Method for treating traumatic brain injury and drug for carrying out said method

Abstract

The invention relates to medicine, and more particularly to experimental pharmacology, and can be used for creating pharmaceutical compositions and dosage forms for the treatment of traumatic brain injury. For the purpose of broadening the existing range of drugs for the treatment of traumatic brain injury, the use of human apolipoprotein A-I or functionally significant fragments thereof is proposed, wherein the claimed drug contains native apolipoprotein A-I obtained from human blood serum, or recombinant human apolipoprotein A-I, or a functionally significant fragment of human apoA-I. The functionally significant fragment of human apoA-I has a sequence consisting of the amino acid sequence of human apoA-I truncated at the N terminal, and more particularly the amino acid sequence of human apoA-I truncated at the N terminal by 2 amino residues (amino acid residues 3-243). The drug is provided in the form of a pharmaceutical composition containing human apolipoprotein A-I or the functionally significant fragment thereof, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is water for injection or saline solution. A method for treating traumatic brain injury includes locally or parenterally administering said drug to a subject in need thereof in therapeutically effective doses.

Description

[0001] Method of treating traumatic brain injury and means for its implementation

[0002] The invention relates to medicine, in particular experimental pharmacology, and can be used to create pharmaceutical compositions and dosage forms intended for the treatment of traumatic brain injury.

[0003] The topic of treating traumatic brain injury (TBI) is currently a very pressing issue in modern medicine. This is due to the extremely low arsenal of pharmacological treatments for this type of injury, especially given its pathophysiological characteristics. In Russia, the incidence of TBI is approximately 4 cases per 1,000 population or 400,000 victims per year. About 10% of them die and the same number become disabled. According to various authors, TBI is the most common cause of death and severe disability in the group of people under 35 years of age. Men suffer TBI 2-3 times more often than women. The most common cause of TBI is road traffic accidents. The second most significant cause of TBI (from 20% to 30%) is falls, especially among young and elderly people. The highest risk of TBI is observed at the age of 15 to 24 years and after 70 years, and in the latter, TBI is mainly associated with falls, industrial, sports and domestic injuries.In the pathogenesis of TBI, the leading role is played by disturbances of the main neurodynamic processes in the central nervous system, causing vascular, cerebrospinal fluid dynamic and endocrine-humoral disorders. The reaction of the cerebral vascular system is manifested by widespread vascular spasm followed by cerebral hyperemia and venous congestion. Disorders of cerebrospinal fluid circulation are associated with the development of cerebrospinal fluid hypertension, disturbances in the permeability of the blood-brain barrier. Weakening of the regulatory functions of the hypothalamic-pituitary system leads to disturbances in the hormonal balance in the body, disturbances in water and salt metabolism, circulatory disorders with the development of cerebral hypoxia and edema-swelling of brain tissue. Currently, TBI is also the dominant injury in military conflicts as a result of mine-explosive contusions.Obviously, all types of TBI have their own specifics depending on their severity, but in terms of drug treatment they are very similar due to a very limited arsenal of drugs. Of the pharmacological agents used to treat TBI, the following are mainly used: diuretics, calcium channel blockers (nifedipine), vasodilators and antihypertensive drugs (cinnarizine, cavinton and inhibitors of angiotensin converting enzyme and its vascular receptors), tranquilizers (relanium, seduxen) and nootropic drugs (nootropil, phenotropil), non-steroidal (ketoprofen) and steroidal (dexamethasone) anti-inflammatory drugs. Basically, the goal of drug therapy for TBI, regardless of its severity, is to reduce craniocerebral pressure and restore cerebral hemodynamics, relieve the inflammatory response to eliminate the phenomena of cerebral hypoxia.

[0004] Patent RU 2477150 C2, published 10.03.2013, Bulletin No. 7 describes a method for treating patients with traumatic brain injury against the background of hypertension syndrome in the acute period. For this purpose, the patient's blood is collected and 50 ml of erythrocytes are then isolated from it. The erythrocytes are saturated with 1 ml of dexamethasone and returned to the patient's circulatory system. The procedure is carried out once a day with an interval of 2-3 days. The course of treatment is 5 procedures. The method allows to reduce the treatment time for this category of patients due to the most complete delivery of the drug, ensured by the saturation of the patient's erythrocytes with it. The disadvantage of the proposed method is the complexity of its implementation and the large number of side effects from the use of dexamethasone.

[0005] Patent RU 2309745 C2, published 10.11.2007, Bulletin No. 31, proposes to use a drug with complex anti-ischemic, antihypoxic and anticonvulsant effects for the treatment of TBI. Zopiclone, a "non-benzodiazepine" benzodiazepine receptor agonist, is used as such a drug. It was shown that in the first three days of the post-traumatic period, zopiclone increased the resistance of the brain to ischemia, hypoxia, hypercapnia and convulsions during trauma by 24.9-42.6% (versus the vasodilator nimodipine or the antihypoxant sodium oxybutyrate). The main disadvantage of zopiclone is its side effect associated with a sedative effect and the development of drug dependence.

[0006] Erythropoietin is used to treat combined craniocerebral trauma to compensate for post-traumatic anemia. Thus, patent RU 2446834 C2, published 10.04.2012, Bulletin No. 10 describes a method for treating victims with severe combined craniocerebral trauma (CCT). For this, the victim undergoes standard, generally accepted intensive therapy. Additionally, the drug EpoCrin (erythropoietin) is administered intravenously by jet stream. On the first day of the post-traumatic period, EpoCrin is administered once at a dose of 10,000 IU. On the second day of the post-traumatic period, depending on the severity of the injury and the threat of death, EpoCrin is administered at a dose of 10,000-20,000 IU. On the third and fourth days, EpoCrin is administered at a dose of 10,000 IU. On the fifth day of the post-traumatic period, Epocrin is administered at a dose of 5000-10000 ME.The developed regimen of epocrine administration, which is included in the intensive care of patients with severe combined TBI, allows to increase the effectiveness of treatment, reduce mortality and the number of cases of complications. The main disadvantage of the proposed method is its narrow pathophysiological focus, in particular, the method is aimed only at compensating for post-traumatic anemia and in no way affects the inflammatory and neurodegenerative processes in the brain that arose after TBI. As a result, its effectiveness is insufficient for the treatment of TBI, and is sufficient only for compensating for post-traumatic anemia.

[0007] Patent RU 2406506 C1, published 20.12.2010, Bulletin No. 35 provides a pharmaceutical composition of choline alfoscerate in the form of an injection solution (cholitilin), which has nootropic activity and cholinomimetic action, a method for the treatment / prevention of CNS diseases and the consequences of traumatic brain injury. The pharmaceutical composition contains choline alfoscerate as an active ingredient in a therapeutically effective amount and povidone (plasdone or kollidone) as a pharmaceutically acceptable carrier in an amount of 0.2-0.8 wt.% based on 100% of the entire composition. The pharmaceutical composition according to the invention is used for the treatment or prevention of CNS diseases and the consequences of traumatic brain injury. The main disadvantage of the pharmaceutical composition is the side effects associated with the cholinomimetic action of cholelithin, such as nausea, abdominal pain and permanent confusion.

[0008] Patent RU 2554502 C9, published 27.06.2015, Bulletin No. 18 provides data on the effectiveness of using M-(2-adamantyl)-hexamethyleneimine hydrochloride (himantane) as a treatment for cerebrovascular accidents and brain injuries. The ability of himantane, when administered 3.5 hours after modeling a hematoma in rats and then administered for 4 days, to reduce animal mortality and improve cognitive and motor performance was established, which indicates its neuroprotective potential in the intracerebral post-traumatic hematoma model. The technical result consists in implementing the declared purpose: it was shown that himantane at a dose of 5 mg / kg when administered intravenously caused an increase in cerebral blood flow in the cerebral cortex of rats after ischemic brain damage, without changing the level of arterial pressure.The drug had no significant effect on the blood supply to the brain of intact animals and did not cause changes in heart rate and respiratory rate. All adamantane derivatives have a large number of side effects, among which the most common are: insomnia, psychomotor agitation, paranoid exogenous psychosis accompanied by visual hallucinations (in predisposed elderly patients), dizziness, decreased visual acuity.

[0009] Patent RU2414901 C2, published 27.03.2011, Bulletin No. 9 describes a drug that has a neuroprotective effect in the acute period of traumatic brain injury. The proposed drug is the well-known anticonvulsant lamotrigine (other names for the substance are lamictal, lamolep) as a drug that prevents ischemic and hypoxic disorders in the acute period of traumatic brain injury. Prevention of secondary disorders of brain function due to ischemia, hypoxia with hypercapnia, hemic hypoxia with simultaneous facilitation of the restoration of the neuropsychic status is shown. Lamotrigine increases the resistance of the brain to the leading factors of its secondary damage by 24.9-42.6% and increases the life expectancy of mice in the post-traumatic period by 109.6%. The disadvantage of the proposed remedy is the large number of side effects from the central nervous system, liver and hematopoietic system.A drug similar to the one described above in the pharmacological group is proposed to be used in patent RU 2318501 C2, published 10.03.2008, Bulletin No. 7. It is proposed to use adepiprole (sodium volproate) as such a drug, which has a multifactorial neuroprotective and nootropic effect in traumatic brain injury. For the first time, a high protective anticonvulsant index of sodium valproate was discovered in the post-traumatic period with major convulsive seizures in the case of its administration from the 2nd day after the injury. A course of treatment with the drug not only restores the learning rate of injured animals to the level of intact animals, but also stimulates the acquisition of behavioral adaptive responses, including delayed reproduction of adaptive reactions (a method for assessing the state of mnemonic functions).The main disadvantage of the claimed drug is also a large number of side effects from the central nervous system and the hematopoietic system; the drug has hepatotoxicity.

[0010] Patent RU 2766785 C1, published 15.03.2022, Bulletin No. 8 describes a method for correcting the pathology of 2-amino-5-ethyl-1,3,4-thiodiazolium-1-acetyl-aminoethanoate in traumatic brain injury. The invention relates to medicine, in particular to experimental pharmacology. The method for correcting neurological deficit in traumatic brain injury involves modeling craniocerebral injury in rats by free fall of a 155 gram load from a height of 0.6 m. To correct the pathology, 2-amino-5-ethyl-1,3,4-thiodiazolium-N-acetyl-aminoethanoate is administered intragastrically through a tube, in a 3-day course, at a dose of 50 mg / kg, once, the first time 30 minutes before modeling the pathology, with subsequent administration for 2 days. The method leads to a pronounced correction of the consequences of traumatic brain injury.2-amino-5-ethyl-1,3,4-thiodiazolium-1-acetyl-aminoethanoate at a dose of 50 mg / kg, administered both before and after traumatic brain injury, helps to reduce neurological symptoms. Data on this effectiveness of 2-amino-5-ethyl-1,3,4-thiodiazolium-N-acetyl-aminoethanoate are also provided in the patents: RU 2756325 C1, published 09.29.2021, Bulletin No. 8, RU 2763012 C1, published 12.24.2021, Bulletin No. 36, RU 2758433 C1, published 12.28.2021, Bulletin No. 31, which presumably has antioxidant and antihypoxic activity and blocks the activation of free radical processes, as well as lipid peroxidation of cell membranes, in acute disorders of regional and general blood circulation.

[0011] In patent RU 2252023 C2 (prototype), published 20.05.2005, Bulletin No. 14, for the treatment of traumatic brain injury, intravenous drip administration of 2-ethyl-6-methyl-3-hydroxypyridine succinate (mexidol) immediately after injury at 200 mg 2 times a day for severe injury and 200 mg 1 time per day for moderate and mild injury is proposed. The method provides a detoxifying effect in the acute period of TBI due to the antitoxic effect of the drug, which was established for the first time - acceleration of the breakdown and removal of toxic metabolites from the cerebrospinal fluid. Mexidol is an antioxidant that has antihypoxic, membrane-protective, nootropic, anticonvulsant and anxiolytic effects. Mexidol is an inhibitor of free radical processes, a membrane protector, with antihypoxic, stress-protective, nootropic, anticonvulsant and anxiolytic effects.The drug increases the body's resistance to various damaging factors (shock, hypoxia and ischemia, cerebrovascular accidents, alcohol intoxication and intoxication with antipsychotic drugs / neuroleptics / ). The mechanism of action of Mexidol® is due to its antioxidant, antihypoxic and membrane-protective effects. The drug inhibits lipid peroxidation, increases superoxide dismutase activity, increases the lipid-protein ratio, reduces membrane viscosity, and increases its fluidity. Mexidol modulates the activity of membrane-bound enzymes (calcium-independent phosphodiesterase, adenylate cyclase, acetylcholinesterase), receptor complexes (benzodiazepine, GABA, acetylcholine), which enhances their ability to bind to ligands, helps maintain the structural and functional organization of biomembranes, transport neurotransmitters and improve synaptic transmission. Mexidol increases the content of dopamine in the brain.It causes an increase in compensatory activation of aerobic glycolysis and a decrease in the degree of inhibition of oxidative processes in the Krebs cycle under hypoxic conditions with an increase in the content of ATP and creatine phosphate, activation of energy-synthesizing functions of mitochondria, stabilization of cell membranes. The drug improves metabolism and blood supply to the brain, improves microcirculation and rheological properties of blood, reduces platelet aggregation. Stabilizes membrane structures of blood cells (erythrocytes and platelets) during hemolysis. It has a hypolipidemic effect, reduces the content of total cholesterol and LINE. The anti-stress effect is manifested in the normalization of post-stress behavior, somatovegetative disorders, restoration of sleep-wake cycles, impaired learning and memory processes, a decrease in dystrophic and morphological changes in various structures of the brain.The main disadvantage of Mexidol is its relatively low effectiveness in cases of traumatic brain injury.

[0012] In recent years, native and synthetic apolipoproteins have attracted increasing attention. Apolipoproteins are structural components of serum lipoproteins that perform important transport and regulatory functions in the body. The advantage of their use as drugs is their natural origin, since they are not foreign to the body's immune system.

[0013] In the article by Kay AD et al., when studying patients with severe traumatic brain injury, a significant loss of apoE-containing lipoproteins from the cerebrospinal fluid and a significant increase in cholesterol were found, which made it possible to propose the concept of a close relationship between apoE and cholesterol metabolism in the context of acute brain injury and the need for treatment aimed at transporting lipids into the CSF for neuronal sprouting and synaptogenesis, however, the proposed concept was not tested by the authors in practice [Kay AD et al. Remodeling of cerebrospinal fluid lipoprotein particles after human traumatic brain injury / / J Neurotrauma. 2003. 20(8):717-723].

[0014] Recent studies have shown that apolipoprotein AI (ApoA-I), the main protein component of high-density lipoprotein particles, plays a critical role in maintaining the integrity of brain blood vessels and reducing the risk of Alzheimer's disease. When radioactively labeled ApoA-I is administered intravenously ( 125I-ApoA-1) wild-type rats were shown to have 125 1-ApoA-1 in various parts of the brain, indicating that it overcomes the blood-brain barrier, including through cholesterol-mediated endocytosis into transformed endothelial cells of human brain microvessels, transcytosis through the endothelium [Zhou AL et al. Apolipoprotein AI Crosses the Blood-Brain Barrier through Clathrin-Independent and Cholesterol-Mediated Endocytosis / / J. Pharmacol. Exp. Ther. 2019. 369:481-488].

[0015] In the review by Sengupta MB, Mukhopadhyay D. attention is drawn to the fact that the role of ApoA-I increases in the secondary phase of traumatic spinal cord injury. It is assumed that ApoA-I is increasingly expressed and secreted as a delayed response to neuronal damage, and this is a self-defense mechanism of the damaged system [Sengupta M.V., Mukhopadhyay D. Possible role of apolipoprotein Al in healing and cell death after neuronal injury / / Front Biosci (Elite Ed). 2016. 8(3):460-477]. However, the review does not consider the use of ApoA-I as a treatment for traumatic brain injury.

[0016] A more detailed study by Li J. et al. showed that after spinal cord injury, a large number of blood macrophages penetrate the lesion site and phagocytose myelin debris, turning into foamy macrophages, which leads to chronic inflammation. The drug D-4F, a peptidomimetic of apolipoprotein AI consisting of D-amino acids, previously known as an agent promoting lipid metabolism of foamy macrophages in atherosclerosis, was studied as a neuroprotective agent in spinal cord injury. It was found that D-4F can promote the removal of myelin debris, reduce the formation of foamy macrophages at the lesion site, and promote neuroprotection and restoration of motor function after spinal cord injury.It is hypothesized that these beneficial functions of D-4F may be related to its ability to enhance the expression of ATP-binding cassette transporter Al (ABCA1), the major transporter that mediates lipid efflux in foamy macrophages, since inhibition of ABCA1 activity can reverse the effect of D-4F in vitro. It is concluded that D-4F may be a promising candidate for the treatment of spinal cord injury by promoting the clearance of myelin debris by foamy macrophages via the ABCA1 pathway [Li J. et al. D-4F, an apolipoprotein AI mimetic, promotes the clearance of myelin debris and the reduction of foamy macrophages after spinal cord injury / / Bioengineered. 2022; 13(5): 11794-11809].A nanocomplex for targeted reparation of vascular-nerve damage is proposed, including a lipid, an apolipoprotein and a target peptide, which in diabetic encephalopathy is capable of predominantly purposefully binding to the cerebrovascular endothelium by binding the target peptide in order to restore vascular endothelial cells, promote the removal of Aβ plaques near blood vessels and restore components of cerebrovascular and neurovascular units, such as microglial cells, astrocytes and neurons. One or more apoproteins from the group of ApoE, ApoA-I, ApoA-II and ApoC-I can be included in the nanocomplex as apoproteins. The nanocomplex reduces cognitive impairment by restoring cerebrovascular functions and cerebral blood flow in diabetic encephalopathy and Alzheimer's disease.Its use in traumatic brain injury has not been studied [WO2021089053 Al NANO COMPLEX FOR TARGETED REPAIRING OF NEUROVASCULAR LESION, AND PREPARATION AND USE THEREOF]. The article by Yan_Ji-G. et al. shows the use of 4F, apolipoprotein AI mimetic, for the treatment and prevention of traumatic brain injury caused by vibration disease in rats exposed to vibration for 8 weeks. It was found that 4F has 5 main preventive effects: increased vasodilation and decreased endothelial damage; increased NO concentration in the blood and decreased superoxide to prevent neuronal damage; increased high-density lipoproteins (L11VP), which improves endothelial restoration; protection of myelin of nerve fibers, which may be associated with an increase in L11VP; reducing secondary inflammation, edema and intraneural pressure, thereby preventing neuronal atrophy and apoptosis [Yan Ji-G. et al.Cumulative Brain Injury from Motor Vehicle-Induced Whole-Body Vibration and Prevention by Human Apolipoprotein AI Molecule Mimetic (4F) Peptide (an Apo AI Mimetic) / / J Stroke Cerebrovasc Dis. 2015. 24(12): 2759-2773]. However, traumatic brain injury in vibration disease is a consequence of prolonged repeated exposure to vibration on the head. The use of 4F - apolipoprotein AI mimetic for the treatment of traumatic brain injury with a single exposure to a traumatic factor, for example, with contusion, has not been studied.

[0017] The technical problem of the present invention is to expand the arsenal of therapeutic means for the treatment of traumatic brain injury, which is a consequence of a single exposure to a traumatic factor (contusion).

[0018] The technical problem posed is solved by using human apolipoprotein AI or its functionally significant fragments for the treatment of traumatic brain injury resulting from a single exposure to a traumatic factor (contusion).

[0019] The technical result is an improvement in locomotor activity and cognitive functions, a reduction in morphological disturbances in the structure of the subject's brain tissue.

[0020] Terminology

[0021] Apolipoprotein AI (ApoA-1) is a native (natural) apolipoprotein of blood plasma, which is part of high-density lipoproteins (HDL).

[0022] A brain contusion, or brain injury, is an injury in the form of macrostructural destruction of brain matter, often with a hemorrhagic component, occurring at the moment of exposure to a traumatic agent. Contusions are classified as mild, moderate, and severe.

[0023] Recombinant ApoA-1 is a protein characterized by the amino acid sequence of natural ApoA-1, obtained by genetic engineering by constructing recombinant DNA, which provides, as part of a plasmid vector, the expression of a synthetic gene including the nucleotide sequence encoding human ApoA-1 in bacterial, mammalian or yeast cells, which can be used as producers of ApoA-1.

[0024] A therapeutically effective amount is a dose of the claimed agent, when administered to a subject, sufficient to obtain the claimed technical result aimed at treating a traumatic brain injury.

[0025] A pharmaceutically acceptable carrier means that the carrier must be compatible with the other ingredients of the composition and not harmful to its recipient, i.e., be non-toxic to cells or mammals at the doses and concentrations in which it is used.

[0026] A functionally significant fragment is a fragment of the complete amino acid sequence of human ApoA-1 (amino acid residues No. 1-243), which has functional autonomy and functional activity, ensuring, as part of the claimed agent, the achievement of the technical results claimed in the invention.

[0027] Traumatic brain injury (TBI) is a complex of contact injuries to the skull, brain matter and its membranes, blood vessels and / or cranial nerves, accompanied by clinical symptoms and, in most cases, morphological changes in the presence of a clear traumatic anamnesis, having a single mechanism and duration of formation. Disclosure of the essence of the invention

[0028] The invention relates to a means for treating traumatic brain injury, comprising human ApoA-1 (ApoA-1) or a functionally significant fragment. Human ApoA-1 has an amino acid sequence of 243 aa with a molecular weight of 28.5 kDa, SEQ ID NO 1 (amino acid residues 1-243)

[0029] In one embodiment, said agent comprises a functionally significant fragment of human ApoA-1.

[0030] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an N-terminally truncated amino acid sequence of human ApoA-1.

[0031] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-1 truncated at the N-terminus by 2 amino acid residues (amino acid residues 3-243).

[0032] In one embodiment, said agent is formulated as a pharmaceutical composition comprising human ApoA-1 or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

[0033] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0034] The above-mentioned agent is also intended for use in the treatment of craniocerebral trauma. The invention also relates to the use of the above-mentioned agent for the treatment of craniocerebral trauma.

[0035] In one embodiment, the above agent comprises a functionally significant fragment of human ApoA-1.

[0036] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an N-terminally truncated amino acid sequence of human ApoA-1.

[0037] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-1 truncated at the N-terminus by 2 amino acid residues (amino acid residues 3-243).

[0038] In one embodiment, said agent is formulated as a pharmaceutical composition comprising human ApoA-1 or a functionally significant fragment thereof and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is water for injection or a physiological solution.

[0039] The invention also relates to a method for treating traumatic brain injury.

[0040] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an N-terminally truncated amino acid sequence of human ApoA-1.

[0041] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-1 truncated at the N-terminus by 2 amino acid residues (amino acid residues 3-243).

[0042] In one embodiment, said agent is formulated as a pharmaceutical composition comprising human ApoA-1 or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

[0043] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline. In one embodiment, the administration of the above agent is carried out locally or parenterally.

[0044] In one embodiment, parenteral administration is performed intravenously or intramuscularly.

[0045] In one embodiment, local administration is performed subcutaneously.

[0046] The subject with signs of traumatic brain injury is a mammal, in particular a human.

[0047] Brief description of the drawings

[0048] Fig. 1. Morphological changes in the cerebral cortex of mice 5 weeks after modeling of traumatic brain injury. Magn. 200.

[0049] Note: a - disruption of cytoarchitecture, pronounced destruction of the external granular layer; b - normo- and hyperchromic neurons in the external granular layer; c - formation of large foci of neuronophagia, their displacement under the pia mater; d - predominance of hyperchromic neurons in the external granular and pyramidal layers, a, b, c - staining with hematoxylin and eosin; d - staining with toluidine blue.

[0050] Fig. 2. Morphological changes in the cerebral cortex of mice 5 weeks after modeling of traumatic brain injury and weekly administration of recombinant anoAl. Note: a - disintegration of the external granular layer; b - diffuse infiltration of the external granular layer by microglial cells; c - formation of a large focus of neuronophagy under the pia mater; d - large focus of neuronophagy. a, d - staining with toluidine blue; b, c - staining with hematoxylin and eosin, a, b, d - magnification 200; c - magnification 400.

[0051] Implementation of the invention

[0052] The invention relates to a means for treating traumatic brain injury and includes native or recombinant human ApoA-I with an amino acid sequence of 243 aa, a molecular weight of 28.5 kDa, or a functionally significant fragment thereof.

[0053] In one embodiment, said agent comprises a functionally significant fragment of human ApoA-I.

[0054] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus (SEQ ID NO: 1, amino acid residues 1-243, ApoA-1-1-243), which has functional autonomy and functional activity that ensures, as part of the claimed agent, the technical results claimed in the invention.

[0055] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-I, truncated at the N-terminus by 2 amino acid residues, with a size of 241 aa and a molecular weight of 28.3 kDa (SEQ ID NO: 2, amino acid residues 3-243, ApoA-1-3-243).

[0056] A functionally significant fragment of human ApoA-I can be obtained using genetic engineering methods by constructing recombinant DNA that provides, as part of a plasmid vector, expression in a bacterial or yeast or other cell of a synthetic gene that has in its composition a nucleotide sequence encoding a functionally significant fragment of human ApoA-I, with subsequent isolation of the recombinant protein synthesized in the cell, encoded by the nucleotide sequence of the synthetic gene, and obtaining on its basis a functionally significant fragment of human ApoA-I using methods known to a specialist, but is not limited to the said genetic engineering method.A functionally significant fragment of human ApoA-I, having the amino acid sequence of human ApoA-1-1-243, shortened from the N-terminus by 2 amino acid residues (rAnoA-I-3-243) was obtained by genetic engineering by cloning a synthetic gene, having in its composition the nucleotide sequence of the human ApoA-1-1-243 gene, in E. coli cells, with subsequent isolation of the target protein and its acid hydrolysis to obtain rApoA-1-3-243, as described in the patent according to Russian Federation Patent No. 2573930.

[0057] In one embodiment, the above agent is formulated as a pharmaceutical composition comprising human ApoA-1-1-243 or hApoA-1-1-243 or a functionally significant fragment thereof, and a pharmaceutically acceptable carrier.

[0058] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0059] In one embodiment, the pharmaceutical composition comprises human hApoA-1-3-243 and water for injection.

[0060] The invention also relates to the use of human ApoA-1-1-243 or human hApoA-1-1-243 or a functionally significant fragment thereof for the treatment of traumatic brain injury.

[0061] In one embodiment, said agent comprises a functionally significant fragment of human ApoA-1.

[0062] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-1 shortened at the N-terminus (SEQ ID NO: 1, amino acid residues 1-243, ApoA-1-1-243), which has functional autonomy and functional activity that ensures, as part of the claimed agent, the technical results claimed in the invention.

[0063] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-1 truncated at the N-terminus by 2 amino acid residues (SEQ ID NO: 2, amino acid residues 3-243, ApoA-1-3-243).

[0064] In one embodiment, the above agent is formulated as a pharmaceutical composition comprising human ApoA-1-1-243 or hApoA-1-1-243 or a functionally significant fragment thereof, and a pharmaceutically acceptable carrier.

[0065] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0066] In one embodiment, the pharmaceutical composition comprises human hApoA-1-3-243 and water for injection. The invention also relates to a method for treating traumatic brain injury.

[0067] In one embodiment, said agent comprises a functionally significant fragment of human ApoA-I.

[0068] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus (SEQ ID NO: 1, amino acid residues 1-243, ApoA-1-1-243), which has functional autonomy and functional activity that ensures, as part of the claimed agent, the technical results claimed in the invention.

[0069] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-I truncated at the N-terminus by 2 amino acid residues (SEQ ID NO: 2, amino acid residues 3-243, ApoA-1-3-243).

[0070] In one embodiment, the above agent is formulated as a pharmaceutical composition comprising human ApoA-1-1-243 or hApoA-1-1-243 or a functionally significant fragment thereof, and a pharmaceutically acceptable carrier.

[0071] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0072] In one embodiment, the pharmaceutical composition comprises human hApoA-1-3-243 and water for injection.

[0073] In one embodiment, the administration of the claimed agent is carried out locally or parenterally.

[0074] In one embodiment, parenteral administration is performed intravenously or intramuscularly.

[0075] In one embodiment, local administration is performed subcutaneously.

[0076] The duration of administration and the therapeutically effective amount of the claimed agent administered may vary widely depending on a variety of factors, including the severity of the traumatic brain injury, subject parameters, including size, weight, age, and other factors well known to those skilled in the art.

[0077] The subject with signs of traumatic brain injury is a mammal, in particular a human.

[0078] Native human ApoA-I is a component of high-density lipoproteins (HDL) in blood plasma. Native human ApoA-I SEQ ID NO 1 (amino acid residues 1-243, ApoA-1-1-243) was obtained by isolating HDL from human plasma by isodense ultracentrifugation in KBr solution [Mills GL et al. Guidebook to Lipoprotein Techniques. Edited by RH Burden and PH van Knippenberg. - Amsterdam: Elsevier, 2000. - 475 p.] followed by delipidation of HDL [Cham BE, Knowlee B. RJ Lipid. Res. - 1976. - Vol. 17, No. 2. P. 176-181] and purification [Pykhtina MB et al. Biopharmaceutical Journal. - 2012. - T. 4. - No. 6. - P. 37-45].

[0079] Recombinant human ApoA-I SEQ ID NO 1 (amino acid residues 1-243, rApoA-1-1-243) was obtained using genetic engineering methods by constructing recombinant DNA that provides, as part of a plasmid vector, the expression of a synthetic gene comprising a nucleotide sequence encoding chimeric human ApoA-I in E. coli cells, for example, according to Russian Patent No. 2573930, or in methylotrophic yeast cells Pichia pasloris, for example, according to Russian Patent No. 2605326], followed by obtaining mature human rApoA-1-1-243 by enzymatic hydrolysis of the corresponding chimeric protein, but is not limited to the methods indicated.

[0080] A functionally significant fragment of human ApoA-I can be obtained using genetic engineering methods by constructing recombinant DNA that provides, as part of a plasmid vector, expression in a bacterial or yeast or other cell of a synthetic gene that has in its composition a nucleotide sequence encoding a functionally significant fragment of human ApoA-I, with subsequent isolation of the recombinant protein synthesized in the cell, encoded by the nucleotide sequence of the synthetic gene, and obtaining on its basis a functionally significant fragment of human ApoA-I using methods known to a specialist, but not limited to the said genetic engineering method.

[0081] A functionally significant fragment of human ApoA-I, having the amino acid sequence of human ApoA-1-1-243, shortened from the N-terminus by 2 amino acid residues (gApoA-1-3-243) was obtained by genetic engineering by cloning a synthetic gene, having in its composition the nucleotide sequence of the human ApoA-1-1-243 gene, in E. coli cells, with subsequent isolation of the target protein and its acid hydrolysis to obtain gApoA-1-3-243, as described in the patent according to Russian Federation Patent No. 2573930. Examples.

[0082] All examples of the invention are carried out using a functionally significant fragment of human ApoA-I, having the amino acid sequence of human ApoA-1-1-243, shortened from the N-terminus by 2 amino acid residues (rApoA-1-3-243).

[0083] Example 1. Obtaining a functionally significant fragment of human ApoA-I gApoA-1-3-243

[0084] A functionally significant fragment of human ApoA-I corresponding to amino acids 3-243 of human ApoA-I (SEQ ID NO 2) (hereinafter referred to as "rApoA-1-3-243") was obtained by genetic engineering by cloning recombinant DNA in a plasmid vector comprising a nucleotide sequence encoding chimeric ApoA-I and providing its synthesis in E. coli cells; subsequent isolation and purification of chimeric ApoA-I. To obtain rApoA-1-3-243, the chimeric ApoA-1 protein was subjected to acid hydrolysis as described in Russian Patent No. 2573930. In particular, the nucleotide sequence of the recombinant DNA includes, from the 5'-end, an ATG codon encoding methionine, then (1) a DNA fragment encoding 6 amino acids of histidine; (2) a DNA fragment encoding 6 aa of the pro-form of human apoA-I protein; (3) a DNA fragment encoding the N-terminal 15 aa fragment of bovine pancreatic RNase A; (2) a DNA fragment encoding 6 aapro-forms of human apoA-I protein; (4) DNA fragment encoding tobacco mosaic virus protease recognition site; (5) mutant gene of mature human apoA-1 protein-1-243. The chimeric ApoA-I protein corresponds to the amino acid sequence encoded by all consecutively indicated nucleotide sequences of recombinant DNA, with sequence (2) being presented twice, the second time after sequence (3).

[0085] The application was carried out in the form of a pharmaceutical composition comprising a functionally significant fragment of human ApoA-I rApoA-1-3-243 and a pharmaceutically acceptable solvent, in which water for injection was used as the pharmaceutically acceptable solvent.

[0086] The administration was performed subcutaneously.

[0087] Example 2. Description of experiments

[0088] Description of the mouse strain used for the experimental implementation of the claimed invention A comparative study of the effectiveness of the use of a functionally significant fragment of human ApoA-I gApoA-1-3-243 was carried out on outbred (non-linear) ICR laboratory mice, which are the most frequently used research objects in the field of oncology, toxicology, teratology, in the development of vaccines, the study of aging and for general purposes.

[0089] Mice weighing 27-30 grams and aged four months were selected for the experiment. The animals were fed regular food with free access to water.

[0090] Four groups of experimental animals were formed:

[0091] K(-) - control group of intact healthy ICR mice, 10 individuals);

[0092] K(+) - control group of mice with traumatic brain injury (10 individuals);

[0093] Experimental group 1 - an experimental group of mice with traumatic brain injury, which were injected with gApoA-1-3-243 once a week for three weeks subcutaneously in the withers area (10 individuals).

[0094] Experimental group 2 is a comparison group of mice with traumatic brain injury, which were injected subcutaneously with Mexidol once a week for three weeks (10 individuals).

[0095] Experimental group 1 was administered hApoA-1-3-243 at a dose of 7 mg / kg body weight, while hApoA-1-3-243 was administered as part of a pharmaceutical composition in which water for injection was used as a pharmaceutically acceptable carrier, at a concentration of hApoA-1-3-243 in solution of 1.2 mg / ml.

[0096] Experimental group 2 was administered Mexidol intravenously at a dose of 7 mg / kg body weight.

[0097] Description of the model of traumatic brain injury

[0098] To conduct comparative studies, a mild traumatic brain injury (TBI) was modeled. For this, the Marmarou model was used [Marmarou A. A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics / / J. neurosurgery, 1994, vol. 80, No. 2, pp. 291-300] in a modification for mice by MJ Kapea et al. [Kanea M. A mouse model of human repetitive mild traumatic brain injury / / J. Neuroscience Methods, 2012, vol. 203, Is. 1, pp. 41-49]. To reproduce mild TBI in mice weighing 27-30 g, a load with a diameter of 20 mm and a weight of 100 g was used at a drop height of 2.0 meters after a blow to the head into a tank 23 cm high, onto a foam cushion for cushioning. To eliminate resistance during impact, the surface of the tank was covered with aluminum foil so that it could withstand the weight of the mouse. After a blow to the head, the foil easily breaks, and the animal falls down with acceleration.The acceleration and fall induced by the impact always involve a 180° horizontal rotation of the mouse body. The method used models the most common form of traumatic brain injury in humans. Existing animal models of TBI produce focal, severe injuries, unlike those observed with repeated and mild concussions, and few are suitable for repeated use. A key aspect of the model used in this study is that the impact to the skull of an unsecured mouse produces rapid acceleration of the freely moving head and trunk, an important characteristic known to occur in human concussion and a factor that is missing from other existing animal models of TBI. The method does not require scalp incisions, cranial helmets, or surgery, and the procedure can be completed in 1–2 minutes. The mice show no signs of seizures, paralysis, or behavioral abnormalities.Skull fractures and intracranial hemorrhage are very rare. Minor motor incoordination and motor hyperactivity resolve over time. Histological analysis reveals mild astrocytic reactivity, no blood-brain barrier disruption, no edema, and no microglial activation. This new animal model is simple and economical, and facilitates characterization of the neurobiological and behavioral sequelae of TBI. The model is ideal for high-throughput screening of potential new treatments for mild concussions experienced by athletes and military personnel. [Kanea M. A mouse model of human repetitive mild traumatic brain injury / / J. Neuroscience Methods, 2012, vol. 203, Is. 1, pp. 41-49].

[0099] Example 3. Results of a study of locomotor activity and cognitive function in mice with traumatic brain injury

[0100] Table 1 shows the effect of hApoA-1-3-243 and mexidol on the indices of locomotor activity and cognitive function in the compared groups of mice 3 weeks after TBI.

[0101] As can be seen from Table 1, the use of gApoA-1-3-243 provides a pronounced therapeutic effect in the increasing consequences of traumatic brain injury even with a double subcutaneous administration at a dose of 7 mg / kg of body weight. This mainly concerns the restoration of impaired locomotor functions, vestibular apparatus disorders and cognitive function (center visits, grooming, wall stands, looking into a hole). According to these indicators, mexidol is significantly inferior to the declared drug and in an equivalent dose has virtually no therapeutic effect. The dose of gApoA-1-3-243 7 mg / kg of body weight is experimentally selected and optimal for the correction of the consequences of traumatic brain injury in an in vivo experiment, at doses below 7 mg / kg of body weight the therapeutic efficacy of the declared drug decreases, and at doses above 7 mg / kg of body weight it increases only slightly.

[0102] Table 1. Effect of hApoA-1-3-243 and mexidol on locomotor activity and cognitive function in the compared groups of mice

[0103] Example 4. Morphological changes in the cerebral cortex of mice with traumatic brain injury

[0104] Experimental groups: 1st - modeling of traumatic brain injury, subcutaneous administration once a week for five weeks of physiological solution (K+); 2nd - modeling of traumatic brain injury, subcutaneous administration once a week for five weeks of gApoA-1-3-243 at a dose of 7 mg / kg in the form of an aqueous solution (water for injection).

[0105] A light-optical study and quantitative assessment of normo- and hyperchromic neurons in the molecular, external granular and pyramidal layers of the cerebral cortex (in a test area of ​​239972.87 µm) were carried out. 2 ). The sample size was at least 1000 cells in non-overlapping fields of view (at least 5 fields of view).

[0106] Histological examination of the preparation of the resected section of the cortex after its fixation with neutral formalin was performed using the standard method with staining with hematoxylin and eosin staining with hematoxylin and toluidine blue. For electron microscopic examination, pieces of tissue from the resected section of the cortex measuring 4–5 mm 3 The samples were pre-fixed in Karnovsky's solution and then in a 1% solution of osmium tetroxide in 0.2 M cacodylate buffer. Dehydration with alcohol and acetone, as well as subsequent embedding in araldite, were carried out according to the standard method. Ultrathin sections were obtained on LKB III and Leica ULTRACUT EM UC7 ultratomes (Leica, Germany), contrasted with uranyl acetate and lead citrate. The study was carried out in JEM-100B and JEM-1400 electron microscopes (Teo1, Japan).

[0107] Morphological changes in the cerebral cortex in the lesion focus 5 weeks after traumatic brain injury and weekly subcutaneous administration of saline were characterized by pronounced cytoarchitectonic disorders, manifested in the degradation of axons and dendrites of neurons and total disintegration of interneuronal associations (Fig. 1, a). In the external granular layer, neurons are reduced in size, polymorphic, and very closely grouped (Fig. 1, b). Hyperchromic cells are located around normochromic neurons, among which microglial cells are present (Fig. 1, c). In the pyramidal layer, neurons are predominantly hyperchromic (shadow cells) with pronounced pericellular edema (Fig. 1, d), scattered throughout the layer; rounded voids at the site of dead neurons are common.

[0108] A distinctive feature of the reorganization of the cerebral cortex in this model of traumatic brain injury was the formation of large foci of neuronophagy (the area of ​​the foci varied from 3400 to 21500 µm 2 ), which were located mainly under the pia mater (see Fig. 1, c). Such foci consisted mainly of microglial cells; in the center of some foci, single neurons were encountered. Extensive acellular zones were located around the foci of neuronophagy. Large-focal neuronophagy in traumatic brain injury may represent one of the mechanisms of resorption of irreversibly damaged neurons with the “pushing out” of mononuclear infiltrates.

[0109] Capillaries and small arteries are few in number and unevenly congested. Pronounced perivascular / pericapillary edema is noted. The general nature of structural changes in the cerebral cortex in the lesion focus 5 weeks after traumatic brain injury and weekly subcutaneous administration of gApoA-1-3-243 was the same as in the previous group, but differed in severity. Disorganization of the outer granular and pyramidal layers of the cortex was noted, which consisted primarily in pronounced degradation of fibrous structures, disintegration of neurons and their chaotic distribution within the layers (Fig. 2, a). In the outer granular layer, neuronophagy processes intensified (Fig. 2, b); microglial cells intensively (diffusely) infiltrated this layer, formed clusters around neurons, and replaced them over large areas. Just as in the previous group, large foci of neuronophagy were formed, which moved under the pia mater (Fig. 2, c, d).In some preparations, 2 to 4 such foci were present simultaneously. In general, it can be said that the processes of resorption of irreversibly damaged neurons in this group occurred more intensively (judging by the large clusters of microglial cells) than in the group without the use of gApoA-1-3-243. This process indicates the beginning of organization, which consists in the formation of newly formed capillaries along the periphery of the damage zone, the transformation of monocytes and microgliocytes into "granular balls" cells filled with myelin decay products is actively launched. Thus, gApoA-1-3-243 reduces the likelihood of massive necrosis, which in turn contributes to progressive cerebral edema and irreversible damage to the functions of the brain.

[0110] According to the quantitative analysis of the degree of cytoplasmic chromatophilia in the gApoA-1-3-243 group, a predominance (by 63.4%, p<0.001) of the proportion of hyperchromic neurons (mainly localized in the pyramidal layer) over normochromic ones located mainly in the external granular layer was established (Table 2). In the pyramidal layer, pronounced degenerative changes in pyramidal neurons were observed, accompanied by pericellular edema. However, the proportion of hyperchromic neurons in mice of the group was 37.3% less (p<0.001) compared to mice that were daily injected subcutaneously with saline (Table 2). This indicator may also indicate a more intensive elimination of irreversibly damaged neurons from the lesion. Such elimination of damaged cells occurs through apoptosis, or programmed cell death. Thus, hApoA-1-3-243 has a neuroprotective effect, since it promotes the death of neurons by apoptosis, rather than necrosis.Despite this, changes in cytoarchitecture persisted, particularly noticeable in the outer granular layer, in which neurons formed scattered clusters in the form of chains and small groups of cells.

[0111] Table 2. Estimation of the number of neurons with different degrees of cytoplasmic chromatophilia

[0112] No significant hemodynamic disturbances were detected in the lesion during this period of the experiment, but uneven capillary congestion and pericapillary edema should be noted.

[0113] Conclusion. Qualitative and quantitative analysis of three layers of the cerebral cortex of mice after traumatic brain injury and corrective treatment with recombinant hApoA-1-3-243 (at a dose of 7 mg / kg) indicates that with the selected method of brain damage, cytoarchitectonic disorders and disintegration of interneuronal associations persist for 5 weeks. The most pronounced structural changes according to such criteria as cytoarchitectonic disorders and the number of hyperchromic neurons were found in the group of animals in which no correction methods were used. Structural changes in the brain in mice receiving recombinant hApoA-1-3-243 as a therapeutic effect had the most pronounced neuronophagy processes, which can be considered as one of the mechanisms of intensive elimination of irreversibly damaged neurons. hApoA-1-3-243 reduces the likelihood of massive necrosis and has a neuroprotective effect.

[0114] The work was carried out using the equipment of the FRC FTM Center for Collective Use “Proteomic Analysis”, supported by funding from the Ministry of Education and Science of Russia (agreement No. 075-15-2021-691) and the FRC FTM Center for Collective Use “Spectrometric Measurements”.

Claims

CLAUSES OF THE INVENTION 1. An agent for the treatment of traumatic brain injury, including human apolipoprotein AI (ApoA-I) or a functionally significant fragment thereof.

2. An agent according to claim 1, which includes a functionally significant fragment of human ApoA-I.

3. The agent according to claim 2, wherein the functionally significant fragment of human ApoA-I has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus.

4. The agent according to claim 3, wherein the functionally significant fragment of human ApoA-I has a sequence that is the amino acid sequence of human ApoA-I, shortened from the N-terminus by 2 amino acid residues (amino acid residues 3-243).

5. The agent according to any one of claims 1-4, formulated in the form of a pharmaceutical composition comprising human ApoA-I or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

6. The agent according to claim 5, wherein the pharmaceutically acceptable carrier is water for injection or saline solution.

7. Use of human apolipoprotein AI or its functionally significant fragments as a treatment for traumatic brain injury.

8. The use according to claim 7, wherein said agent comprises native apolipoprotein AI obtained from human blood serum.

9. The use according to claim 7, wherein said agent comprises recombinant human apolipoprotein AI.

10. The use according to claim 7, wherein the functionally significant fragment of human ApoA-I has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus.

11. The use according to claim 10, wherein the functionally significant fragment of human ApoA-I has a sequence that is the amino acid sequence of human ApoA-I shortened from the N-terminus by 2 amino acid residues (amino acid residues 3-243).

12. Use according to any one of paragraphs 7-11, wherein said agent is formulated as a pharmaceutical composition comprising human apolipoprotein AI or a functionally significant fragment thereof and a pharmaceutically acceptable carrier. 23 13. Use according to item 12, wherein the pharmaceutically acceptable carrier is water for injection or a saline solution.

14. A method for treating a traumatic brain injury, comprising administering to a subject in need thereof a means according to any of paragraphs 1-6.

15. The method according to 14, wherein said agent comprises a functionally significant fragment of human ApoA-1.

16. The method according to claim 15, wherein the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-1 shortened at the N-terminus.

17. The method according to claim 16, wherein the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-1 shortened from the N-terminus by 2 amino acid residues (amino acid residues 3-243).

18. The method according to any one of claims 14-17, wherein said agent is formulated as a pharmaceutical composition comprising human ApoA-1 or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

19. The method according to claim 18, wherein the pharmaceutically acceptable carrier is water for injection or saline solution.

20. The method according to any one of paragraphs 14-19, wherein the administration is carried out locally or parenterally.

21. The method according to item 20, wherein parenteral administration is carried out intravenously or intramuscularly.

22. The method according to item 20, wherein local administration is carried out subcutaneously.

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