Medicine for treating craniocerebral injury and application thereof

Abstract

The invention relates to the field of medicine, in particular to experimental pharmacology, and can be used for preparing a pharmaceutical composition and a dosage form for treating craniocerebral injury. In order to expand the reserve of treatment means for craniocerebral injury, the invention provides application of human apolipoprotein A-I or functional important fragments thereof, and the means comprise natural apolipoprotein A-I (extracted from human serum) extracted from human serum, and functional fragments of recombinant human apolipoprotein A-I or human ApoA-I. The functional fragment ApoA-I has a human ApoA-I amino acid sequence truncated from an N terminal, and specifically, the human ApoA-I amino acid sequence has two amino acid residues (amino acid residues 3-243) truncated from the N terminal. The preparation is prepared in the form of a pharmaceutical composition, and comprises human serum lipoprotein AI or a functional fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is water for injection or normal saline. A method of treating craniocerebral injury comprises administering the formulation to a patient in need of treatment in a therapeutically effective dose in a topical or parenteral administration manner.

Description

Technical Field

[0001] This invention relates to the medical field, particularly experimental pharmacology, and can be used to develop pharmaceutical compositions and dosage forms for the treatment of traumatic brain injury. Background Technology

[0002] Currently, the treatment of traumatic brain injury (TBI) is an extremely urgent issue in modern medicine. This is because pharmacological methods for treating this type of injury are very limited, especially considering its pathophysiological characteristics. In Russia, the incidence of TBI is approximately 4 cases per 1000 people, meaning 400,000 people are injured annually. About 10% of these injuries result in death, and another similar proportion result in disability. According to various authors, TBI is the most common cause of death and severe disability in people under 35 years of age. Furthermore, men are 2-3 times more likely to suffer TBI than women. The most common cause of TBI is road traffic accidents. The second most common cause is falls (accounting for 20% to 30%), especially among young people and the elderly. The highest risk of TBI is found in people aged 15 to 24 and those over 70 years of age, with TBI in those over 70 primarily related to falls, occupational injuries, sports injuries, and everyday life injuries.

[0003] In the pathogenesis of traumatic brain injury, disruptions in the main neurodynamic processes of the central nervous system play a dominant role, leading to abnormalities in vascular, cerebrospinal fluid dynamics, and endocrine-humoral aspects. The cerebrovascular system's response manifests as widespread vasospasm, followed by cerebral congestion and venous stasis. Cerebrospinal fluid circulation disorders are associated with the development of cerebrospinal fluid hypertension and abnormal blood-brain barrier permeability. Weakened hypothalamic-pituitary regulatory function leads to hormonal imbalances, water-electrolyte disturbances, and abnormal blood circulation, subsequently causing cerebral hypoxia and cerebral edema.

[0004] Currently, in military conflicts, concussion injuries from landmine explosions are a major type of injury, and traumatic brain injury is also a significant one. Clearly, all types of traumatic brain injury have their own unique characteristics depending on their severity, but from a medical treatment perspective, they are remarkably similar due to the very limited availability of medications.

[0005] Pharmacological drugs used to treat traumatic brain injury mainly include diuretics, calcium channel blockers (nifedipine), vasodilators and antihypertensive drugs (cinnarizine, vinpocetine, and angiotensin-converting enzyme inhibitors and their angiogenic receptor inhibitors), sedatives (diazepam, escitalopram), nootropics (piracetam, fenracetam), nonsteroidal anti-inflammatory drugs (ketoprofen), and steroidal anti-inflammatory drugs (dexamethasone). Regardless of the severity of brain injury, the main goal of drug treatment for traumatic brain injury is to reduce intracranial pressure, restore cerebral hemodynamics, and suppress the inflammatory response to eliminate cerebral hypoxia.

[0006] Patent RU 2477150 C2 (published March 10, 2013, Publication No. 7) describes a treatment method for patients with traumatic brain injury accompanied by hypertension syndrome in the acute phase. The procedure is as follows: blood is collected from the patient, and 50 ml of red blood cells are separated. These red blood cells are then saturated with 1 ml of dexamethasone and subsequently reinfused into the patient's circulatory system. This procedure is performed once daily, with an interval of 2-3 days, for a total of 5 treatments. This method utilizes the saturation effect of the patient's own red blood cells to achieve more complete drug delivery, thereby helping to shorten the treatment cycle for such patients. However, the disadvantages of this method are its complexity and the significant side effects associated with the use of dexamethasone.

[0007] Patent RU 2309745 C2 (published November 10, 2007, Publication No. 31) discloses a drug for treating traumatic brain injury (TBI) with comprehensive anti-ischemic, anti-hypoxic, and anticonvulsant effects. Studies have shown that zopiclone, as this drug, is a non-benzodiazepine benzodiazepine receptor agonist. Research indicates that in the first three days after trauma, zopiclone improves the tolerance of post-traumatic brain tissue to ischemia, hypoxia, hypercapnia, and seizures by 24.9%–42.6% compared to the vasodilator nimodipine or the antihypertensive drug sodium oxybutyrate. The main drawbacks of zopiclone are its sedation-related side effects and the potential for drug dependence.

[0008] Erythropoietin is used to treat concomitant traumatic brain injury (TBI) to compensate for post-traumatic anemia. For example, patent RU2446834 C2 (published April 10, 2012, Publication No. 10) describes a treatment method for patients with severe concomitant TBI. In this method, in addition to standard intensive treatment, patients receive intravenous administration of the growth factor (erythropoietin). On the first day after trauma, a single dose of 10,000 IU of erythropoietin is administered; on the second day, 10,000-20,000 IU of erythropoietin is administered, depending on the severity of the trauma and the risk of death; on the third and fourth days, 10,000 IU of erythropoietin is administered; and on the fifth day, 5,000-10,000 IU of erythropoietin is administered. This erythropoietin dosing regimen incorporated into the intensive treatment of patients with severe concomitant TBI improves treatment efficacy and reduces mortality and the incidence of complications. However, the main drawback of this method is its narrow pathophysiological focus. Specifically, it only aims to compensate for post-traumatic anemia and has no effect on brain inflammation and neurodegenerative processes that occur after traumatic brain injury. Therefore, its efficacy is insufficient to treat traumatic brain injury and can only meet the need for compensating for post-traumatic anemia.

[0009] In patent RU 2406506 C1 (published December 20, 2010, Publication No. 35), this invention provides a pharmaceutical composition of α-glycerophosphate choline (such as "choline ester" related preparations) in injectable solution form, having nootropic and cholinergic activity, and a method for treating / preventing central nervous system diseases and sequelae of traumatic brain injury. This pharmaceutical composition contains a therapeutically effective amount of α-glycerophosphate choline as the active ingredient, using povidone (plasdone or collidone) as a pharmaceutically acceptable carrier, with the povidone content accounting for 0.2%-0.8% of the total mass of the composition. The pharmaceutical composition of this invention is used for treating or preventing central nervous system diseases and sequelae of traumatic brain injury. The main disadvantage of this pharmaceutical composition is that its side effects are primarily related to cholinergic activity, such as nausea, abdominal pain, and persistent confusion.

[0010] Patent RU 2554502 C9 (published June 27, 2015, Publication No. 18) describes the efficacy data of quinine cyclo-(2-adamantyl)-hexamethyleneimine hydrochloride (i.e., "amantadine") in treating cerebral circulatory disorders and brain injury. Studies have confirmed that in a rat model of intracerebral hematoma, administration of amantadine 3.5 hours after model establishment, followed by continuous administration for 4 days, reduced animal mortality and improved cognitive and motor function indicators. This indicates that amantadine has neuroprotective potential in a post-traumatic brain hematoma model. The technical effect lies in achieving the claimed use: studies have shown that intravenous injection of amantadine at a dose of 5 mg / kg increases cerebral blood flow to the cerebral cortex in rats with ischemic brain injury without altering arterial blood pressure levels. The drug has no significant effect on cerebral blood supply in healthy animals and does not cause changes in heart rate or respiratory rate. All adamantane derivatives have a number of side effects, the most common of which are: insomnia, psychomotor agitation, paranoid exogenous psychosis with visual hallucinations (in susceptible elderly patients), dizziness, and decreased vision.

[0011] Patent RU 2414901 C2 (published March 27, 2011, Publication No. 9) describes a drug with neuroprotective effects in the acute phase of traumatic brain injury. This patent proposes the use of the known anticonvulsant lamotrigine (other names for this substance include levofloxacin, etc.) as a drug to prevent ischemic and hypoxic disorders in the acute phase of traumatic brain injury. Studies have shown that lamotrigine can prevent secondary brain dysfunction caused by ischemia, hypoxia accompanied by hypercapnia, and hemorrhagic hypoxia, while also helping to alleviate the recovery process of neuropsychiatric status. Lamotrigine can increase the brain's tolerance to major secondary injury factors by 24.9%-42.6% and prolong the survival time of mice after trauma by 109.6%. However, the disadvantage of this drug is that it can produce many side effects on the central nervous system, liver, and hematopoietic system. Patent RU 2318501 C2 (published March 10, 2008, Publication No. 7) proposes a drug belonging to the same pharmacological group as the aforementioned drug. This patent suggests the use of Depakine (sodium valproate), a drug with multifactorial neuroprotective and cognitive-enhancing effects in cases of traumatic brain injury. Studies have shown for the first time that, in cases of severe post-traumatic seizures, administration of sodium valproate starting on the second day after trauma exhibits a high anticonvulsant protective index during the post-traumatic period. Treatment with this drug, according to the prescribed course, not only restores the learning speed of traumatized animals to normal levels but also promotes the acquisition of behavioral adaptive responses, including delayed re-enactment adaptive responses (a method for assessing memory function). The main drawbacks of this drug are also its numerous side effects on the central nervous system and hematopoietic system, as well as its hepatotoxicity.

[0012] Patent RU 2766785 C1 (published March 15, 2022, Publication No. 8) describes a method for correcting the pathology of traumatic brain injury using 2-amino-5-ethyl-1,3,4-thiadiazole-N-acetaminophen. This invention belongs to the medical field, specifically relating to experimental pharmacology: the method for correcting neurological deficits caused by traumatic brain injury includes establishing a craniocerebral injury model in rats by free fall of a 155-gram weight from a height of 0.6 meters. To correct the pathological state, 2-amino-5-ethyl-1,3,4-thiadiazole-N-acetaminophen is administered to rats via gavage for 3 days at a dose of 50 mg / kg once daily. The first dose is given 30 minutes before the simulated pathological condition, and administration continues for the next 2 days. 2-Amino-5-ethyl-1,3,4-thiadiazole-N-acetaminophen, administered before and after traumatic brain injury at a dose of 50 mg / kg, can alleviate neurological symptoms. Similar efficacy data for 2-amino-5-ethyl-1,3,4-thiadiazole-N-acetaminophen are also documented in the following patents: RU 2756325 C1 (published September 29, 2021, Publication No. 8), RU 2763012 C1 (published December 24, 2021, Publication No. 36), and RU2758433 C1 (published December 28, 2021, Publication No. 31). These patents suggest that the compound possesses antioxidant and anti-hypoxic activities, capable of blocking the activation of free radical processes and the peroxidation of cell membrane lipids, making it particularly suitable for acute regional and systemic circulatory disturbances.

[0013] In patent RU 2252023 C2 (prototype) (published May 20, 2005, Publication No. 14), a treatment method for traumatic brain injury was proposed: immediate intravenous infusion of 2-ethyl-6-methyl-3-hydroxypyridine succinate (Mexidol) after trauma, 200 mg twice daily for severe injuries, and 200 mg once daily for moderate to mild injuries. This method can achieve a detoxification effect in the acute phase of traumatic brain injury, thanks to the newly discovered antitoxic effect of the drug—accelerating the decomposition and excretion of toxic metabolites in the cerebrospinal fluid. Mexidol is an antioxidant with anti-hypoxia, membrane protection, nootropic, anticonvulsant, and anti-anxiety effects. It is also a free radical process inhibitor, and as a membrane protectant, it possesses anti-hypoxia, anti-stress, nootropic, anticonvulsant, and anti-anxiety effects. This drug enhances the body's resistance to various damaging factors (shock, hypoxia and ischemia, cerebral circulatory disorders, alcohol poisoning, and antipsychotic / neuroblocker poisoning). Mexidol®'s mechanism of action is based on its antioxidant, anti-hypoxic, and membrane-protective effects. It inhibits lipid peroxidation, increases superoxide dismutase activity, improves the lipid-to-protein ratio, reduces cell membrane viscosity, and increases its fluidity. Mexidol regulates the activity of membrane-bound enzymes (calcium-independent phosphodiesterase, adenylate cyclase, acetylcholinesterase) and receptor complexes (benzodiazepines, gamma-aminobutyric acid (GABA), acetylcholine), enhancing their binding affinity to ligands. This helps maintain the structural and functional integrity of biological membranes, neurotransmitter transport, and improves synaptic transmission. Mexidol can increase dopamine levels in the brain. Under hypoxic conditions, it enhances the activation of compensatory aerobic glycolysis, reduces the inhibition of oxidation processes in the tricarboxylic acid cycle, increases the levels of adenosine triphosphate (ATP) and creatine phosphate, activates mitochondrial energy synthesis, and stabilizes cell membranes. This drug can improve brain metabolism and blood supply, improve microcirculation and blood rheology, and reduce platelet aggregation. During hemolysis, it can stabilize blood cell membrane structures (red blood cells and platelets). It has a lipid-lowering effect, reducing the levels of total cholesterol and low-density lipoprotein (LDL). Its anti-stress effects are manifested in: restoring post-stress behavior and autonomic nervous system dysfunction to normal, restoring damaged sleep-wake cycles, learning and memory processes, and reducing malnutrition and morphological changes in various brain structures. The main drawback of Mexidol is its relatively low efficacy in treating traumatic brain injury.

[0014] In recent years, both natural and synthetic apolipoproteins have attracted increasing attention. Apolipoproteins are structural components of serum lipoproteins and play important transport and regulatory roles in the body. Their advantage as pharmaceuticals lies in their natural origin, as they are not foreign substances to the body's immune system.

[0015] In an article by Kay AD et al., a study of patients with severe traumatic brain injury found a significant loss of apolipoprotein E (apoE)-containing lipoproteins in the cerebrospinal fluid, along with a significant increase in cholesterol. This led researchers to propose that apolipoprotein E is closely linked to cholesterol metabolism in the context of acute brain injury, and that treatments targeting lipid transport in the central nervous system (CNS) are necessary to promote neuronal regeneration and synapse formation. However, the researchers have not yet verified this proposal in practice [Kay AD et al. Remodeling of cerebrospinal fluidlipoprotein particles after human traumatic brain injury / / J Neurotrauma.2003. 20(8):717-723].

[0016] Recent studies have shown that apolipoprotein AI (ApoA-I), as a major protein component of high-density lipoprotein particles, plays a crucial role in maintaining cerebral vascular integrity and reducing the risk of Alzheimer's disease. Intravenous injection of radioactively labeled apolipoprotein AI (¹²) into wild-type rats... 5 Following I-ApoA-I), the study found¹² 5 I-ApoA-I is present in multiple sites of the rat brain, indicating that this protein can cross the blood-brain barrier. This includes entry into human brain vascular endothelial cells (after transformation) via cholesterol-mediated endocytosis, and transcytosis within endothelial cells. [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].

[0017] In a review by Sengupta MB and Mukhopadhyay D., researchers noted that apolipoprotein AI (ApoA-I) plays an enhanced role in the secondary phase of spinal cord injury. It is speculated that the expression and secretion of ApoA-I are significantly increased as a delayed response to neuronal damage, reflecting a self-protective mechanism of the damaged system. [Sengupta M.V., Mukhopadhyay D. Possible role of apolipoprotein AI in healing and cell death after neuronal injury / / Front Biosci (Elite Ed). 2016. 8(3):460-477]. However, this review did not explore the possibility of using ApoA-I as a treatment for traumatic brain injury.

[0018] A more detailed study by Li J. et al. showed that after spinal cord injury, a large number of hematogenous macrophages invade the injury site and engulf myelin sheath fragments, transforming into foam macrophages, which in turn leads to chronic inflammation. D-4F is an apolipoprotein AI mimic peptide composed of D-amino acids, previously known to promote lipid metabolism in foam macrophages in atherosclerosis. Researchers studied it as a neuroprotective agent after spinal cord injury and found that D-4F can promote the clearance of myelin sheath fragments, reduce the formation of foam macrophages at the injury site, and contribute to neuroprotection and motor function recovery after spinal cord injury. The study speculates that these beneficial effects of D-4F may be related to its ability to enhance the expression of ATP-binding cassette transporter A1 (ABCA1)—ABCA1 is the main transporter mediating lipid efflux from foam macrophages, because in in vitro experiments, inhibiting ABCA1 activity reversed the effects of D-4F. The study concludes that D-4F may be a promising candidate drug for the treatment of spinal cord injury, as it can promote the clearance of myelin sheath fragments by foam macrophages through the ABCA1 pathway. [Li J. etal. D-4F, an apolipoprotein AI mimetic, promotes the clearance of myelindebris and the reduction of foamy macrophages after spinal cord injury / / Bioengineered.2022; 13(5): 11794-11809].

[0019] A nanocomposite for targeted repair of vascular-neural injury is proposed, comprising lipids, apolipoproteins, and targeting peptides. In diabetic encephalopathy, this nanocomposite can preferentially bind to cerebral vascular endothelium through the binding of the targeting peptides, thereby repairing vascular endothelial cells, promoting the clearance of amyloid (Aβ) plaques near blood vessels, and repairing components of cerebral vascular and neurovascular units (such as microglia, astrocytes, and neurons). The nanocomposite may contain one or more of the following apolipoprotein groups: ApoE, ApoA-I, ApoA-II, and ApoC-I. This nanocomposite alleviates cognitive impairment by restoring cerebral vascular function and cerebral blood flow in diabetic encephalopathy and Alzheimer's disease. However, its application in traumatic brain injury has not been investigated. [WO 2021089053 A1 NANO COMPLEX FORTARGETED REPAIRING OF NEUROVASCULAR LESION, AND PREPARATION AND USE THEREOF].

[0020] In an article by Yan Ji-G. et al., the application of apolipoprotein AI mimic peptide 4F in the treatment and prevention of vibration-induced traumatic brain injury was investigated in rats exposed to vibration for 8 weeks. The study found that 4F has five main preventive effects: promoting vasodilation and reducing endothelial damage; increasing blood nitric oxide (NO) concentration and reducing superoxide levels, thereby preventing neuronal damage; increasing high-density lipoprotein (HDL) levels and improving endothelial repair; protecting the myelin sheath of nerve fibers (which may be related to increased HDL); and 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 A-IMolecule Mimetic (4F) Peptide (an Apo AI Mimetic) / / J Stroke CerebrovascDis. 2015. 24(12):2759-2773]. However, traumatic brain injury caused by vibration is the result of long-term, repetitive vibration to the head. The application of apolipoprotein AI mimetic peptide 4F in traumatic brain injury caused by a single injury factor (such as contusion) has not yet been studied.

[0021] The technical problem of this invention is to expand the range of drugs for treating craniocerebral injuries caused by a single traumatic factor (contusion).

[0022] This technical problem can be solved by using human apolipoprotein AI (ApoA-I) or its functionally active fragments to treat traumatic brain injury caused by a single injury factor (contusion).

[0023] Its technical effects include improving the subjects' motor activity and cognitive function, and reducing morphological damage to brain tissue structures.

[0024]

the term

[0025] Apolipoprotein AI (ApoA-I) is a natural plasma apolipoprotein and a component of high-density lipoprotein (HDL).

[0026] Brain contusion, also known as cerebral concussion, is an injury characterized by macroscopic damage to the brain tissue, often accompanied by hemorrhage, occurring instantaneously upon the application of the causative agent. Clinically, it can be classified as mild, moderate, or severe contusion.

[0027] Recombinant apolipoprotein AI (recombinant ApoA-I) is a protein whose amino acid sequence is identical to that of natural apolipoprotein AI. It is obtained through genetic engineering: recombinant DNA is constructed and expressed in a plasmid vector containing a synthetic gene encoding the nucleotide sequence of human apolipoprotein AI. The expression process takes place in bacterial, mammalian, or yeast cells, which can serve as production strains (or cells) of apolipoprotein AI.

[0028] Therapeutic effective dose—refers to the dose that, when administered to a subject, is sufficient to achieve the claimed technical effect for treating traumatic brain injury.

[0029] A pharmaceutically acceptable carrier is one that is compatible with other components in the composition and will not cause harm to the recipient, meaning it is non-toxic to cells or mammals at the dose and concentration used.

[0030] Functionally active fragments refer to fragments of the complete amino acid sequence (amino acid residues 1-243) of human apolipoprotein AI (ApoA-I), which have functional autonomy and functional activity, and can achieve the technical effects claimed in this invention in the composition of the drug described in this invention.

[0031] Traumatic brain injury (TBI) refers to complex contact injuries to the skull, brain tissue and its meninges, blood vessels and / or cranial nerves, accompanied by clinical symptoms. In most cases, it is combined with a clear history of trauma and morphological changes, and has a unified pathogenesis and formation time. Summary of the Invention

[0032] This invention relates to a medicament for treating traumatic brain injury, comprising human apolipoprotein AI (ApoA-I) or a functionally important fragment thereof. The amino acid sequence of human apolipoprotein AI consists of 243 amino acid residues, with a molecular weight of 28.5 kDa, corresponding to SEQ ID NO 1 (amino acid residues 1-243).

[0033] In one embodiment, the drug contains a functionally important fragment of human apolipoprotein AI (ApoA-I).

[0034] In one embodiment, the functionally important fragment of human apolipoprotein ApoA-I has a human ApoA-I amino acid sequence truncated from the N-terminus.

[0035] In one embodiment, the functional active fragment of human apolipoprotein ApoA-I has a human ApoA-I amino acid sequence truncated by 2 amino acid residues from the N-terminus (i.e., amino acid residues 3-243).

[0036] In one embodiment, the drug is formulated as a pharmaceutical composition comprising human ApoA-I or a functionally important fragment thereof, and a pharmaceutically acceptable carrier.

[0037] In one implementation, the pharmaceutically acceptable carrier is water for injection or physiological saline.

[0038] The above-mentioned drugs can also be used to treat traumatic brain injury, and the present invention also relates to the application of the above-mentioned drugs to treat traumatic brain injury.

[0039] In one embodiment, the drug contains a functionally important fragment of human ApoA-I.

[0040] In one embodiment, the functionally important fragment of human ApoA-I has a human apoA-I amino acid sequence truncated from the N-terminus.

[0041] In one embodiment, the functionally important fragment of human ApoA-I has a human ApoA-I amino acid sequence shortened by 2 amino acid residues (amino acid residues 3-243) from the N-terminus.

[0042] In one embodiment, the agent is formulated into a pharmaceutical composition comprising human ApoA-I or a functionally important fragment thereof and a pharmaceutically acceptable carrier.

[0043] In one implementation, the pharmaceutically acceptable carrier is water for injection or physiological saline.

[0044] The present invention also relates to a method for treating craniocerebral injury.

[0045] In one embodiment, the functionally important fragment of ApoA-I has a human ApoA-I amino acid sequence truncated from the N-terminus.

[0046] In one embodiment, the functionally important fragment of ApoA-I has a human apoA-I amino acid sequence shortened by 2 amino acid residues (amino acid residues 3-243) from the N-terminus.

[0047] In one embodiment, the agent is formulated into a pharmaceutical composition comprising human ApoA-I or a functionally important fragment thereof and a pharmaceutically acceptable carrier.

[0048] In one implementation, the pharmaceutically acceptable carrier is water for injection or physiological saline.

[0049] In one embodiment, the above-mentioned drug is administered either locally or parenterally.

[0050] In one embodiment, parenteral administration is performed via intravenous injection or intramuscular injection.

[0051] In one implementation, local administration is performed via subcutaneous injection.

[0052] Subjects exhibiting symptoms of traumatic brain injury were mammals, particularly humans. Attached Figure Description

[0053] Figure 1. Morphological changes in the cerebral cortex 5 weeks after the establishment of a mouse model of traumatic brain injury (200x magnification). Note: a - Disordered cell structure, with significant destruction of the outer granular layer;

[0054] b - Normally stained and deeply stained neurons are present in the outer granular layer;

[0055] c - Large neuronal phagocytic lesions form and migrate beneath the pia mater;

[0056] d - Deeply stained neurons are dominant in the outer granular layer and pyramidal cell layer.

[0057] a, b, and c are hematoxylin-eosin staining; d is toluidine blue staining.

[0058] Figure 2. Morphological changes in the cerebral cortex after weekly administration of recombinant apolipoprotein AI (ApoA-I) for 5 weeks following the establishment of a mouse model of traumatic brain injury. Note: a - Disintegration of the outer granular layer;

[0059] b - Diffuse infiltration of microglia is present in the outer granular layer;

[0060] c - A large neuronal phagocytic lesion forms beneath the pia mater;

[0061] d - Large neuronal phagocytic lesions.

[0062] a and d are toluidine blue staining; b and c are hematoxylin-eosin staining. a, b, and d are magnified 200 times; c is magnified 400 times. Detailed Implementation

[0063] This invention relates to a medicament for treating traumatic brain injury, comprising natural or recombinant human apolipoprotein AI (ApoA-I) – whose amino acid sequence consists of 243 amino acid residues and has a molecular weight of 28.5 kDa, or a functionally active fragment thereof.

[0064] In one embodiment, the drug comprises a functionally active fragment of human ApoA-I.

[0065] In one embodiment, the functionally active fragment of human ApoA-I has a truncated human ApoA-I amino acid sequence (SEQ ID NO 1, amino acid residues 1-243, i.e., ApoA-I-1-243) from the N-terminus, which has functional autonomy and activity and can achieve the technical effects claimed in the present invention in the composition of the drug of the present invention.

[0066] In one embodiment, the functional active fragment of human apolipoprotein AI (ApoA-I) has a human ApoA-I amino acid sequence truncated by 2 amino acid residues from the N-terminus, consisting of 241 amino acid residues and a molecular weight of 28.3 kDa (corresponding to amino acid residues 3-243 of SEQ ID NO 2, i.e., ApoA-I-3-243).

[0067] The functionally active fragment of human apolipoprotein AI (ApoA-I) can be obtained through genetic engineering methods: recombinant DNA is constructed and expressed in a plasmid vector to synthesize a gene (containing a nucleotide sequence encoding the functionally active fragment of human ApoA-I) in bacteria, yeast, or other cells. Subsequently, the recombinant protein encoded by the nucleotide sequence of the synthesized gene is isolated from the cell, and the functionally active fragment of human ApoA-I is obtained based on this using methods known to those skilled in the art, but not limited to the aforementioned genetic engineering methods. The functionally active fragment of human ApoA-I (rApoA-I-3-243) having an amino acid sequence truncated by two amino acid residues from the N-terminus of human ApoA-I-1-243 (amino acid sequence from position 1 to 243) is obtained through genetic engineering methods: a synthetic gene containing the nucleotide sequence of human ApoA-I-1-243 is cloned into *E. coli* cells, followed by isolation of the target protein and acid hydrolysis to obtain rApoA-I-3-243, as described in Russian Federation Patent No. 2573930.

[0068] In one embodiment, the above-mentioned drug is formulated as a pharmaceutical composition comprising human ApoA-I-1-243, recombinant human ApoA-I-1-243 (rApoA-I-1-243) or a functionally active fragment thereof, and a pharmaceutically acceptable carrier.

[0069] In one implementation, the pharmaceutically acceptable carrier is water for injection or physiological saline.

[0070] In one embodiment, the pharmaceutical composition comprises recombinant human ApoA-I-3-243 (rApoA-I-3-243) and water for injection.

[0071] This invention also relates to the application of human ApoA-I-1-243, recombinant human ApoA-I-1-243 or its functionally active fragments in the treatment of traumatic brain injury.

[0072] In one embodiment, the drug comprises a functionally active fragment of human ApoA-I.

[0073] In one embodiment, the functionally active fragment of human apolipoprotein AI (ApoA-I) has a human ApoA-I amino acid sequence truncated from the N-terminus (SEQ ID NO 1, i.e., amino acid residues 1-243, ApoA-I-1-243), which has functional autonomy and functional activity, and can achieve the technical effects claimed by the present invention in the composition of the drug of the present invention.

[0074] In one embodiment, the functional active fragment of human apolipoprotein AI (ApoA-I) has a human ApoA-I amino acid sequence truncated by 2 amino acid residues from the N-terminus (SEQ ID NO 2, i.e., amino acid residues 3-243, ApoA-I-3-243).

[0075] In one embodiment, the above-mentioned drug is formulated as a pharmaceutical composition comprising human ApoA-I-1-243, recombinant human ApoA-I-1-243 (rApoA-I-1-243) or a functionally active fragment thereof, and a pharmaceutically acceptable carrier.

[0076] In one implementation, the pharmaceutically acceptable carrier is water for injection or physiological saline.

[0077] In one embodiment, the pharmaceutical composition comprises recombinant human ApoA-I-3-243 (rApoA-I-3-243) and water for injection.

[0078] The present invention also relates to a treatment method for craniocerebral injury.

[0079] In one embodiment, the drug comprises a functionally active fragment of human apolipoprotein AI (ApoA-I).

[0080] In one embodiment, the functionally active fragment of human ApoA-I has a human ApoA-I amino acid sequence truncated from the N-terminus (SEQ ID NO 1, i.e., amino acid residues 1-243, ApoA-I-1-243), which has functional autonomy and functional activity, and can achieve the technical effects claimed by the present invention in the composition of the medicament described in the present invention.

[0081] In one embodiment, the functional active fragment of human ApoA-I has a human ApoA-I amino acid sequence truncated by 2 amino acid residues from the N-terminus (SEQ ID NO 2, i.e., amino acid residues 3-243, ApoA-I-3-243).

[0082] In one embodiment, the above-mentioned drug is formulated as a pharmaceutical composition comprising human ApoA-I-1-243, recombinant human ApoA-I-1-243 (rApoA-I-1-243) or a functionally active fragment thereof, and a pharmaceutically acceptable carrier.

[0083] In one implementation, the pharmaceutically acceptable carrier is water for injection or physiological saline.

[0084] In one embodiment, the pharmaceutical composition comprises recombinant human apolipoprotein AI-3-243 and water for injection.

[0085] In one embodiment, the drug is administered either locally or parenterally.

[0086] In one embodiment, parenteral administration is performed via intravenous injection or intramuscular injection.

[0087] In one implementation, local administration is performed via subcutaneous injection.

[0088] The duration of administration and the therapeutically effective dose can vary widely, depending on a variety of factors, including the severity of the traumatic brain injury, the individual parameters of the subject (including body size, weight, and age), and other factors well known to those skilled in the art.

[0089] Subjects exhibiting symptoms of traumatic brain injury were mammals, particularly humans.

[0090] Natural human apolipoprotein AI (ApoA-I) is a component of high-density lipoprotein (HDL) in plasma.

[0091] The natural human apolipoprotein AI SEQ ID NO 1 (amino acid residues 1-243, ApoA-I-1-243) was obtained by separating high-density lipoprotein (HDL) from human plasma using density gradient ultracentrifugation in potassium bromide (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 defatting of HDL [Cham В. Е., Knowlee В. RJ Lipid. Res. - 1976. - Vol.17, No. 2. P. 176-181] and purification [Пыхтина М.Б.и др.Биофармацевтический журнал. - 2012. - Т. 4. - №6. - С 37-45].

[0092] Human recombinant apolipoprotein AI (ApoA-I) SEQ ID NO 1 (amino acid residues 1-243, rApoA-I-1-243) is obtained by genetic engineering methods: constructing recombinant DNA and expressing a synthetic gene containing the encoding human chimeric ApoA-I nucleotide sequence in a plasmid vector (the expression system can be Escherichia coli cells, such as those described in Russian Federation Patent No. 2573930; or methyltrophic Pichia pastoris cells, such as those described in Russian Federation Patent No. 2605326), and then obtaining mature human rApoA-I-1-243 by enzymatic digestion of the corresponding chimeric protein, but not limited to the above methods.

[0093] The functionally active fragment of human apolipoprotein AI (ApoA-I) can be obtained by genetic engineering methods: constructing recombinant DNA, enabling the expression of a synthetic gene in bacteria, yeast or other cells in a plasmid vector (the synthetic gene contains a nucleotide sequence encoding a functionally active fragment of human ApoA-I), then isolating the recombinant protein encoded by the nucleotide sequence of the synthetic gene synthesized in the cell, and obtaining the functionally active fragment of human ApoA-I based on this method using methods known to those skilled in the art, but not limited to the above-described genetic engineering methods.

[0094] The human ApoA-I functional fragment (rApoA-I-3-243) with an amino acid sequence truncated by two amino acid residues from the N-terminus of human ApoA-I-1-243 (the amino acid sequence of human apolipoprotein AI at positions 1-243) is obtained through genetic engineering methods: a synthetic gene containing the nucleotide sequence of the human ApoA-I-1-243 gene is cloned into E. coli cells, and then the target protein is isolated and acid-hydrolyzed to obtain rApoA-I-3-243, as specifically described in Russian Federation Patent No. 2573930.

[0095]

Example

[0096] All embodiments of the present invention use the functional active fragment of human apolipoprotein AI (ApoA-I), which has an amino acid sequence (rApoA-I-3-243) that is truncated by 2 amino acid residues from the N-terminus of human ApoA-I-1-243.

[0097] Example 1. Preparation of the human ApoA-I functional active fragment rApoA-I-3-243

[0098] The functional active fragment of human apolipoprotein AI (ApoA-I), corresponding to amino acids 3-243 of human ApoA-I (SEQ ID NO 2) (hereinafter referred to as "rApoA-I-3-243"), was obtained through genetic engineering: recombinant DNA containing the nucleotide sequence encoding chimeric ApoA-I was cloned into a plasmid vector, which then synthesized the chimeric protein in E. coli cells; subsequently, the chimeric ApoA-I was isolated and purified. To obtain rApoA-I-3-243, the chimeric ApoA-I protein was acid-hydrolyzed, specifically as described in Russian Federation Patent No. 2573930.

[0099] Specifically, the nucleotide sequence of the recombinant DNA, starting from the 5' end, includes: the ATG codon (encoding methionine), followed by (1) a DNA fragment encoding a 6-amino acid residue histidine; (2) a DNA fragment encoding a 6-amino acid residue precursor form of human ApoA-I protein; (3) a DNA fragment encoding a 15-amino acid residue N-terminal fragment of bovine pancreatic ribonuclease A; (4) a DNA fragment encoding a 6-amino acid residue precursor form of human ApoA-I protein (this sequence is repeated, with the second occurrence following sequence (3)); (5) a DNA fragment encoding the recognition site of the tobacco mosaic virus protease; and (6) a mutant gene of the mature human ApoA-I-1-243 protein. The amino acid sequence of the chimeric ApoA-I protein corresponds to the amino acid sequence encoded sequentially by all the above-mentioned nucleotide sequences in the recombinant DNA, with sequence (2) appearing twice, the second occurrence following sequence (3).

[0100] When applied, it is in the form of a pharmaceutical composition comprising the functionally active fragment rApoA-I-3-243 of human apolipoprotein AI (ApoA-I) and a pharmaceutically acceptable solvent, wherein the pharmaceutically acceptable solvent is water for injection.

[0101] The administration method is subcutaneous injection.

[0102] Example 2. Experiment Description

[0103] Description of the mouse strains used in the experiments of this invention:

[0104] A comparative study on the application effects of the functionally active fragment rApoA-I-3-243 of human apolipoprotein AI (ApoA-I) was conducted on ICR inbred (non-purebred) mice. This strain is one of the most commonly used research subjects in oncology, toxicology, teratology, vaccine development, aging research, and general experiments.

[0105] Mice weighing 27-30 grams and 4 months old were used in the experiment. The animals were fed a standard diet and had free access to water.

[0106] The experimental animals were divided into 4 groups:

[0107] K (-)—— Blank control group: 10 healthy ICR mice;

[0108] K (+)—— Positive control group: 10 mice with traumatic brain injury model;

[0109] Experimental Group 1: Ten mice with traumatic brain injury model were subcutaneously injected with rApoA-I-3-243 once a week (injection site: scapular region) for 3 consecutive weeks.

[0110] Experimental group 2 (control group): Ten mice with traumatic brain injury model were injected subcutaneously with mexidol once a week for three consecutive weeks.

[0111] In experimental group 1, the dose of rApoA-I-3-243 was 7 mg / kg body weight, and the administration method was a pharmaceutical composition using water for injection as a pharmaceutically acceptable carrier, with a concentration of 1.2 mg / ml of rApoA-I-3-243 in the solution.

[0112] Experimental group 2 received intravenous mexidol at a dose of 7 mg / kg body weight.

[0113] Description of traumatic brain injury models

[0114] To conduct a comparative study, a mild traumatic brain injury (TBI) model was constructed. The model used was based on the Marmarou model [Marmarou A. A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics / / J. neurosurgery, 1994, vol. 80, №. 2, pp. 291-300], and referenced the modified scheme for mice by MJ Kanea 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 grams, a 20 mm diameter, 100 g weight was dropped from a height of 2.0 m to impact the mouse's head. The impact occurred inside a container 23 cm high, with a sponge pad at the bottom to cushion the impact. To eliminate impact resistance, the container surface was covered with aluminum foil, strong enough to support the mouse's weight—the foil would easily break upon head impact, causing the mouse to fall rapidly. During the acceleration and fall triggered by the impact, the mouse's body would inevitably undergo a 180° horizontal rotation. This method simulates the most common types of traumatic brain injury in humans. Existing animal models of traumatic brain injury often result in focal, severe damage, which is inconsistent with the reality of recurrent mild concussions, and only a few models are suitable for repeated simulations. The key feature of the model used in this study is the impact on the unfixed mouse's head, causing rapid acceleration of the freely moving head and trunk. This characteristic is well-known to be closely related to the occurrence of concussions in humans, a factor absent in other existing animal models of traumatic brain injury. This method requires no incision of the scalp, installation of skull protectors, or surgical intervention, and the entire procedure can be completed within 1-2 minutes. Mice do not exhibit symptoms such as seizures, paralysis, or behavioral abnormalities; skull fractures and intracranial hemorrhage are extremely rare. Mild motor incoordination and hyperkinesis resolve spontaneously over time. Histological analysis showed that the model elicited only a mild astrocyte response, without blood-brain barrier disruption, edema, or microglia activation. This novel animal model is easy to operate, inexpensive, and convenient for studying the neurobiological and behavioral consequences of traumatic brain injury. It is ideal for efficiently screening potential new treatments for mild concussions—injuries that are more common in athletes and military personnel.[Kanea М. A mouse model of human repetitive mild traumatic brain injury / / J. Neuroscience Methods, 2012, vol. 203, Is. 1, pp. 41-49].

[0115] Example 3. Results of a study on motor activity and cognitive function in mice with traumatic brain injury.

[0116] Table 1 shows the effects of rApoA-I-3-243 and mexidol on motor activity and cognitive function indicators in mice of each group 3 weeks after traumatic brain injury.

[0117] As shown in Table 1, even with two subcutaneous injections at a dose of 7 mg / kg body weight, rApoA-I-3-243 still demonstrated significant therapeutic effects in the progressive consequences of traumatic brain injury. This was primarily reflected in the recovery of impaired motor function, vestibular function, and cognitive function (including indicators such as central area activity, grooming behavior, standing against a wall, and exploring nests). In these indicators, mexidol was significantly inferior to the drug described in this invention and had almost no therapeutic effect at equivalent doses. The 7 mg / kg body weight dose of rApoA-I-3-243 was the optimal dose selected experimentally for correcting the consequences of traumatic brain injury in vivo: when the dose was below 7 mg / kg body weight, the therapeutic effect of the drug decreased; while when the dose was above 7 mg / kg body weight, the efficacy only slightly improved.

[0118] Table 1. Effects of rApoA-I-3-243 and mexilide on motor activity and cognitive function indices in mice of different groups.

[0119]

[0120] Example 4. Morphological changes in the cerebral cortex after traumatic brain injury in mice.

[0121] Experimental groups: Group 1 – Establishing a traumatic brain injury model, with subcutaneous injection of normal saline once a week for five weeks (K+ group); Group 2 – Establishing a traumatic brain injury model, with subcutaneous injection of 7 mg / kg of rApoA-I-3-243 aqueous solution (prepared with water for injection) once a week for five weeks.

[0122] Light microscopy was performed to observe staining changes (normal / darkly stained) in neurons of the molecular layer, outer granular layer, and pyramidal cell layer of the cerebral cortex (detection area: 239972.87 μm). 2 The sample size is at least 1000 cells, taken from non-overlapping fields of view (at least 5 fields of view).

[0123] For histological studies of excised cerebral cortex tissue specimens, fixation with neutral formalin was performed first, followed by standard processing methods, including hematoxylin-eosin staining and hematoxylin-toluidine blue staining. For electron microscopy, the excised cortical tissue was cut into 4-5 mm cubes, pre-fixed in Carnowski solution, and then fixed in a 1% osmium tetroxide solution prepared with 0.2 M dimethylarsyl buffer. Dehydration with ethanol and acetone was performed according to standard methods, followed by embedding in epoxy resin. Ultrathin sections were prepared using an LKB III and a Leica ULTRACUT EM UC7 ultramicrotome (Leica GmbH, Germany) and stained with uranium acetate and lead citrate. Observation was performed under a JEM-100B and JEM-1400 electron microscope (JEOL Ltd., Japan).

[0124] Five weeks after traumatic brain injury, with weekly subcutaneous saline injections, the morphological changes in the cerebral cortex injury area showed significant cellular structural disorder, specifically the degradation of neuronal axons and dendrites, and the complete disintegration of interneuronal connections (Fig. 1, a). Neurons in the outer granular layer were reduced in size, exhibited diverse morphologies, and were tightly aggregated (Fig. 1, b). Darkly stained cells, including microglia, were distributed around normally stained neurons (Fig. 1, c). Neurons in the pyramidal cell layer were predominantly darkly stained (shadow cells), accompanied by significant pericellular edema (Fig. 1, d), and these neurons were dispersed throughout the cell layer; in addition, circular vacuoles formed due to neuronal death were widely visible in the field of vision.

[0125] In this model of traumatic brain injury, a notable feature of cortical remodeling is the formation of large-area neurophagocytic foci (lesion area ranging from 3400 to 21500 μm). 2 These lesions are mainly distributed below the pia mater (see Figure 1, c). These lesions are primarily composed of microglia, with scattered neurons visible in the center of some lesions. The neurophagocytic foci are surrounded by extensive acellular areas. The large-area neurophagocytic foci that appear in traumatic brain injury may be one of the mechanisms for clearing irreversibly damaged neurons, allowing mononuclear cell infiltrates to be "drained" outside the brain.

[0126] The number of capillaries and arterioles is small, and the congestion is uneven. Obvious perivascular / pericapillary edema is visible.

[0127] Five weeks after traumatic brain injury, with weekly subcutaneous injections of rApoA-I-3-243, the overall characteristics of structural changes in the cerebral cortex injury area were consistent with the previous group (saline injection group), but the severity differed. Disordered structures were observed in the outer granular layer and pyramidal cell layer, primarily manifested as significant degradation of fiber structures, neuronal disintegration, and their disordered distribution within each layer (Fig. 2, a). Neurophagocytosis in the outer granular layer was intensified (Fig. 2, b); microglia extensively (diffusely) infiltrated this layer, forming aggregates around neurons and replacing neurons in large areas. Similar to the previous group, this group also developed large areas of neurophagocytic foci, which migrated beneath the pia mater (Fig. 2, c, d). Two to four such foci were present simultaneously in some specimens. Overall, the clearance of irreversibly damaged neurons in this group (as evidenced by the large aggregation of microglia) was more severe than in the group without rApoA-I-3-243. According to quantitative analysis, in the rApoA-I-3-243 group, cytoplasmic staining revealed a significantly higher proportion of deeply stained neurons (mainly located in the pyramidal cell layer) than normally stained neurons (mainly located in the outer granular layer), with the former accounting for 63.4% more (p<0.001) (Table 2). Obvious pyramidal cell degeneration and pericellular edema were observed in the pyramidal cell layer. However, compared to mice receiving daily subcutaneous injections of saline, the proportion of deeply stained neurons in this group was reduced by 37.3% (p<0.001) (Table 2). This indicator also suggests that the clearance process of irreversibly damaged neurons from the injury area was more active. This clearance of damaged cells is achieved through apoptosis (i.e., programmed cell death). Therefore, rApoA-I-3-243 exhibits neuroprotective effects because it induces neuronal death through apoptosis rather than necrosis.

[0128] Nevertheless, changes in cell structure still exist, especially in the outer granular layer, where neurons form dispersed cell clusters in the form of chains and small cell groups.

[0129] Table 2. Assessment of the number of neurons with different degrees of cytoplasmic staining

[0130]

[0131] During this experimental phase, no obvious hemodynamic disturbances were found at the site of injury, but attention should be paid to uneven capillary congestion and pericapillary edema.

[0132] Conclusion: Qualitative and quantitative analysis of the three layers of the cerebral cortex in mice after traumatic brain injury (TBI) treated with recombinant rApoA-I-3-243 (7 mg / kg) showed that, with the selected TBI treatment, cellular architecture disorder and disintegration of neuronal connections persisted for 5 weeks. In the group without any corrective treatment, the structural changes, including cellular architecture disorder and the number of deeply stained neurons, were the most significant. The brain structural changes in mice treated with recombinant rApoA-I-3-243 exhibited the most pronounced neurophagocytic process, which can be considered one of the mechanisms for the potent clearance of irreversibly damaged neurons. rApoA-I-3-243 can reduce the likelihood of large-scale necrosis and exert a neuroprotective effect.

[0133] This research was conducted using the following equipment: the core facilities of the Proteomics Analysis Equipment Sharing Center of the Russian Federal Research Center for Basic and Translational Medicine (Agreement No. 075-15-2021-691), funded by the Ministry of Education and Science of the Russian Federation, and the facilities of the Spectroscopic Measurement Equipment Sharing Center of the Russian Federal Research Center for Basic and Translational Medicine.

Claims

1. A drug for treating traumatic brain injury, comprising human apolipoprotein AI (ApoA-I) or a functionally important fragment thereof.

2. The drug according to claim 1, comprising a functionally important fragment of ApoA-I.

3. The medicament according to claim 2, wherein the functionally important fragment of human ApoA-I has a human ApoA-I amino acid sequence truncated from the N-terminus.

4. The medicament according to claim 3, wherein the functionally important fragment of human ApoA-I has an amino acid sequence representing human ApoA-I, which is truncated by two amino acid residues (amino acid residues 3-243) from the N-terminus.

5. The medicament according to any one of claims 1-4, formulated as a pharmaceutical composition comprising human ApoA-I or a functionally important fragment thereof and a pharmaceutically acceptable carrier.

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

7. Use human apolipoprotein AI or its key functional fragments as a means of treating traumatic brain injury.

8. The application according to claim 7, wherein the drug comprises a natural apolipoprotein AI derived from human serum.

9. The application according to claim 7, wherein the drug comprises recombinant human apolipoprotein AI.

10. The application according to claim 7, wherein the functionally important fragment of human ApoA-I has a human ApoA-I amino acid sequence truncated from the N-terminus.

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

12. The application according to any one of claims 7-11, wherein the pharmaceutical formulation is a pharmaceutical composition comprising human apolipoprotein AI or a functionally important fragment thereof and a pharmaceutically acceptable carrier.

13. The application according to claim 12, wherein the pharmaceutically acceptable carrier is water for injection or physiological saline.

14. A method for treating traumatic brain injury, comprising administering a drug to a subject in need according to any one of claims 1-6.

15. The method of claim 14, wherein the medicament comprises a functionally important fragment of human ApoA-I.

16. The method of claim 15, wherein the functionally important fragment of human ApoA-I has a human ApoA-I amino acid sequence truncated from the N-terminus.

17. The method of claim 16, wherein the functionally important fragment of human ApoA-I has a human ApoA-I amino acid sequence shortened by 2 amino acid residues (amino acid residues 3-243) from the N-terminus.

18. The method according to any one of claims 14-17, wherein the pharmaceutical formulation is a pharmaceutical composition comprising human apolipoprotein AI or a functionally important fragment thereof and a pharmaceutically acceptable carrier.

19. The method of claim 18, wherein the pharmaceutically acceptable carrier is water for injection or physiological saline.

20. The method according to any one of claims 14-19, wherein the administration is performed topically or parenterally.

21. The method of claim 20, wherein the administration is performed parenterally by intravenous or intramuscular injection.

22. The method of claim 20, wherein the drug is administered locally by subcutaneous injection.

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