cerebral artery until there is a difficulty in further advancement. The occluder is fixed in the vessel by ligation of the common carotid artery with the upper ligature above the site of perforation of the vessel wall. Then, the common carotid artery is ligated from the opposite side to reduce the blood supply to the brain and the wound is sutured.

As an occluder, a sterile thread from chromed catgut 5.0 is used. The thread is cut into fragments of 30 mm and at the last dye, for example, methylene blue, a distance of 20 mm is noted.

One of the early criteria for the correctness of the performed technique is a violation of the blood supply to the eyes of the left side, due to occlusion a. ophthalmica. After 48 hours, complete melting of the eye tissue is observed. For the experiment, rats of both sexes weighing 150-200 g are taken. After modeling an ischemic stroke, the blood supply to the tissues of the eye of the left side is evaluated immediately after the operation by the proposed method. After 24, 48 and 72 hours, neurological changes are assessed.

The method allows you to bring the model closer to the natural conditions for the development of ischemic stroke, to ensure high reproducibility and reduce morbidity during the experiment.

Endovascular focal cerebral cerebral ischemia model by intraluminal occlusion of the middle cerebral artery (FCI)

Another model for reproducing ischemic stroke is the model of endovascular focal cerebral cerebral ischemia through intraluminal occlusion of the middle cerebral artery, which, unlike the previous model, is a model of a focal ischemic stroke located in a known place of damage - the middle cerebral artery basin.

This model was first described in 1986 by J. Koizumi and Y. Yoshida and subsequently modified and reproduced by Zea Longa E.L. in 1989. Subsequently, the model was adapted to mice. The essence of the model is the formation of cerebral ischemia in the basin of the middle cerebral artery (SMA) by the monofilament in the endovascular way.

To reproduce the pathology, the animal is injected into anesthesia. Allocate the common carotid artery and its branches. Temporarily ligate the common carotid artery. Monofilament is passed through the external carotid artery into the internal carotid artery at a distance of approximately 20-25 mm from the bifurcation of the common carotid artery. An important factor is the processing of monofilament with silicone gel to prevent perforation of cerebral vessels. After the monofilament is performed, it is fixed with a ligature on the external carotid artery, blood flow is restored by removing the ligature from the general SA and the wound is sutured in layers.

The advantages of this model are that the formation of pathology involves a cerebral infarction of a clear localization and does not require craniotomy.

In addition to directly intraluminal occlusion of the middle cerebral artery, there is a modification of the model using reperfusion. In this case, the intraluminal occlusion of the middle cerebral artery is temporary, after which the blood flow is restored by removing the ligature from the common carotid artery. The external carotid artery, through which the filament was inserted, is pre-ligated. The time to reperfusion is set by the performers in accordance with the technical equipment of the laboratory, as well as to obtain data of focal cerebral ischemia at various time intervals of ischemia.

This model, used in experimental practice, is of great importance for the development of methods for the treatment of ischemic strokes and more than compensates for the high cost, long training time and technical complexity of reproducing the model.

Subarachnoid hemorrhage (SAH) model by intraluminal cerebral perforation

Subarachnoid hemorrhage - hemorrhage into the subarachnoid space (the cavity between the arachnoid and the pia mater). According to ICD 10, this condition refers to hemorrhagic strokes.

The main cause of SAH is rupture of an arterial aneurysm, usually of the midbrain artery. It is this state that reproduces the SAH model by intraluminal perforation of cerebral vessels.

This model was first described in 1995 by J. B. Bederson. In fact, it is a modification of focal cerebral ischemia according to the Zea-Longa method. Induction of pathology is carried out similarly to FCI, except that the thread is passed at a distance of 27-30 mm from the bifurcation of the common carotid artery. Thus, perforation of the cerebral artery is achieved. Then the filament is removed. Ligature is applied to the external carotid artery. They restore blood circulation in the common carotid artery, after which the surgical wound is sutured in layers.

The model is characterized by high mortality, which is 40-60% in the first three days after modeling. In surviving animals, clouding of the cornea is often observed as a result of occlusion of the branch of the middle cerebral artery - the visual artery.

It is important to note that the model is characterized by a clear localization of the lesion, which ensures its high repeatability.

The development of surgical interventions to simulate diseases is an essential condition for the successful completion of a pharmacological experiment. The development of various stroke models usually requires about 144 laboratory assistants / days. Such preparation for the actual experiment can significantly reduce mortality, select informative indicators for assessing pathology, and calculate the time required for the study. Particular attention when modeling strokes should be given to methods and means for anesthesia.

Adequate modeling of the pathology will make it possible to objectively evaluate the effectiveness of new drugs for the treatment of strokes at the stage of preclinical studies.

EXAMPLES

Example 1. Obtaining IGF-1-long

Gene synthesis and genetic engineering methods

The synthesis of the gene encoding human IGF-1-long was carried out by elongation of partially overlapping oligonucleotides obtained using automated chemical synthesis. Restriction and ligation reactions, selection of transformants, cultivation of bacterial cells, isolation and electrophoresis of plasmid DNA was carried out using standard genetic engineering methods. The synthesized gene was inserted into the pET28a (+) vector (Novagen, USA) at the NdeI and XhoI sites obtained with the pET28-IGF-1 plasmid transformed E. coli cells. E. coli DH10 B [F ^- mcrA Δ (mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu ) 7697 galU galK λ ^- rpsL nupG / pMON14272 / pMON7124] (Thermo Fisher Scientific, USA). To produce the recombinant protein with plasmid pET28-IGF-1, E. coli BL21 Star (DE3) cells (Invitrogen, USA), F ^- ompT hsdS _B (r _B ^- , m _B ^- ) gal dcm rne131 (DE3) were transformed. Cell transformation was performed by electroporation using a Gene Pulser Xcell ™ Electroporation Systems (Bio-Rad, USA) pulse generator according to the manufacturer's instructions.

Cell cultivation and analysis of the content of recombinant IGF-1-long

The cells of the producer strain were cultured in two ways - using isopropyl-β-D-thiogalactopyranoside (IPTG, IPTG) and lactose as inducers of the synthesis of the target protein.

In the first variant, the E. coli BL21 cell culture (pET28-IGF-1) was grown in a thermostatic shaker in 1 L of LB medium containing 100 μg / ml kanamycin at a temperature of +37 ° C and a rotation speed of 250 rpm. After the culture reached an optical density of A600 of 0.6 optical units, an inductor — IPTG — was added to a final concentration of 1 mM and cultivation was continued for 5 hours.

For Studier lactose induction, a separate colony of producer strain cells was inoculated in 1 L of PYP medium with 0.2% lactose and 100 μg / ml kanamycin and grown at +16 ° C overnight. 1 ml culture samples were centrifuged at 4000 g for 10 min, after which cell pellets were lysed by the addition of a five-fold lysing solution (10% SDS, 10 mM dithiothreitol, 30% glycerol, 0.2 M Tris-Hcl, pH 6.8 and 0 , 05% bromophenol blue) and boiled for 10 minutes. Lemmli electrophoresis was performed in 12.5% SDS page under denaturing reducing conditions, followed by Coomassie G-250 staining and gel densitometry. As a negative control, a cell culture of the producer strain was used without induction of IGF-1 protein synthesis.

Isolation and purification of recombinant IGF-1-long

E. coli BL21 cells (pET28-IGF-1) grown under the conditions of induction of protein synthesis by lactose were precipitated by centrifugation at 10,000 g, resuspended in physiological saline (1 × PBS) in a ratio of 5 ml of 1 × PBS / g of wet biomass, after which they were destroyed using an ultrasonic wave generator (Heidolph Sonik 100, Germany) and centrifuged at 10000 g for 12 min at +4 ° С. The resulting precipitate was resuspended in the initial volume of the washing solution containing 2 M urea, 20 mM Tris-HCl, pH 7.6, incubated for 30 minutes at room temperature, and then centrifuged at 10,000 g for 12 min at 4 ° C. The supernatant was removed, the precipitate of washed inclusion bodies was dissolved in a solubilizing solution (8 M urea, 20 mm Tris-HCl, pH 8.0).

Refolding was carried out by diluting the solubilized protein 8 times with a solution of 20 mm Tris-HCl, pH 8.0; 0.01% Tween 20, after which it was filtered through a 0.2 μm filter. The resulting solution was applied to a 5 ml metal chelate column (His-Trap Chelating, GE Healthcare, USA), equilibrated with start buffer (20 mM Tris, pH 8.0; 0.01% Tween 20). A Discovery 100 chromatograph (Bio-Rad, USA) was used. After washing the column with 10 volumes of the start buffer, the adsorbed protein was eluted with an imidazole gradient (0 mM - 200 mM) in the start buffer. The fractions containing the target protein were combined, the pH of the protein solution was adjusted to a value of 8.8 by the addition of 10 mM NaOH and re-filtered through a 0.2 μm filter. The protein solution was applied to a GE Healthcare SP Sepharose Fast Flow column equilibrated with start buffer-2 (20 mM Tris-HCl, pH 8.8; 0.01% Tween 20). After applying and washing the column with start buffer 2, the protein was eluted with a linear NaCl gradient. Fractions containing the target protein were pooled and dialyzed against 1 × PBS. The bacterial endotoxin content in the preparations was determined by the LAL test (GeneScript, USA). The protein solution was stored at a temperature of -20 ° C with the addition of glycerol up to 10%.

Example 2. Evaluation of morphological changes in brain tissue

For the experiment, a model of endovascular focal cerebral cerebral ischemia was used by intraluminal occlusion of the middle cerebral artery (FCI).

Then, after stroke induction, rats, divided into groups of 16 individuals, were daily administered intranasally with preparations containing the corresponding active substances. Experimental groups 2-4 were injected with IGF-1-long (SEQ ID NO: 5) in the form of a solution containing 0.9% NaCl and 0.3% reopoliglukin. As a negative control, we used a group of individuals who were not given any drugs (group 1), and as a positive control we used a group of individuals who were given the standard drug for stroke Piritinol (group 5).

Histological analysis was performed as follows.

For light microscopy, the rat brain is fixed in 96% ethanol. Paraffin sections are stained with methylene blue, hematoxylin-eosin and Nissl's method. Histopathological changes are then evaluated on a 6-point scale (Clonidine Decreases Ptasma Catecholamines and Improves Outcome From Incomplete Ischemia in the Rat, William E. Hoffman, Ph D, Mary Ann Cheng, MD, Chinamma Thomas, MD, Verna L. Baughman, MD, and Ronald F. Albrecht, MD Departments of Anesthesiology and Pathology, Humana / Michael Reese Hospital, University of Illinois, Chicago, Chicago, Illinois. ANESTH ANALG 1991; 73; 460-4).

Table 1. Scoring histological changes in rat brain

Points Histological changes 0 The absence of dead neurons 1 Death of single neurons 2 Small focal heart attacks 3 Focal heart attacks 4 Heart attacks with damage to more than 50% of the hemisphere 5 Total hemisphere infarction

Table 2. The effect of the administered drug IGF-1-long on histological changes in the brain of experimental rats.

Group 1 2 3 4 5 Time after stroke induction, day Control, without the introduction of drugs for stroke Introduction
180 mcg IGF-1-long per 20 grams of individual weight Administration of 18 μg IGF-1-long per 20 grams of individual weight Introduction
180 mcg IGF-1-long plus 500 mcg arginine per 20 grams of individual weight Control, 5 mg per 20 grams of individual weight 1 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 2 4 4 4 2 3 2 4 5 4 2 3 2 3 6 5 2 2 2 3 7 5 2 2 2 3

Example 3. Pharmacokinetic properties of IGF-1-long

The lifetime of the fusion protein of the invention was evaluated after administration to rats of 100 μg of protein (IGF-1-long (SEQ ID NO: 5) and IGF-1 without the long domain (SEQ ID NO: 2)) in the form of the same preparations as indicated in example 2, and measuring the level of protein in serum. As shown in Table 3, in comparison with the unmodified domain of the long form, a significant extension of the half-life of the hybrid protein is achieved, which ultimately leads to a prolonged action of the protein.

Table 3. Change in concentration of IGF-1 and IGF-1-long

Time, day the level of IGF-1 in the blood, ng / ml, with the introduction of 100 μg per animal the level of IGF-1-long in the blood, ng / ml, with the introduction of 100 μg per animal 1 765 658 2 223 634 3 183 580 4 196 530 5 179 227 6 187 192

Claims (16)

1. A hybrid protein with neuroprotective activity, containing the sequence of insulin-like growth factor 1 (IGF-1) or its isoform with neuroprotective activity, to which is attached from the C-terminus, directly or via a linker, the sequence SEQ ID NO: 3. 2. The hybrid protein of claim 1, wherein IGF-1 has the sequence of SEQ ID NO: 1 or 2. 3. A nucleic acid encoding a fusion protein according to claim 1 or 2. 4. An expression vector containing a nucleic acid according to claim 3. 5. A host cell that produces a fusion protein according to claim 1 or 2, containing a vector according to claim 4. 6. A pharmaceutical composition for the treatment of stroke, comprising the fusion protein of claim 1 or 2 in an effective amount and a pharmaceutically acceptable carrier. 7. The pharmaceutical composition of claim 6, further comprising an amino acid selected from arginine, lysine, glutamine, or any mixture thereof. 8. The pharmaceutical composition according to claim 6 or 7, wherein the amount of each amino acid used is 0.1-50 mg / ml. 9. The pharmaceutical composition according to any one of paragraphs. 6-8, where the stroke is an ischemic or hemorrhagic stroke. 10. A method of treating a stroke, comprising intranasally administering to a patient a fusion protein according to claim 1 or 2 in an amount of 0.1-3 mg in each nasal passage. 11. The method according to p. 10, where the first introduction of the hybrid protein is carried out within 120 minutes from the start of the attack. 12. The method according to p. 10 or 11, wherein said hybrid protein is administered daily 1-3 times a day for from 7 to 30 days. 13. The method according to any one of paragraphs. 10-12, in which the patient, together with the specified hybrid protein is additionally administered an amino acid selected from arginine, lysine, glutamine, or any mixture thereof. 14. The method according to any one of paragraphs. 10-13, where the stroke is an ischemic or hemorrhagic stroke. 15. The use of a hybrid protein according to claim 1 or 2 for the manufacture of a medicament for the treatment of stroke. 16. The use of claim 15, wherein the stroke is an ischemic or hemorrhagic stroke.