RU2848765C1 - Peptide-amino acid complexes for restoring the deficiency of the endogenous component of animal blood and treating diseases or health disorders in animals associated with neuroinflammation - Google Patents

Abstract

FIELD: biotechnology and medicine.

SUBSTANCE: peptide-amino acid complex of the formula AA-Zn-HAEE is proposed for restoring endogenous HAEE deficiency in animals, as well as a pharmaceutical composition using the specified peptide-amino acid complex for treating animal diseases and health disorders associated with neuroinflammation. AA (amino acid) is an amino acid selected from Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met, Thr; Zn is a divalent zinc (II) cation; NAEE is a synthetic peptide, acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu.

EFFECT: stable peptide-amino acid complexes with preservation of the effective "unfolded" conformation of the HAEE peptide and their use in a lower dose to restore endogenous HAEE deficiency in the treatment of diseases and health disorders in animals associated with neuroinflammation.

4 cl, 15 dwg, 5 tbl, 5 ex

Description

The invention relates to peptide-amino acid complexes which are described by the formula AA-Zn-HAEE, where AA (amino acid) is an amino acid selected from Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met, Thr; Zn is a dicharged zinc cation (Zn); HAEE is a synthetic peptide acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu. The present invention also relates to pharmaceutical compositions based on peptide-amino acid complexes and their use for restoring the physiologically normal concentration of endogenous HAEE in the blood of animals and for the treatment of diseases or health disorders in animals associated with neuroinflammation.

Prior art

Numerous diseases and health disorders in mammals, in particular humans, can be accompanied by neuroinflammation: traumatic brain injury [Simon, D.W. et al. (2017) The far-reaching scope of neuroinflammation after traumatic brain injury. Nature Reviews Neurology 13 (3), 171-191], spinal cord injury [Hellenbrand, D.J. et al. (2021) Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. Journal of Neuroinflammation 18 (1), 284], stroke [Anrather, J. and Iadecola, C. (2016) Inflammation and Stroke: An Overview. Neurotherapeutics 13 (4), 661-670], COVID-19 [Vanderheiden, A. and Klein, R.S. (2022) Neuroinflammation and COVID-19. Curr Opin Neurobiol 76, 102608], delirium [Cerejeira, J. et al. (2012) The Cholinergic System and Inflammation: Common Pathways in Delirium Pathophysiology. Journal of the American Geriatrics Society 60(4), 669-675; Lyman, M. et al. (2014) Neuroinflammation: the role and consequences. Neurosci Res 79, 1-12], diabetes [Olasehinde, T.A. et al. (2022) Neuroinflammatory Biomarkers in Diabetic Encephalopathy: Linking Cholinergic and Cognitive Dysfunction. In Biomarkers in Diabetes (Patel, V.B. and Preedy, V.R. eds), pp. 1-20, Springer International Publishing], tumors of the peripheral nerves and central nervous system [Balkwill, F. and Mantovani, A. (2001) Inflammation and cancer: back to Virchow? The lancet 357 (9255), 539-545; Gysler, S.M. and Drapkin, R. (2021) Tumor innervation: peripheral nerves take control of the tumor microenvironment. The Journal of Clinical Investigation 131 (11); Shalapour, S. and Karin, M. (2019) Pas de Deux: Control of Anti-tumor Immunity by Cancer-Associated Inflammation. Immunity 51 (1), 15-26], as well as a number of autoimmune diseases (for example, multiple sclerosis [Filippi, M. et al. (2018) Multiple sclerosis. Nature Reviews Disease Primers 4 (1), 43; Kamma, E. et al. (2022) Central nervous system macrophages in progressive multiple sclerosis: relationship to neurodegeneration and therapeutics. Journal of Neuroinflammation 19 (1), 45], systemic lupus erythematosus) [Gottschalk, T.A. et al. (2015) Pathogenic Inflammation and Its Therapeutic Targeting in Systemic Lupus Erythematosus. Front Immunol 6, 550]) and all neurodegenerative diseases (e.g., Alzheimer's disease [Leng, F. and Edison, P. (2021) Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here? Nature Reviews Neurology 17 (3), 157–172], Pick's disease (or frontotemporal dementia) [Chu, M. et al. (2023) Peripheral inflammation in behavioral variant frontotemporal dementia: associations with central degeneration and clinical measures. Journal of Neuroinflammation 20 (1), 65], Parkinson's disease [Troncoso-Escudero, P. et al. (2018) Outside in: Unraveling the Role of Neuroinflammation in the Progression of Parkinson's Disease. Front Neurol 9, 860], amyotrophic lateral sclerosis [Robberecht, W. and Philips, T. (2013) The changing scene of amyotrophic lateral sclerosis. Nature Reviews Neuroscience 14 (4), 248–264], cerebral amyloid angiopathy [de Souza, A. and Tasker, K. (2023) Inflammatory Cerebral Amyloid Angiopathy: A Broad Clinical Spectrum. J Clin Neurol 19 (3), 230–241]. The inevitable result of neuroinflammation is the degradation and death of neurons (neurodegeneration) [Zhang, W. et al. (2023) Role of neuroinflammation in neurodegeneration development. Signal Transduction and Targeted Therapy 8 (1), 267]. As a consequence of neurodegeneration, patients experience a decrease in quality and life expectancy, and may develop acquired dementia [Ahmad, M.A. et al. (2022) Neuroinflammation: A Potential Risk for Dementia. Int J Mol Sci 23 (2)]. Therefore, the development of drugs and therapeutic technologies aimed at regulating neuroinflammation is relevant [van der Flier, W.M. et al. (2023) Towards a future where Alzheimer's disease pathology is stopped before the onset of dementia. Nature Aging 3 (5), 494-505].

Neurodegenerative diseases have a number of common development factors and a number of other generalizing mechanisms (Litvinenko I.V., Krasakov I.V., Bisaga G.N. et al. Modern conception of the pathogenesis of neurodegenerative diseases and therapeutic strategy. Zhurnal Neurologii i Psikhiatrii imeni S.S. Korsakova. 2017;117(6-2):3-10. (In Russ.). doi: 10.17116/jnevro2017117623-10), which may link Alzheimer's disease (AD), vascular dementia, neurodegeneration in multiple sclerosis (MS), Parkinson's disease, as well as Alzheimer's disease in conditions of activation of systemic inflammation (Williams-Gray S.N., Wijeyekoon R., Yarnall A.J. et al. ICICLE-PD study group. Serum immune markers and disease progression in an incident Parkinson's disease cohort (ICICLE-PD). Movement Disorders. 2016;31(7):995-1003. https://doi.org/10.1002/mds.26563). Inflammation is a typical pathological process in mammals, particularly humans. A typical pathological process is characterized by a certain set of characteristic features. It is currently believed that the neurodegenerative process is secondary to the inflammatory process (Mostafa A., Jalilvand S., Shoja Z. Et al. Multiple sclerosis-associated retrovirus, Epstein-Barr virus, and vitamin D status in patients with relapsing-remitting multiple sclerosis. Journal of Medical Virology. 2017. doi: 10.1002/jmv.24774; Litvinenko I.V., Krasakov I.V., Bisaga G.N. et al. Modern conception of the pathogenesis of neurodegenerative diseases and therapeutic strategy. Zhurnal Neurologii i Psychiatrii imeni S.S. Korsakova. 2017;117(6-2):3-10. (InRuss.). doi: 10.17116/jnevro2017117623-10).

Alzheimer's disease is one of the most common neurodegenerative diseases among the elderly and is characterized by neuritic plaques, the main component of which is the peptide beta-amyloid (Aβ). The N-terminal region of beta-amyloid 1-16 is a metal-binding domain, where zinc (Zn) and copper (Cu) are attached to the Aβ peptide (Guilloreau L., Damian L., Coppel Y., et al. (2006). Structural and thermodynamic properties of CuII amyloid-β16/28 complexes associated with Alzheimer's disease. JBIC Journal of Biological Inorganic Chemistry, 11(8), 1024-1038, doi: 10.1007/s00775-006-0154-l). Reducing the level of zinc ions in the blood by binding zinc ions with specific chelating agents and/or blocking the interactions of the Aβ peptide with zinc ions can prevent pathologies associated with these interactions (Ayton S., Lei P., Bush A. Biometals and Their Therapeutic Implications in Alzheimer's Disease. Neurotherapeutics. 2015. 12(1): p. 109-120). High concentrations of zinc cause precipitation of the AR peptide and affect the formation of extracellular AR aggregates (amyloid plaques) in specific parts of the brain (hippocampus, cortex, thalamic nuclei), which are associated with the development of Alzheimer's disease (Frederickson C. J., Bush A. I. (2001). Synaptically released zinc: physiological functions and pathological effects. Biometals, 14(3), 353-366, doi: 10.1023/A: 1012934207456).

Currently, there are no effective drugs in clinical medical practice that could cure diseases and health disorders associated with neuroinflammation in mammals, particularly humans, without any side effects.

For example, in the United States in July 2024, donanemab, sold under the brand name Kisunla, was approved for medical use. It is a monoclonal antibody and is intended for the treatment of people with mild cognitive impairment or mild dementia (reviewed by Khartabil, N.; Awaness, A. (2025) Targeting Amyloid Pathology in Early Alzheimer's: The Promise of Donanemab-Azbt. Pharmacy, 13, 23). Its drawbacks include strong side effects: when taking donanemab in high doses, some people developed a form of cerebral edema (swelling of the brain) called "amyloid-related imaging abnormalities with edema or effusion" (ARIA-E).

A number of peptides are known from scientific and patent literature that could be used to treat such diseases without serious side effects.

A compound known from PCT/FR2011/052477 and Russian patent No. 2588143 is the peptide Ac-HAEE-NH ₂ (hereinafter referred to as HAEE). In the abbreviation HAEE, the letters stand for: H – histidine, A – alanine, E – glutamic acid. The HAEE peptide is capable of inhibiting the interaction of Aβ peptides with Zn (II) ions, causing a decrease or prevention of Aβ peptide aggregation in the presence of Zn (II) ions at physiological concentrations of Zn (II) ions of 100-400 μM. Higher concentrations of Zn (II) ions, on the order of 1 mmol (mM), suppress the interactions between HAEE and the 1-16 region of Aβ. Incubation of Aβ aggregates with HAEE for at least 1 hour at 20, 40, 80, 100, and 150 μM HAEE was shown to result in disaggregation of the aggregates. A synthetic analogue of the 35-HAEE-38 region of the α4 subunit of the α4β2 nicotinic acetylcholine receptor (α4β2-nAChR), a tetrapeptide of HAEE, specifically binds to the 11-EVHH-14 region of beta-amyloid (Aβ) in the absence of zinc ions [patent WO 2012/056157; Russian Federation patent No. 2588143; Barykin, E.R. et al. (2020) Tetrapeptide Ac-HAEE-NH ₂ Protects α4β2 nAChR from Inhibition by Ap Int J Mol Sci 21 (17)] and in the presence of zinc ions [Mitkevich, VA et al. (2023) Zn-dependent β-amyloid Aggregation and its Reversal by the Tetrapeptide HAEE. Aging Dis 14 (2), 309-318]. HAEE effectively suppresses the formation of amyloid plaques in animal models of Alzheimer's disease [Tsvetkov, PO et al. (2015) Peripherally Applied Synthetic Tetrapeptides HAEE and RADD Slow Down the Development of Cerebral β-Amyloidosis in AβPP/PS1 Transgenic Mice. J Alzheimers Dis 46 (4), 849-53: Mitkevich, VA et al. (2023) Zn-dependent β-amyloid Aggregation and its Reversal by the Tetrapeptide HAEE. Aging Dis 14 (2), 309-318].

It is known from Russian Patent No. 2679059 that, despite the inhibition of amyloid plaque formation, the effect of known drugs, in particular HAEE, on improving cognitive function in Alzheimer's disease is, on average, very limited. There is a need to develop alternative effective agents for the treatment of Alzheimer's disease, including by reducing or preventing the binding of Zn(II) ions to the Aβ peptide. A peptide with the amino acid sequence (in standard single-letter codes) H[isoD][pS]GYEVHH (where isoD denotes an isomerized aspartic acid residue, and pS refers to a phosphorylated serine residue) with open or protected ends of the main polypeptide chain has been proposed as an alternative agent for the treatment of Alzheimer's disease.

From the patent literature, complexes of HAEE with metal ions and HAEE salts are known [RU Patent No. 2784249; RU Patent No. 2784319; RU Patent No. 2784326; RU Patent No. 2784425; RU Patent No. 2784732; RU Patent No. 2784746; RU Patent No. 2785354], as well as a ternary complex of HAEE with Zn and human serum albumin [RU Patent No. 2709539].

A zinc complex of the HAEE peptide and a pharmaceutical composition for the treatment of neurodegenerative diseases are known from Russian patent No. 2784319. The composition contains as an active substance a zinc complex of the formula [Zn(HAEE)] _m X _n , where Zn is a divalent zinc (II) cation, m = 1 for singly and divalent anions, m = 3 for trivalent anions, HAEE is a synthetic peptide acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu, X is a pharmaceutically acceptable anion, n = 1 for divalent anions, n = 2 for singly and trivalent anions. The work notes the importance of maintaining the "unfolded" conformation of the HAEE peptide, which leads to a high therapeutic effect. It was shown that this conformation remains unchanged in the case of exposure of the zinc complex of the HAEE peptide in blood plasma, while the native HAEE peptide in blood plasma loses the “unfolded” conformation.

A disadvantage of this invention is the potential interaction of the [Zn(HAEE)] ²⁺ ion with other molecules that may be present in both the pharmaceutical composition and blood plasma. This ion is prone to coordination, particularly with peptide molecules in blood plasma, as evidenced by the known HAEE complex with human albumin, which is coordinated by a zinc ion (RU Patent No. 2709539). Interactions with albumin and blood transport proteins alter the spatially coordinated structure of HAEE, which may alter bioavailability and even pharmacological activity, which depends on the spatial arrangement of the peptide functional groups.

Thus, the present invention differs from the analog from Russian Patent No. 2784319 by the presence of an amino acid in the complex structure, which preserves the spatial structure of HAEE in its active form in biological fluids, resulting in conformational stability and activity up to interaction with the target (receptors, ion channels, transport systems). The loss of the "unfolded" conformation is achieved by interaction of HAEE with other molecular structures in blood plasma in the absence of a zinc ion. At the same time, the known zinc complex of the HAEE peptide can also interact, while maintaining the electrostatic interaction between [HAEE-Zn] ²⁺ , with other molecules contained in plasma, which may partially impede its passage to the pharmacodynamic target.

Russian Patent No. 2709539 discloses that short charged peptides like HAEE have a very short plasma lifetime (from a few seconds to several minutes) and have difficulty crossing the blood-brain barrier (BBB), significantly reducing their therapeutic efficacy. To overcome these shortcomings, the authors of Russian Patent No. 2709539 developed a pharmaceutical composition for delivering HAEE across the BBB for the treatment of neurodegenerative diseases, including Alzheimer's dementia (Alzheimer's disease). This composition improves the pharmacokinetic properties and increases the bioavailability of HAEE.

This composition contains an effective amount of a substance in the form of a complex of HAEE with zinc and human serum albumin (HAEE-Zn-HSA) in the form of a solution for administration with a pharmaceutically acceptable carrier selected from neutral carriers and diluents, such as saline. The resulting pharmaceutical composition increases the bioavailability of the substance in the brain by fivefold, doubles its half-life, and improves cognitive function in experimental animals by 20%.

Russian Patent No. 2709539 indicates that the HAEE peptide forms a specific ternary complex (HAEE-Zn-HSA) with human albumin (HSA) in the presence of zinc ions. However, no interactions between HAEE and HSA were observed in the absence of zinc ions. This complex is characterized by intermolecular binding between HAEE and HSA molecules, in which the zinc cation serves as the coordinating cation. This complex has not been obtained in solid form.

The invention according to Russian patent No. 2709539 was chosen as the closest analogue, since it represents the only known complex for HAEE of two substances coordinated through a zinc ion.

It is known from Russian patent No. 2709539 that the pharmaceutical composition HAEE-Zn-HSA is stable in a suitable aqueous buffer system, in the pH range from 4 to 8, in the presence or absence of various salts.

It is known that the charge of albumin in a neutral environment is -15, in an alkaline environment it reaches -140, and in a slightly acidic environment it is 0 (isoelectric state at pH = 5.97) and albumin precipitates (Biochemistry with exercises and problems. Edited by A.I. Glukhov, E.S. Severin. GEOTAR-Media, Moscow. 2019. P. 19), therefore this complex should not exist in a slightly acidic environment at pH 4-5.5.

Russian Patent No. 2709539 discloses that, to prepare the HAEE-Zn-HSA pharmaceutical composition suitable for preclinical studies, 5 mg of HAEE, 600 mg of human albumin, and 0.5 mg of zinc chloride ( _ZnCl2 ) were dissolved in 20 milliliters of saline. A lyophilized preparation of the synthetic HAEE peptide (more than 98% purity) was used as the HAEE source. With a known molecular weight of HAEE equal to 525.52 g/mol, the ratio of components in the HAEE-Zn-HSA complex is approximately HAEE: Zn: HSA ~ 1: 2.6: 1 by mole.

A disadvantage of this invention is the high content of human albumin, which is 120 times higher by weight than the content of the active substance HAEE, which can lead to the formation of a complex with a non-stoichiometric ratio of its components and an impurity of a separate HSA phase.

Another drawback of this invention is the use of chloride anion as a counterion in the preparation of the HAEE-Zn-HSA ternary complex. We found that chloride anions make such complexes hygroscopic, which can reduce their stability during long-term storage under high humidity conditions.

High concentrations of human albumin can have various negative effects on the body of both experimental animals and humans (Gales B.J., Erstad B.L. (1993). Adverse reactions to human serum albumin. Annals of Pharmacotherapy, 27(1), 87–94, doi: 10.1177/106002809302700119; Kremer H., Baron-Menguy C., Tesse A. et al. (2011). Human serum albumin improves endothelial dysfunction and survival during experimental endotoxemia: concentration-dependent properties. Critical care medicine, 39(6), 1414–1422, doi: 10.1097/CCM.0b013e318211ff6e). Some samples of HSA, unlike bovine serum albumin (BSA), did not bring mouse embryos to the blastocyst stage when cultured (Leveille M.C., Carnegie J., Tanphaichitr N. (1992). Effects of human sera and human serum albumin on mouse embryo culture. Journal of assisted reproduction and genetics, 9(1), 45-52, doi: 10.1007/BFO1204114). HSA can induce an immune response in mice after its administration (Favoretto B.C., Ricardi R., Silva, S.R. et al. (2011). Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunized with human serum albumin. Toxicon, 57(4), 600-607, doi: 10.1016/j.toxicon.2010.12.023).

It is known that HSA can be used to improve cognitive function in the treatment of Alzheimer's disease, but only when administered intracerebroventricularly (intracranial). Administration of HSA into blood plasma may reduce its effectiveness (Ezra, A., Rabinovich-Nikitin, I., Rabinovich-Toidman, P., & Solomon, B. (2016). Multifunctional effect of human serum albumin reduces Alzheimer's disease-related pathologies in the 3xTg mouse model. Journal of Alzheimer's Disease, 50(1), 175-188, doi: 10.3233/JAD-150694).

Therefore, reducing the amount of human albumin or the possibility of completely eliminating it from the drug, replacing complex and traumatic intracerebroventricular administration with parenteral, oral or intranasal administration, while simultaneously enhancing the therapeutic effect, is an important medical task in the treatment of diseases and health disorders associated with neuroinflammation in mammals, in particular humans.

Thus, the objectives of the invention are to obtain peptide-amino acid complexes AA-Zn-HAEE as effective and safe means for restoring the level of endogenous HAEE in diseases and health disorders associated with neuroinflammation in animals, to obtain pharmaceutical compositions based on such complexes and to use them to restore the level of endogenous HAEE in diseases and health disorders associated with neuroinflammation in animals.

According to the present invention, peptide-amino acid complexes are claimed which are described by the formula AA-Zn-HAEE, where AA is an amino acid selected from Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met, Thr; Zn is a dicharged zinc (II) cation; HAEE is a synthetic peptide acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu; methods for their production, pharmaceutical compositions based on the peptide-amino acid complexes and their use for the treatment of neurodegenerative diseases of animals associated with neuroinflammation and related to a decrease in the level of endogenous HAEE are claimed.

In the complexes, the molar content of the amino acid, zinc (II) cation and HAEE peptide is equimolar (1:1:1).

It has been previously shown that the HAEE peptide in solution, in blood plasma, and in the form of a zinc complex differ in the time of release from a chromatographic column due to differences in the hydrophobic interactions of the HAEE peptide with the column material, which are determined by the prevailing spatial structure of the HAEE peptide (RU Patent No. 2784319).

In this study, it was demonstrated that when the native HAEE peptide, its zinc complex without the amino acid, or the AA-Zn-HAEE complexes obtained in this study were introduced into animal blood plasma, the steric spatial structure of the peptide differed in each case. This was documented by the time of substance release from an HPLC chromatography column (Table 1).

While the chromatographic peak retention time for the native HAEE peptide was 1.30 min and 2.78 min for the zinc complex, the retention time for the peptide-amino acid complexes obtained in the present invention was 1.9-2.5 min. It is assumed that at the retention time of 2.78 min, the HAEE peptide binds to plasma elements through interactions unusual for it (in the absence of zinc ions) and changes its active "unfolded" conformation to an inactive one; the zinc complex, having a strong affinity for non-specific binding to plasma substances, such as HSA, has an active "unfolded" conformation, whereas the peptide-amino acid complexes have minimal interaction with plasma substances, i.e., are essentially inert, and the conformation of the HAEE peptide in such a complex with amino acids does not change.

Due to this, it is precisely this conformation of HAEE in peptide-amino acid complexes that is most likely most similar to endogenous HAEE and exhibits maximum therapeutic efficacy, as demonstrated in experimental in vivo models. The endogeneity of the HAEE peptide was previously demonstrated in Russian patents No. 2826728 and No. 2826726.

This was an unexpected result, as the HAEE-Zn-HSA complex and the [Zn(HAEE)] ²⁺ complex were previously thought to be fully therapeutically effective. However, it turned out that the peptide-amino acid complexes are more effective in treating neurodegenerative diseases and animal health disorders associated with neuroinflammation and reduced endogenous HAEE levels, as confirmed by in vivo experiments and indirectly by conformational changes in the HAEE peptide.

It is known that disruption of the 3D structure of oligopeptides can lead to a decrease in their functions and a reduction in therapeutic effect (Sikora, K., Jaskiewicz, M., Neubauer, D., Migon, D., & Kamysz, W. (2020). The role of counter-ions in peptides—an overview. Pharmaceuticals, 13(12), 442.). It has been previously shown that for optimal interaction with protein partners, the HAEE peptide must be in an “unfolded” conformation (Barykin E. P., Garifulina A. I., Tolstova A. P., Anashkina A. A., Adzhubei A. A., Mezentsev Y. V., Shelukhina I. V., Kozin S. A., Tsetlin V. L, Makarov A. A. Tetrapeptide Ac-HAEE-NH(2) Protects α4β2 nAChR from Inhibition by Aβ // International journal of molecular sciences. - 2020. - Vol. 21, No. 17. - P. 6272; Zolotarev Y. A., Mitkevich V. A., Shram S. I., Adzhubei A. A., Tolstova A. P., Talibov O. B., Dadayan A. K., Myasoyedov A. A. NF, Makarov AA, Kozin SA. Pharmacokinetics and Molecular Modeling Indicate nAChRα4-Derived Peptide HAEE Goes Through the Blood-Brain Barrier. Biomolecules. 2021 Jun 18;11(6):909). In the native HAEE peptide, the imidazole group of the histidine amino acid residue can form stable polar bonds with the carboxyl groups of the side chains of glutamic acid amino acid residues, which does not contribute to maintaining the biologically significant "unfolded" conformation of this peptide and, as a result, reduces the beneficial therapeutic effects of HAEE.

Thus, one of the advantages of the present invention is the increase in the therapeutic efficacy of the HAEE peptide through the production and use of its peptide-amino acid complexes due not only to the preservation of the 3D structure of the HAEE peptide in such complexes in the “unfolded” conformation, but also, apparently, due to the nature-like conformation, close to that of endogenous HAEE.

Previously, Russian Patent No. 2784319 demonstrated that administration of the HAEE-Zn-HSA complex, obtained using the method described in Russian Patent No. 2709539, to the blood plasma of mice resulted in stress, changes in appearance, and even death. Administration of the zinc complex of the HAEE peptide to the blood plasma of healthy mice did not result in any behavioral or physical impairment. Compared to control animals administered saline, administration of the peptide-amino acid complexes not only did not result in stress or changes in appearance, but also resulted in a slight increase in animal weight and a calm response to the administration of the peptide-amino acid complex. This indicates the absence of side effects and the beneficial effects of the peptide-amino acid complexes.

Peptide-amino acid complexes can be used to treat neurodegenerative diseases and other similar pathologies accompanied by neuroinflammatory processes, and effectively influence neurodegenerative diseases caused by inflammatory causes, at least by restoring impaired cognitive functions and restoring the level of endogenous HAEE.

Peptide-amino acid complexes can be used in solid or liquid form. Liquid forms have been shown to have accelerated stability for at least 2 years (6 months at 40°C). Solid forms of peptide-amino acid complexes have been shown to have stability for at least 4 years (1 year of accelerated storage at 40°C).

The technical result of the invention is

- obtaining new peptide-amino acid complexes of the general formula AA-Zn-HAEE, where AA (amino acid) is an amino acid selected from Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met, Thr; Zn is a dicharged zinc (II) cation; HAEE is a synthetic peptide acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu;

- the possibility of using peptide-amino acid complexes in the treatment of diseases and health disorders in animals associated with neuroinflammation, in particular, those caused indirectly by inflammatory factors;

- the possibility of using peptide-amino acid complexes to restore the deficiency of endogenous HAEE;

- high stability of peptide-amino acid complexes in solid and liquid form;

- the most effective “unfolded” conformation of the HAEE peptide, which allows it to be used in a lower dose;

- in the absence of side effects, a positive effect on healthy animals.

According to the method used, peptide-amino acid complexes are obtained in amorphous form, as confirmed by X-ray phase analysis. A characteristic feature of peptide-amino acid complexes is the presence of two or three broad maxima in the amorphous phase. For the peptide-glycine complex, such maxima are observed in the region of 13.5, 20, and 23.5 2Θ° (Fig. 1).

HAEE has characteristic intense maxima in the Fourier transform IR spectra at 1644, 1530, 1396 and 1249 cm ^-1 (Fig. 2). The IR spectra of a number of amino acids are represented by a set of high-intensity vibrations in the region of 1700-600 cm ^-1 (Figs. 3-6). Examples of IR spectra of peptide-amino acid complexes of HAEE with various amino acids are shown in Figs. 7-10. The IR spectra of the peptide-amino acid complexes have characteristic intense maxima in the region of 1120-1040 cm ^-1 , which distinguishes them from the HAEE molecule (Fig. 2). High-intensity vibrations in the region of 1700-600 cm ^-1 inherent in crystalline free amino acids are not manifested in the peptide-amino acid complexes. The maxima in the region of 1120-1040 cm ^-1 can be attributed to the CN val., C-O val. and -NH ₂ -groups of amino acids.

Peptide-amino acid complexes can be prepared by mixing aqueous solutions of HAEE and the amino acid in equimolar ratios, then adding a water-soluble zinc(II) salt in an equimolar ratio, stirring for 10-30 minutes, and drying under vacuum. If the corresponding amino acid is insufficiently soluble in water, the solution can be heated to 60-70°C.

A rotary evaporator or sublimator can be used to dry peptide-amino acid complexes under vacuum. In the case of freeze-drying, the solution is pre-frozen; however, this method has limitations when attempting to use high concentrations, due to the limited solubility of amino acids, such as leucine and methionine.

A water-soluble zinc salt can be selected from sulfate, nitrate, acetate, or chloride. To obtain peptide-amino acid complexes without a counterion, anion-exchange chromatography or dialysis is performed before the vacuum drying step. Sulfate, nitrate, and acetate are preferred; zinc chloride is also acceptable, but the hygroscopicity of such peptide-amino acid complexes should be carefully monitored without removing the chloride counterion.

If the claimed peptide-amino acid complexes need to be obtained without a counterion, the costly anion-exchange chromatography or dialysis step can be avoided by using organic zinc salts with corresponding amino acids, such as zinc glycinate, zinc methionate, etc., instead of the aforementioned inorganic zinc salts. The solubility of these compounds must be taken into account. The recommended concentration of the peptide-amino acid complex solution should not exceed 3% by weight, preferably 1-2% by weight, and preferably 0.5-1% by weight.

Peptide-amino acid complex powder or lyophilisate is obtained only in amorphous form. The presence of crystalline phases of the corresponding amino acids and/or absorption maxima in the IR spectra of free amino acids results in a mixture of several substances and requires a repeat of the preparation method and a new drying procedure.

Another object of the invention is pharmaceutical compositions containing an effective amount of a peptide-amino acid complex and excipients. The pharmaceutical composition may be solid—in the form of a powder, lyophilisate, capsules, or tablets—or liquid—as a solution, mixture, drops, spray, or syrup. Solid compositions of peptide-amino acid complexes contain an effective amount and, optionally (for a lyophilisate), excipients.

The effective amount of peptide-amino acid complexes in the pharmaceutical composition depends on the type of nosology, body weight, route of administration, and therefore can vary from 0.1 to 50 mg, preferably from 0.2 to 30 mg, and desirably from 0.3 to 10 mg.

Excipients are selected from those known to be used for this purpose, for example, those described in the Handbook of Pharmaceutical Excipients, Sixth edition, Edited by R.C. Rowe, 2009, 917 p. The preparation of solid pharmaceutical compositions does not require the use of wet granulation.

Peptide-amino acid complexes, both pharmaceutical substances and pharmaceutical compositions based on them, exhibit high stability of the active ingredient when stored in sealed vials. Accelerated storage of peptide-amino acid complexes and pharmaceutical compositions based on them for 6 months at 40°C and 75% humidity demonstrated no loss of the active ingredient: before storage, the concentration of each complex was 100.0±2.0%, and after storage, the concentration of the corresponding complex was 99.4±1.8%.

The effective amount of peptide-amino acid complexes in the pharmaceutical substance depends on the type of nosology, body weight, route of administration, and therefore can vary from 0.1 to 50 mg, preferably from 0.2 to 30 mg, and desirably from 0.3 to 10 mg.

The effective amount of peptide-amino acid complexes in therapeutic treatment depends on the type of nosology, body weight, route of administration, and therefore can vary from 0.1 to 50 mg, preferably from 0.2 to 30 mg, and desirably from 0.3 to 10 mg.

Taking into account the listed solid and liquid pharmaceutical compositions, the method of administration of the pharmaceutical composition of peptide-amino acid complexes can be parenteral, oral and intranasal.

Another object of the invention is the use of peptide-amino acid complexes to restore the physiologically normal concentration of endogenous HAEE in blood plasma.

Another object of the invention is the use of peptide-amino acid complexes for the treatment of diseases and health disorders in animals associated with neuroinflammation, which consists of administering peptide-amino acid complexes as part of the described pharmaceutical compositions to an animal in an effective amount.

The potential for therapeutic treatment of animal diseases and health disorders associated with neuroinflammation has been demonstrated in several in vivo animal models. A key factor in these trials is the endogeneity of the HAEE peptide and the decline in its physiological levels in the presence of nervous system disorders, as previously demonstrated in Russian patents Nos. 2826728 and 2826726. In these patents, the HAEE peptide was administered in its native form, as salts, and as complexes with metal cations. This resulted in the restoration of the physiological norm of the endogenous HAEE peptide, which, as a marker, characterizes the body's recovery status in neurodegenerative disorders. This restoration was accompanied by an improvement in the health of the experimental animals and a reduction in the clinical manifestations of the corresponding diseases or disorders.

It was also shown that administration of peptide-amino acid complexes, compared to previously known forms of the HAEE peptide, enhanced the effectiveness of restoring HAEE levels in various in vivo animal models. When the dose of the peptide-amino acid complex was reduced by 2.5 times, its effectiveness in animal models was comparable to that of previously known HAEE salts and complexes (sodium salt, zinc complex).

Description of figures

Fig. 1 Diffraction pattern of the peptide-amino acid complex Gly-Zn-HAEE.

Fig. 2 Fourier transform IR spectrum of the HAEE peptide.

Fig. 3 Fourier transform IR spectrum of the amino acid Gly.

Fig. 4 Fourier transform IR spectrum of the amino acid Met.

Fig. 5 Fourier transform IR spectrum of the amino acid Ala.

Fig. 6 Fourier transform IR spectrum of the amino acid Phe.

Fig. 7 Fourier transform IR spectrum of the peptide-amino acid complex Gly-Zn-HAEE.

Fig. 8 Fourier transform IR spectrum of the peptide-amino acid complex Met-Zn-HAEE.

Fig. 9 Fourier transform IR spectrum of the peptide-amino acid complex Ala-Zn-HAEE.

Fig. 10 Fourier transform IR spectrum of the peptide-amino acid complex Phe-Zn-HAEE.

Fig. 11 Numbering of hydrogen atoms in the HAEE peptide.

Fig. 12 ¹ H NMR spectrum for the HAEE peptide.

Fig. 13 ¹ H-NMR spectrum for the peptide-amino acid complex Gly-Zn-HAEE.

Fig. 14 ¹ H-NMR spectrum for the peptide-amino acid complex Met-Zn-HAEE.

Fig. 15. Isolated ion current chromatograms of fragments b1, b2, b3, and a1 of the internal standard with the inclusion of two stable carbon isotopes [ ¹³ C] at a final concentration of 1 ng/ml and the HAEE peptide after extraction from a human blood plasma sample. The calculated HAEE concentration based on the relative response of the area under the peak of the quantifier ion [ ¹³ C ₂ ]-b1 with m/z = 182.0814 was 0.322 ng/ml (A). Deconvolution plots of the mass spectrum of the fragments of the internal standard with the inclusion of stable carbon isotopes [ ¹³ C] and the corresponding HAEE fragment ions in the blood plasma sample after extraction (B). Composite fragmentation spectra of HAEE and the internal standard with the inclusion of stable carbon isotopes [ ^13C ], as well as the residual signal of the parent ion of the internal standard with m/z = 528 and HAEE with m/z = 526 in the blood plasma sample after extraction. The distance between the fragments and the parent ions of the internal standard with HAEE is on average 2.0067 atomic mass units (without correction for mass defect), which corresponds to the inclusion of two carbon isotopes [ ^13C ] in the internal standard molecule (B).

Description of devices

Plasma HAEE concentrations were measured by HPLC-MS on a XEVO G2-XS Q-TOF mass spectrometer (Waters, Ireland) with an Acquity UPLC H-Class Plus modular HPLC system. The parent quasi-molecular ion and fragment ions were recorded on the mass detector after preliminary extraction of the deproteinized fraction of the biological material (blood plasma) on an ion-exchange solid-phase carrier.

Powder X-ray diffraction (XRD) analysis was performed on a Rigaku Ultima TV diffractometer (Japan), with a tube voltage of 40 kV, a tube current of 30 mA, and a Cu anode material. The goniometer was a vertical Θ/Θ type, with the sample stationary. The goniometer radius was 185 mm/285 mm. The maxima in the diffraction pattern were accumulated over 1 hour. The 2Θ angle ranged from 3 to 70 degrees.

Infrared spectra (IR spectra) were obtained on a Spectrum Two FTIR spectrometer (Perkin Elmer, USA) with an attachment

diffuse reflectance in the range of 4000-600 cm ^-1 with a resolution of 2 cm ^-1 , number of scans - 10.

¹ H NMR spectra were obtained on a Bruker Avarice IIIHD 500 NMR spectrometer, operating frequency 500.13 MHz for ¹ H nuclei.

Drying of frozen samples was performed in penicillin vials using a Heto FD 2.5 sublimator at a pressure of 0.1–0.2 mbar.

The accelerated storage test was conducted in a Memmert HCP50 chamber at 40°C and 75% humidity. Samples were taken monthly for six months, and the active ingredient content was determined using HPLC.

Embodiments of the invention

The invention is illustrated by the following examples, but is not limited thereto.

Example 1. Preparation of peptide-amino acid complexes AA-Zn-HAEE

To obtain peptide-amino acid complexes, zinc acetate, chloride, sulfate, nitrate, glycinate, aspartate, and histidinate were used as a source of zinc at a concentration of 0.01-0.1 M.

To obtain peptide-amino acid complexes, powders of Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met and Thr were used as a source of amino acids.

To obtain the complex, an aqueous solution of 0.01 M HAEE peptide and amino acid solutions with concentrations of 0.01-0.1 M were prepared. Aliquots of the HAEE and corresponding amino acid solutions were mixed in an equimolar ratio, stirred for 5-10 min, and an equimolar amount of 0.1 M zinc salt solution was added and stirred again for 10-15 min. Detailed reagents and their quantities are provided in Table 2. Next, lyophilization or rotary evaporation was performed. Lyophilization was performed on pre-frozen samples at -20°C, shelves were cooled with liquid nitrogen, and the vacuum was 0.2-0.1 mbar.

When using zinc glycinate, aspartate and histidinate, no amino acid solution was added, since the compounds already contain the amino acid.

For water-soluble amino acids, it is preferable to use aqueous solutions. For sparingly water-soluble amino acids, such as Hyp, Pro, and Trp, it is preferable to use ethanol. When using sparingly soluble amino acids, their solutions were prepared at 60°C to increase solubility (Table 2, examples #3 and #7) or their ethanol solutions were used (Table 2, examples #14-16).

All dried samples of peptide-amino acid complexes were white. Product yield was 95-99%.

Figure 11 shows the numbering of hydrogen atoms in the HAEE peptide, which are identified in the ¹ H-NMR spectrum of the free HAEE peptide (Fig. 12) and in the ¹ H-NMR spectra of the peptide-amino acid complexes Gly-Zn-HAEE and Met-Zn-HAEE (Figs. 13, 14).

Table 3 presents the ¹ H NMR chemical shift values (according to Figs. 12–14) for protons in the HAEE peptide, in peptide-amino acid complexes, and in free amino acids, which show shifts of proton signals from 0.03 to 0.2 ppm in the complexes compared to free amino acids and the HAEE peptide. Changes in proton chemical shifts in the ¹ H NMR spectra are associated with changes in proton coordination during the formation of AA-Zn-HAEE structures, which characterizes the existence of peptide-amino acid complexes not only in the solid phase but also in aqueous solutions. Table 3 shows that, in the Gly-Zn-HAEE and Met-Zn-HAEE complexes, there is a mutual influence between the HAEE molecule and the amino acid (Gly, Met) coordinated to the HAEE via a zinc ion. This leads to the formation of new hydrogen bonds in the peptide-amino acid complexes and, accordingly, shifts in the proton chemical shifts relative to the free HAEE peptide molecules and amino acids. The greatest shift in the chemical shifts in the HAEE peptide is observed at the CH(12) group of the imidazole histidine fragment and the α-groups of the glutamine fragments, which is associated with the coordination of the -COOH groups of glutamic acid.

All samples of peptide-amino acid complexes are characterized by an amorphous form. The diffraction pattern (Fig. 1) shows a typical amorphous phase of a peptide-amino acid complex, using Gly-Zn-HAEE as an example. Several amorphous halos can be identified in the region of 13.5, 20, and 23.5 2Θ°.

The Fourier transform IR spectra of the peptide-amino acid complexes are characterized by the appearance of maxima in the region of 1120-1040 cm ^-1 , which distinguishes the peptide-amino acid complexes from free HAEE and the zinc complex HAEE known from Russian Patent No. 2784319. As an example, Figs. 7-10 show the Fourier transform IR spectra of the peptide-amino acid complexes Gly-Zn-HAEE, Met-Zn-HAEE, Ala-Zn-HAEE, and Phe-Zn-HAEE, in which the characteristic maxima are in the region of 1120-1040 cm ^-1 , which can be attributed to the CN val., C-O val., and -NH ₂ groups of amino acids.

For comparison, Fig. 2 shows the IR spectrum of the HAEE peptide, which has only characteristic maxima at 1644, 1530, 1396, and 1249 cm ^-1 . The IR spectra of free amino acids Gly, Met, Ala, and Phe are shown in Figs. 3-6 and are represented by a multitude of high-intensity vibrations in the region of 1700-600 cm ^-1 . After combining the amino acids and the HAEE peptide into a single AA-Zn-HAEE complex, vibrations of the amino acid bonds appear only in the region of 1120-1040 cm ^-1 , which confirms the formation of peptide-amino acid complexes.

The resulting peptide-amino acid complexes were intravenously administered to mice in vivo (wild-type mice), blood plasma was isolated, the peptide fraction of the blood plasma was isolated, which was then loaded onto an HPLC chromatography column (3.6 ng/ml, 0.5% formic acid, 50% methanol), and the HAEE release time from the column was recorded using a mass spectrometric detector. The experiment used free HAEE peptide (n = 5), a zinc complex without the amino acid (n = 5), and the AA-Zn-HAEE complexes obtained in the present invention (n = 90, 5 animals for each peptide-amino acid complex). It was found that for each complex, the release time of HAEE from the column varies statistically significantly and apparently depends on the coordination strength of HAEE, its steric 3D spatial structure in the complex, the size of the molecule, as well as the number and coordination of hydrophobic and electrostatic interactions with the column support (Table 1).

For HAEE intravenously administered to mice in its native form and then extracted from blood samples, the chromatographic peak emergence time was 1.30 min. Under the same experimental conditions, the chromatographic peak emergence time for the zinc complex of HAEE was 2.78 min. It is assumed that the change in the HAEE molecule emergence time from the chromatographic column reflects a change in HAEE conformation. For the peptide-amino acid complexes, the chromatographic peak emergence time was 1.9-2.5 min (Table 1).

The obtained data indicate that after interaction of the HAEE peptide and its complexes with mouse blood plasma elements, the conformations of the native HAEE peptide and the HAEE peptide in the zinc complex differ significantly from the conformation of HAEE in the peptide-amino acid complexes. Thus, the AA-Zn-HAEE peptide-amino acid complexes after interaction with blood plasma elements have a different conformation from the HAEE peptide and the HAEE zinc complex. This, in turn, indicates the stability of the HAEE molecule conformation in the peptide-amino acid complexes, which may enhance the therapeutic efficacy of the proposed complexes.

Example 2. Pharmaceutical compositions based on the zinc complex of the HAEE peptide

Pharmaceutical compositions for parenteral use may contain antimicrobial preservatives, stabilizers, emulsifiers, solubilizers, and other excipients with similar effects. Excipients that enhance the stability of the active ingredients include ascorbic, hydrochloric, tartaric, citric, and acetic acids; sodium carbonate and bicarbonate; sodium hydroxide; potassium or sodium sulfite; sodium hydrosulfite or metabisulfite; sodium thiosulfate; disodium EDTA; sodium citrate; mono- or dibasic sodium phosphate; and antimicrobial preservatives such as methyl parahydroxybenzoate and propyl parahydroxybenzoate; chlorobutanol, cresol, and phenol.

Infusion dosage forms should generally be isotonic with mammalian blood and should not contain antimicrobial preservatives.

The dose of the active ingredient in the pharmaceutical composition is calculated individually and can vary from 0.2 mg to 40 mg per animal per day, with 0.3 to 30 mg being more preferred. Other ratios of the active ingredient in the pharmaceutical composition are also possible.

Pharmaceutical compositions based on peptide-amino acid complexes of the formula AA-Zn-HAEE in the form of a lyophilisate for the preparation of a solution for parenteral use:

1. Gly-Zn-HAEE, lyophilisate, 1.75 mg.

2. Leu-Zn-HAEE, lyophilisate, 2 mg.

3. Tyr-Zn-HAEE, lyophilisate, 2 mg.

4. Glu-Zn-HAEE, lyophilisate, 0.5 mg.

5. Gln-Zn-HAEE, lyophilisate, 0.2 mg.

6. Asp-Zn-HAEE, lyophilisate, 1.5 mg.

7. Phe-Zn-HAEE, lyophilisate, 5 mg.

8. Ala-Zn-HAEE, lyophilisate, 2 mg.

9. Lys-Zn-HAEE, lyophilisate, 1 mg.

10. His-Zn-HAEE, lyophilisate, 10 mg.

11. Cys-Zn-HAEE, lyophilisate, 7.5 mg.

12. Val-Zn-HAEE, lyophilisate, 2 mg.

13. Pro-Zn-HAEE, lyophilisate, 2.5 mg.

14. Hyp-Zn-HAEE, lyophilisate, 0.1 mg.

15. Trp-Zn-HAEE, lyophilisate, 15 mg.

16. Ile-Zn-HAEE, lyophilisate, 2 mg.

17. Met-Zn-HAEE, lyophilisate, 1.5 mg.

18. Thr-Zn-HAEE, lyophilisate, 2.5 mg.

19. Gly-Zn-HAEE 2 mg, methyl parahydroxybenzoate 0.5 mg, disodium edetate 0.5 mg.

20. Met-Zn-HAEE, 1 mg.

Pharmaceutical compositions in the form of a solution for parenteral use (AA denotes any amino acid in the complex):

1. Peptide-amino acid complex AA-Zn-HAEE 1.75 mg, water up to 100%.

2. Peptide-amino acid complex AA-Zn-HAEE 3 mg, isotonic aqueous solution up to 100%.

3. Peptide-amino acid complex AA-Zn-HAEE, 1 mg, 0.2% sodium sulfite, isotonic aqueous solution up to 100%.

4. Peptide-amino acid complex AA-Zn-HAEE 2 mg, 10 zinc sulfate, isotonic aqueous solution 0.5 ml.

5. Peptide-amino acid complex AA-Zn-HAEE 1.5 mg, 1 M hydrochloric acid or 1 M sodium hydroxide - to pH 6.7-7.1, water for injection 1 ml.

Pharmaceutical compositions in the form of a solution for oral administration (AA denotes any amino acid in the complex): - peptide-amino acid complex AA-Zn-HAEE 0.5-15 mg, sorbitol 100 mg; glycerol 50 mg; methyl parahydroxybenzoate - 1.0 mg; propyl parahydroxybenzoate - 0.25 mg; sodium citrate dihydrate - 5 mg; zinc sulfate - 5 mg, potassium sorbate - 4 mg; citric acid to pH 6; purified water to 1 ml.

Pharmaceutical compositions in the form of nasal drops (AA denotes any amino acid in the complex):

1. Peptide-amino acid complex AA-Zn-HAEE 3 mg, methyl parahydroxybenzoate (nipagin) - 1 mg, purified water 0.5 ml.

2. Peptide-amino acid complex AA-Zn-HAEE 5 mg, benzalkonium - 0.15 mg, disodium EDTA - 0.5 mg, potassium dihydrogen phosphate - 4 mg, sodium hydrogen phosphate - 5 mg, sodium chloride - 9 mg, purified water - up to 1 ml. Pharmaceutical compositions in the form of a nasal spray (AA denotes any amino acid in the complex):

- peptide-amino acid complex AA-Zn-HAEE 0.2 mg, Avicel RC-591 (microcrystalline cellulose, sodium carmellose) - 2 mg, glycerol 2 mg, citric acid - 0.2 mg, sodium citrate - 0.24 mg polysorbate-80 - 0.01 mg, benzalkonium chloride - 0.02 mg, water for injection - up to 100 mg. The composition is given for a single injection dose of about 0.1 ml.

Pharmaceutical compositions in the form of syrup (AA denotes any amino acid in the complex):

- peptide-amino acid complex AA-Zn-HAEE 20 mg, sucrose 20 g, flavoring 0.1 g, methyl parahydroxybenzoate 0.07 g, caramel 15 mg, purified water to 50 ml.

Pharmaceutical compositions in the form of gelatin capsules for oral administration (AA denotes any amino acid in the complex):

- peptide-amino acid complex AA-Zn-HAEE 0.5-15 mg, microcrystalline cellulose - 200 mg; povidone K-30 - 15 mg, lactose - 100 mg, colloidal silicon dioxide - 50 mg; magnesium stearate - 2 mg; sodium croscarmellose - 30 mg.

Microcrystalline cellulose, sodium carboxymethyl starch, magnesium stearate, colloidal silicon dioxide.

Pharmaceutical compositions in the form of film-coated tablets (AA denotes any amino acid in the complex):

- peptide-amino acid complex AA-Zn-HAEE 2-20 mg, lactose monohydrate - 100 mg, low-substituted hyprolose - 15 mg, povidone - 5 mg, magnesium stearate - 0.5 mg.

Peptide-amino acid complexes in the form of pharmaceutical substances, lyophilisates, and pharmaceutical compositions were subjected to accelerated storage testing for 6 months at 40°C and 75% humidity. The results are presented in Table 4. The data obtained show that, during this accelerated storage period, all complexes exhibited a statistically significant absence of degradation, with up to 100% of the complexes retained. Thus, the stability of the peptide-amino acid complexes in solid and liquid form over 2 years of normal storage was confirmed.

Example 3. Determination of concentrations of endogenous peptide HAEE in the blood plasma of healthy volunteers and mammals (data were previously published in Russian patents No. 2826728 and 2826726)

Venous blood was collected from healthy volunteers (age range of the generalized sample from 37 to 86 completed years) and from the following mammalian species: (1) domestic goat (age range of the generalized sample from 5 to 10 completed years); (2) domestic bull (age range of the generalized sample from 8 to 13 completed years); (3) domestic horse (age range of the generalized sample from 8 to 14 completed years); (4) domestic sheep (age range of the generalized sample from 4 to 7 completed years); (5) house mouse (age range of the generalized sample from 6 to 9 completed months). Within half an hour after blood collection, aliquots of blood plasma (EDTA-2K+) were prepared and frozen for long-term storage.

The concentration of HAEE in blood plasma is measured using a method combining the separation of high-performance liquid chromatography and registration on a mass detector (HPLC-MS) of the parent quasi-molecular ion and fragment ions after preliminary extraction on an ion-exchange solid-phase carrier of the deproteinized fraction of the biological material (blood plasma). To 100 μl of blood plasma, 10 μl of a solution of a chemically identical, but different in isotopic composition, internal standard HAEE, differing by Am = +2.0067 a.u. or another mass depending on the isotopic composition of the internal standard, were added to 100 μl of blood plasma to a final concentration of 1 ng/ml (1000 pg/ml). Seven volumes of a mixture of organic solvents (methanol, acetonitrile and water) in a ratio of 7:2:1 were added to the mixture of blood plasma and the internal standard. After the addition of the mixture of organic solvents, the formation of a white or milky suspension was observed. The suspension was incubated at 40°C for 10 min with constant stirring at 1100 rpm. After incubation, the suspension was clarified by centrifugation at 15,000 g in an angle rotor at 10°C for 10 min. The supernatant (750 μl) was then quantitatively collected and transferred to 5 ml of a cooled aqueous solution of 2.5% ammonium hydroxide to reduce the final concentration of organic solvents (methanol and acetonitrile) and sensitize the target (isolated) HAEE component and the internal standard under the conditions of the basic reaction medium. The resulting solution of the blood plasma fraction in a 2.5% ammonium hydroxide solution (approximately 5.7 ml) was applied to a strong mixed anion-exchange solid-phase carrier, previously washed sequentially in methanol (1 ml), acetonitrile (1 ml), water (1 ml) and stabilized in a 2.5% aqueous ammonium hydroxide solution (2 ml). After applying the blood plasma fraction (approximately 5.7 ml) to the stabilized carrier, the solid-phase carrier was equilibrated with the bound HAEE and an internal standard in a 2.5% aqueous ammonium hydroxide solution (2 ml).

The carrier was then washed successively in water (1 ml) and in a 60% aqueous methanol solution (1 ml). Coelution of HAEE and the internal standard in a single fraction was accomplished with a solution of 3% formic acid in acetonitrile (0.6 ml). The collected fraction was dried under vacuum in a concentrator at a temperature not exceeding 45°C until a dry residue (complete evaporation of the organic solvent) was obtained. The resulting dry residue was reconstituted in 30 µl of an aqueous 0.1% formic acid solution for subsequent instrumental analysis by HPLC-MS.

The signal of the endogenous peptide HAEE in blood plasma (EDTA-2K+) was recorded by high-performance liquid chromatography combined with mass spectrometry (HPLC-MS) in the positive electrostatic ionization mode and tandem or targeted scanning for precursor and fragment ions. Quantitative assessment was performed based on the area or height of the chromatographic peak of the isolated ion current of the precursor ion or the basic fragment ion-quantifier of the endogenous peptide HAEE and the internal standard [C ₂ ]-HAEE, which differs from HAEE in isotopic composition due to the inclusion of two isotopes [ ¹³ C ₂ ] of the carbon atom, i.e., differs from HAEE by +2.00671 atomic mass units (Fig. 15).

Thus, an aliquot of blood plasma, after the necessary laboratory procedures aimed at obtaining a fraction enriched in HAEE and the internal standard, was analyzed by quantitative mass spectrometry using high-performance liquid chromatography and an internal isotope-labeled standard.

The mean concentration of endogenous HAEE peptide in the studied plasma samples of healthy volunteers (N=10) was determined to be 430±52 pg/ml. This value reflects the physiologically normal concentration of endogenous HAEE peptide in human plasma.

It was also found that the endogenous HAEE peptide was present in the blood plasma of the studied intact mammals (goat, bull, horse, sheep, mouse) in the following physiologically normal concentrations: (1) 5023±227 pg/ml for goat; (2) 1680±75 pg/ml for bull; (3) 1589±176 pg/ml for horse; (4) 2545±271 pg/ml for sheep; (5) 1179±86 pg/ml for mouse.

Example 4. Determination of the concentration of the endogenous HAEE peptide in the blood plasma of patients with confirmed diagnoses of diseases or health disorders associated with neuroinflammation (data were previously published in Russian patents No. 2826728 and 2826726)

Using the quantitative method for analyzing the concentration of the endogenous HAEE peptide in accordance with Example 3 of the present invention, the concentrations of the endogenous HAEE peptide in the blood plasma of patients with confirmed diagnoses of the following diseases or health disorders associated with neuroinflammation were determined: (1) Alzheimer's disease; (2) Parkinson's disease; (3) amyotrophic lateral sclerosis; (4) multiple sclerosis; (5) type II diabetes mellitus; (6) traumatic brain injury; (7) post-COVID syndrome (Table 5).

Based on the measurements performed (Table 5), the following values of average concentrations of the endogenous HAEE peptide in blood plasma were determined: (1) 74±14 pg/ml for patients diagnosed with Alzheimer's disease; (2) 82±18 pg/ml for patients diagnosed with Parkinson's disease; (3) 95±19 pg/ml for patients diagnosed with amyotrophic lateral sclerosis; (4) 186±3 pg/ml for patients diagnosed with multiple sclerosis; (5) 192±37 pg/ml for patients diagnosed with type II diabetes mellitus; (6) 205±59 pg/ml for patients with the consequences of traumatic brain injury; (7) 244±38 pg/ml for patients diagnosed with post-COVID syndrome.

Thus, all patients with confirmed diagnoses of diseases or health disorders associated with neuroinflammation exhibit a significant deficiency of endogenous HAEE peptide in blood plasma compared to the physiologically normal concentration of endogenous HAEE peptide, which, according to the data from Example 3, is 430±52 pg/ml.

Example 5. The use of peptide-amino acid complexes in comparison with known forms of the HAEE peptide to restore the deficiency of endogenous HAEE peptide in the blood plasma of animals associated with neuroinflammation

The possibility of therapeutic treatment of animal diseases or disorders associated with neuroinflammation by restoring physiologically normal concentrations of endogenous HAEE peptide has been demonstrated using animal models (Table 5), including:

(1) Alzheimer's disease model (Jankowsky, J. L., Slunt, H. H., Ratovitski, T., Jenkins, N. A., Copeland, N. G., & Borchelt, D. R. (2001). Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomolecular engineering, 17(6), 157-165);

(2) model of Parkinson's disease (Kuroiwa H, Yokoyama H, Kimoto H, Kato H, Araki T. Biochemical alterations of the striatum in an MPTP-treated mouse model of Parkinson's disease. Metab Brain Dis. 2010 Jun;25(2): 177-83. doi: 10.1007/s11011-010-9195-9);

(3) a model of amyotrophic lateral sclerosis (Deikin A.V., Kovrazhkina E.A., Ovchinnikov R.K., Bronovitsky E.V., Razinskaya O.D., Smirnov A.P., Ermolkevich T.G., Elyakov A.B., Popov A.N., Fedorov E.N., Lytkina O.A., Kukharsky M.S., Tarasova T.V., Shelkovnikova T.A., Ustyugov A.A., Ninkina N.N., Goldman I.L., Sadchikova E.R., Bachurin S.O., Skvortsova V.I. A model of amyotrophic lateral sclerosis based on a line of transgenic mice expressing a mutant form of the human FUS protein. S.S. Korsakov Journal of Neurology and Psychiatry. 2014;114(8):62-69);

(4) model of multiple sclerosis (Levy, N, Yaniv Assaf, and D Frenkel. 2010. 'Characterization of brain lesions in a mouse model of progressive multiple sclerosis', Experimental neurology, 226: 148-58);

(5) model of type II diabetes mellitus (Gilbert, E. R., Z. Fu, and D. Liu. 2011. 'Development of a nongenetic mouse model of type 2 diabetes', Exp Diabetes Res, 2011:416254);

(6) traumatic brain injury model (Yang, X. M., X. H. Chen, J. F. Lu, C. M. Zhou, J. Y. Han, and C. H. Chen. 2018. 'In vivo observation of cerebral microcirculation after experimental subarachnoid hemorrhage in mice', Neural RegenRes, 13: 456–62).

The therapeutic effect on all experimental mice, which were used as the above-mentioned models of diseases or disorders, was exerted in the following manner. Each animal aged 5-6 months was administered intravenously one dose of the peptide-amino acid complex preparation prepared in physiological solution daily for 5 days in comparison with the known forms of the HAEE peptide. Nine peptide-amino acid complexes were tested: (1) Gly-Zn-HAEE; (2) Glu-Zn-HAEE; (3) Asp-Zn-HAEE; (4) Phe-Zn-HAEE; (5) Ala-Zn-HAEE; (6) Arg-Zn-HAEE; (7) His-Zn-HAEE; (8) Cys-Zn-HAEE; (9) Met-Zn-HAEE.

The amount of peptide-amino acid complex in terms of the mass of the HAEE peptide and the mass of the animal in one dose of the drug was 0.02 mg/kg; the following were used as a comparison drug: 0.05 mg/kg for the native HAEE peptide; 0.05 mg/kg for the monosubstituted sodium salt of HAEE; 0.05 mg/kg for the zinc complex of HAEE.

Peptide-amino acid complexes Gly-Zn-HAEE, Glu-Zn-HAEE, Asp-Zn-HAEE, Phe-Zn-HAEE, Ala-Zn-HAEE, Arg-Zn-HAEE, His-Zn-HAEE, Cys-Zn-HAEE, Met-Zn-HAEE were obtained according to the method of preparation described in Example 1.

Monosubstituted sodium salt of HAEE and zinc complex of HAEE were prepared according to Russian patent No. 2784326 and Russian patent No. 2784319, respectively.

For each of the above animal models, 12 groups of mice (n = 10) were formed. Three control groups of animals received comparison drugs (HAEE, sodium salt of HAEE, and zinc complex of HAEE). Nine experimental groups of animals received one of the following drugs: Gly-Zn-HAEE, Glu-Zn-HAEE, Asp-Zn-HAEE, Phe-Zn-HAEE, Ala-Zn-HAEE, Arg-Zn-HAEE, His-Zn-HAEE, Cys-Zn-HAEE, Met-Zn-HAEE.

The effectiveness of the therapeutic effect on each of the 12 groups of mice for each of the animal models studied was assessed based on an analysis of changes in the concentrations of the endogenous HAEE peptide in the blood plasma at three time points: (1) the day before the start of treatment; (2) 3 days after the start of treatment (after three administrations of the corresponding drug); (3) 10 days after the start of treatment.

For 5-6 month old mice used as an animal model of Alzheimer's disease (Jankowsky, J. L., Slunt, H. H., Ratovitski, T., Jenkins, N. A., Copeland, N. G., & Borchelt, D. R. (2001). Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomolecular engineering, 17(6), 157-165), the following measurement results were obtained.

The level of endogenous peptide HAEE the day before the start of treatment was:

(1) 426±90 pg/ml - for the group receiving the HAEE peptide;

(2) 477±84 pg/ml - for the group receiving the mono-substituted sodium salt

NAEE;

(3) 451±73 pg/ml - for the group receiving zinc complex HAEE;

(4) 442±65 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 455±62 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 464±70 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 481±92 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 435±58 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 444±67 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 478±83 pg/ml - for the group receiving His-Zn-HAEE;

(11) 428±79 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 439±68 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 3 days after the start of treatment was:

(1) 587±126 pg/ml - for the group receiving HAEE;

(2) 649±114 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 620±89 pg/ml - for the group receiving zinc complex HAEE;

(4) 592±98 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 626±91 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 656±110 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 643±105 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 672±130 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 610±95 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 580±90 pg/ml - for the group receiving His-Zn-HAEE;

(11) 613±102 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 639±108 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 10 days after the start of treatment was:

(1) 872±202 pg/ml - for the group receiving HAEE;

(2) 959±163 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 899±160 pg/ml - for the group receiving zinc complex HAEE;

(4) 888±172 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 901±161 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 950±178 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 967±159 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 912±126 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 944±139 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 951±147 pg/ml - for the group receiving His-Zn-HAEE;

(11) 932±144 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 990±150 pg/ml - for the group receiving Met-Zn-HAEE.

Thus, in mice aged 5-6 months with an experimental model of Alzheimer's disease, the concentration of endogenous HAEE peptide in blood plasma ranged from 426±90 pg/ml to 481±92 pg/ml, which indicates a significant deficiency of endogenous HAEE peptide compared to the physiologically normal concentration of endogenous HAEE peptide in the blood plasma of mice, which, according to data from Example 3 of the present invention, is 1179±86 pg/ml.

A course of peptide-amino acid complexes statistically significantly restored plasma levels of the endogenous HAEE peptide in patients with Alzheimer's disease to near-normal physiological levels, even at a dosage 2.5 times lower than that of its analogs—the monobasic sodium salt and zinc HAEE complex. Furthermore, increasing HAEE levels to near-normal levels correlated with an improvement in clinical symptoms in Alzheimer's disease, including behavioral reflexes. No statistically significant differences in biological activity were observed between the different peptide-amino acid complexes.

For mice aged 5-6 months used as an animal model of Parkinson's disease (Kuroiwa H, Yokoyama H, Kimoto H, Kato H, Araki T. Biochemical alterations of the striatum in an MPTP-treated mouse model of Parkinson's disease. Metab Brain Dis. 2010 Jun; 25(2):177-183. doi: 10.1007/sl 1011-010-9195-9), the following measurement results were obtained.

The level of endogenous peptide HAEE the day before the start of treatment was:

(1) 440±100 pg/ml - for the group receiving the HAEE peptide;

(2) 464±73 pg/ml - for the group receiving the mono-substituted sodium salt

NAEE;

(3) 437±85 pg/ml - for the group receiving zinc complex;

(4) 454±75 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 423±72 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 450±81 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 418±56 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 472±85 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 465±65 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 433±77 pg/ml - for the group receiving His-Zn-HAEE;

(11) 484±82 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 421±69 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 3 days after the start of treatment was:

(1) 615±135 pg/ml - for the group receiving HAEE;

(2) 640±98 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 601±120 pg/ml - for the group receiving zinc complex HAEE;

(4) 630±88 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 646±92 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 661±99 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 639±101 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 656±110 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 680±105 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 595±87 pg/ml - for the group receiving His-Zn-HAEE;

(11) 633±104 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 654±128 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 10 days after the start of treatment was:

(1) 875±208 pg/ml - for the group receiving HAEE;

(2) 893±142 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 862±187 pg/ml - for the group receiving zinc complex HAEE;

(4) 892±132 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 880±129 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 970±167 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 990±174 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 949±180 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 979±131 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 974±177 pg/ml - for the group receiving His-Zn-HAEE;

(11) 917±165 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 900±140 pg/ml - for the group receiving Met-Zn-HAEE.

Thus, in mice aged 5-6 months with an experimental model of Parkinson's disease, the concentration of the endogenous HAEE peptide in the blood plasma ranged from 418±56 pg/ml to 484±82 pg/ml, which indicates a significant deficiency of the endogenous HAEE peptide compared to the physiologically normal concentration of the endogenous HAEE peptide in the blood plasma of mice, which, according to the data from Example 3 of the present invention, is 1179±86 pg/ml.

A course of peptide-amino acid complexes statistically significantly restored plasma levels of the endogenous HAEE peptide in Parkinson's disease to near-normal physiological levels, even at a dosage 2.5 times lower than that of its analogs—the monobasic sodium salt and zinc HAEE complex. Furthermore, the increase in HAEE levels to near-normal physiological levels correlated with an improvement in clinical symptoms in Parkinson's disease, including compensation for autonomic nervous system dysfunction. No statistically significant differences in biological activity were observed between the different peptide-amino acid complexes.

For mice aged 5-6 months used as an animal model of amyotrophic lateral sclerosis (Deikin A.V., Kovrazhkina E.A., Ovchinnikov R.K., Bronovitsky E.V., Razinskaya O.D., Smirnov A.P., Ermolkevich T.G., Elyakov A.B., Popov A.N., Fedorov E.N., Lytkina O.A., Kukharsky M.S., Tarasova T.V., Shelkovnikova T.A., Ustyugov A.A., Ninkina N.N., Goldman I.L., Sadchikova E.R., Bachurin S.O., Skvortsova V.I. Model of amyotrophic lateral sclerosis based on a line of transgenic mice expressing a mutant form of human FUS protein. S.S. Korsakov Journal of Neurology and Psychiatry. 2014;114(8):62-69), the following measurement results were obtained.

The level of endogenous peptide HAEE the day before the start of treatment was:

(1) 699±73 pg/ml - for the group under the influence of the HAEE peptide;

(2) 728±39 pg/ml - for the group under the influence of monosubstituted sodium salt of HAEE;

(3) 722±68 pg/ml - for the group under the influence of zinc complex HAEE;

(4) 745±77 pg/ml – for the group under the influence of Gly -Zn-HAEE;

(5) 732±75 pg/ml - for the group under the influence of Glu-Zn-HAEE;

(6) 705±69 pg/ml - for the group under the influence of Asp-Zn-HAEE;

(7) 721±86 pg/ml - for the group under the influence of Phe-Zn-HAEE;

(8) 772±81 pg/ml - for the group under the influence of Ala-Zn-HAEE;

(9) 700±68 pg/ml - for the group under the influence of Arg-Zn-HAEE;

(10) 761±92 pg/ml - for the group under the influence of His -Zn-HAEE;

(11) 748±84 pg/ml - for the group under the influence of Cys-Zn-HAEE;

(12) 712±65 pg/ml - for the group under the influence of Met-Zn-HAEE.

The level of endogenous peptide HAEE 3 days after the start of treatment was:

(1) 957±101 pg/ml - for the group under the influence of the HAEE peptide;

(2) 996±68 pg/ml - for the group under the influence of monosubstituted sodium salt of HAEE;

(3) 980±78 pg/ml - for the group under the influence of zinc complex HAEE;

(4) 990±112 pg/ml - for the group under the influence of Gly -Zn-HAEE;

(5) 964±98 pg/ml - for the group under the influence of Glu-Zn-HAEE;

(6) 975±99 pg/ml - for the group under the influence of Asp-Zn-HAEE;

(7) 972±110 pg/ml - for the group under the influence of Phe-Zn-HAEE;

(8) 944±101 pg/ml - for the group under the influence of Ala-Zn-HAEE;

(9) 995±125 pg/ml - for the group under the influence of Arg-Zn-HAEE;

(10) 930±79 pg/ml - for the group under the influence of His -Zn-HAEE;

(11) 967±119 pg/ml - for the group under the influence of Cys-Zn-HAEE;

(12) 940±115 pg/ml - for the group under the influence of Met-Zn-HAEE.

The level of endogenous peptide HAEE 10 days after the start of treatment was:

(1) 1401±224 pg/ml - for the group under the influence of the HAEE peptide;

(2) 1509±127 pg/ml - for the group under the influence of monosubstituted sodium salt of HAEE;

(3) 1515±163 pg/ml - for the group under the influence of zinc complex HAEE;

(4) 1432±191 pg/ml - for the group under the influence of Gly -Zn-HAEE;

(5) 1492±180 pg/ml - for the group under the influence of Glu-Zn-HAEE;

(6) 1570±149 pg/ml - for the group under the influence of Asp-Zn-HAEE;

(7) 1483±166 pg/ml - for the group under the influence of Phe-Zn-HAEE;

(8) 1521±210 pg/ml - for the group under the influence of Ala-Zn-HAEE;

(9) 1513±200 pg/ml - for the group under the influence of Arg-Zn-HAEE;

(10) 1500±195 pg/ml - for the group under the influence of His -Zn-HAEE;

(11) 1456±173 pg/ml - for the group under the influence of Cys-Zn-HAEE;

(12) 1507±168 pg/ml - for the group under the influence of Met-Zn-HAEE.

Thus, in mice aged 5-6 months with an experimental model of amyotrophic lateral sclerosis, the concentration of endogenous HAEE peptide in blood plasma ranged from 699±73 pg/ml to 761±92 pg/ml, which indicates a deficiency of endogenous HAEE peptide compared to the physiologically normal concentration of endogenous HAEE peptide in the blood plasma of mice, which, according to data from Example 3 of the present invention, is 1179±86 pg/ml.

A course of peptide-amino acid complexes statistically significantly restored and even slightly increased plasma levels of endogenous HAEE peptide in patients with amyotrophic lateral sclerosis to physiological levels, even at a dosage 2.5 times lower than that of analogs—sodium HAEE monobasic salt and zinc HAEE complex. Furthermore, increasing HAEE levels to physiological levels correlated with an improvement in clinical symptoms in amyotrophic lateral sclerosis, including a reduction in motor dysfunction and neuroinflammation (astrogliosis). No statistically significant differences in biological activity were observed between the different peptide-amino acid complexes.

For 5-6 month old mice used as an animal model of multiple sclerosis (Levy, H, Yaniv Assaf, and D Frenkel. 2010. 'Characterization of brain lesions in a mouse model of progressive multiple sclerosis', Experimental neurology, 226: 148-58), the following measurement results were obtained.

The level of endogenous peptide HAEE the day before the start of treatment was:

(1) 730±70 pg/ml - for the group receiving HAEE;

(2) 726±62 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 713±58 pg/ml - for the group receiving zinc complex HAEE;

(4) 705±72 pg/ml - for the group receiving Gly -Zn-HAEE:

(5) 722±75 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 715±68 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 712±83 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 742±88 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 709±72 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 731±82 pg/ml - for the group receiving His-Zn-HAEE;

(11) 725±79 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 714±66 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 3 days after the start of treatment was:

(1) 1007±73 pg/ml - for the group receiving HAEE;

(2) 1007±99 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 979±64 pg/ml - for the group receiving zinc complex HAEE;

(4) 994±72 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 1031±95 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 989±69 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 1021 ±96 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 972±83 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 1000±80 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 1060±95 pg/ml - for the group receiving His-Zn-HAEE;

(11) 1041 ±87 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 1014±76 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 10 days after the start of treatment was:

(1) 1431±131 pg/ml - for the group receiving HAEE;

(2) 1463±194 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 1386±162 pg/ml - for the group receiving zinc complex HAEE;

(4) 1154±127 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 1231±117 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 1203±112 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 1225±122 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 1176±119 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 1214±139 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 1332±191 pg/ml - for the group receiving His-Zn-HAEE;

(11) 1287±144 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 1358±154 pg/ml - for the group receiving Met-Zn-HAEE.

Thus, in mice aged 5-6 months with an experimental model of multiple sclerosis, the concentration of the endogenous HAEE peptide in the blood plasma ranged from 705±72 pg/ml to 742±88 pg/ml, which indicates a deficiency of the endogenous HAEE peptide compared to the physiologically normal concentration of the endogenous HAEE peptide in the blood plasma of mice, which, according to data from Example 3 of the present invention, is 1179±86 pg/ml.

A course of peptide-amino acid complexes statistically significantly restored plasma levels of the endogenous HAEE peptide to physiological levels in patients with multiple sclerosis, even at a dosage 2.5 times lower than that of analogs—sodium HAEE monobasic salt and zinc HAEE complex. Furthermore, increasing HAEE levels to physiological levels correlated with an improvement in clinical symptoms in multiple sclerosis, including a reduction in limb muscle weakness and an increase in horizontal and vertical activity. No statistically significant differences in biological activity were observed between the different peptide-amino acid complexes.

For 5-6 month old mice used as an animal model of type 2 diabetes mellitus (Gilbert, E. R., Z. Fu, and D. Liu. 2011. 'Development of a nongenetic mouse model of type 2 diabetes', Exp Diabetes Res, 2011: 416254), the following measurement results were obtained.

The level of endogenous peptide HAEE the day before the start of treatment was:

(1) 668±168 pg/ml - for the group receiving HAEE;

(2) 628±186 pg/ml - for the group receiving the monosubstituted sodium salt of HAEE;

(3) 699±177 pg/ml - for the group receiving zinc complex HAEE;

(4) 692±153 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 669±165 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 654±189 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 661±176 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 675±181 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 632±165 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 640±171 pg/ml - for the group receiving His-Zn-HAEE;

(11) 657± 159 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 670±175 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 3 days after the start of treatment was:

(1) 902±223 pg/ml - for the group receiving HAEE;

(2) 859±274 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 980±251 pg/ml - for the group receiving zinc complex HAEE;

(4) 890±221 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 865±189 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 874±199 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 926±211 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 934±200 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 981±265 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 970±248 pg/ml - for the group receiving His-Zn-HAEE;

(11) 976±279 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 910±256 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 10 days after the start of treatment was:

(1) 1386±351 pg/ml - for the group receiving HAEE;

(2) 1250±415 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 1419±344 pg/ml - for the group receiving zinc complex HAEE;

(4) 1190±321 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 1246±397 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 1312±401 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 1164±399 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 1255±383 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 1321±367 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 1273±386 pg/ml - for the group receiving His-Zn-HAEE;

(11) 1371±409 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 1250±375 pg/ml - for the group receiving Met-Zn-HAEE.

Thus, in mice aged 5-6 months with an experimental model of type II diabetes mellitus, the concentration of endogenous HAEE peptide in blood plasma ranged from 628±186 pg/ml to 699±177 pg/ml, which indicates a significant deficiency of endogenous HAEE peptide compared to the physiologically normal concentration of endogenous HAEE peptide in the blood plasma of mice, which, according to data from Example 3 of the present invention, is 1179±86 pg/ml.

A course of peptide-amino acid complexes statistically significantly restored and even slightly exceeded the endogenous HAEE peptide level in plasma in patients with type 2 diabetes mellitus to physiological levels, even at a dosage 2.5 times lower than that of its analogs—sodium HAEE monobasic salt and zinc HAEE complex. Furthermore, the increase in HAEE levels to physiological levels correlated with an improvement in clinical symptoms in type 2 diabetes mellitus, including a reduction in hyperglycemia and weight loss compared to the control group. No statistically significant differences in biological activity were observed between the different peptide-amino acid complexes.

For 5-6 month old mice used as an animal model of traumatic brain injury (Yang, X. M., X. H. Chen, J. F. Lu, C. M. Zhou, J. Y. Han, and C. H. Chen. 2018. 'In vivo observation of cerebral microcirculation after experimental subarachnoid hemorrhage in mice', Neural Regen Res, 13: 456-62), the following measurement results were obtained.

The level of endogenous peptide HAEE the day before the start of treatment was:

(1) 574±173 pg/ml - for the group receiving HAEE;

(2) 760±136 pg/ml - for the group receiving mono-substituted sodium salt

NAEE;

(3) 746±123 pg/ml - for the group receiving zinc complex HAEE;

(4) 682±131 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 698±148 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 657±169 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 631±162 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 700±151 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 642±145 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 690±170 pg/ml - for the group receiving His-Zn-HAEE;

(11) 676±149 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 680±155 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 3 days after the start of treatment was:

(1) 795±251 pg/ml - for the group receiving HAEE;

(2) 1052±172 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 1026±175 pg/ml - for the group receiving zinc complex HAEE;

(4) 884±181 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 988±171 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 975±169 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 843±183 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 894±197 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 1010±245 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 864±190 pg/ml - for the group receiving His-Zn-HAEE;

(11) 891 ±218 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 806±195 pg/ml - for the group receiving Met-Zn-HAEE.

The level of endogenous peptide HAEE 10 days after the start of treatment was:

(1) 1138±389 pg/ml - for the group receiving HAEE;

(2) 1534±314 pg/ml - for the group receiving monosubstituted sodium salt of HAEE;

(3) 1510±305 pg/ml - for the group receiving zinc complex HAEE;

(4) 1200±281 pg/ml - for the group receiving Gly -Zn-HAEE;

(5) 1197±273 pg/ml - for the group receiving Glu-Zn-HAEE;

(6) 1430±265 pg/ml - for the group receiving Asp-Zn-HAEE;

(7) 1380±276 pg/ml - for the group receiving Phe-Zn-HAEE;

(8) 1294±297 pg/ml - for the group receiving Ala-Zn-HAEE;

(9) 1416±347 pg/ml - for the group receiving Arg-Zn-HAEE;

(10) 1265±290 pg/ml - for the group receiving His-Zn-HAEE;

(11) 1342±318 pg/ml - for the group receiving Cys-Zn-HAEE;

(12) 1344±297 pg/ml – for the group receiving Met-Zn-HAEE.

Thus, in mice aged 5-6 months with an experimental model of traumatic brain injury, the concentration of the endogenous HAEE peptide in the blood plasma ranged from 574±173 pg/ml to 760±136 pg/ml, which indicates a deficiency of the endogenous HAEE peptide compared to the physiologically normal concentration of the endogenous HAEE peptide in the blood plasma of mice, which, according to data from Example 3 of the present invention, is 1179±86 pg/ml.

A course of peptide-amino acid complexes increased plasma levels of the endogenous HAEE peptide in patients with normal traumatic brain injury, even at a dosage 2.5 times lower than that of analogs—the monobasic sodium salt and zinc complex of HAEE. Furthermore, increasing HAEE levels to physiological levels correlated with an improvement in clinical symptoms in traumatic brain injury, including the absence of fatalities and the preservation of the number of neurons with normal morphology in histological sections. No statistically significant differences in biological activity were observed between the different peptide-amino acid complexes.

Taken together, the above data indicate that, in diseases or health conditions associated with neuroinflammation, endogenous HAEE deficiency is effectively restored by therapeutic use of AA-Zn-HAEE peptide-amino acid complexes at lower doses compared to known forms of HAEE preparations. In each case, increasing endogenous HAEE concentrations increased the clinical signs of a positive therapeutic effect.

Thus, the claimed invention provides the possibility of restoring the physiologically normal concentration of endogenous HAEE in the blood in diseases or health disorders of animals associated with neuroinflammation and characterized by a decrease in the level of endogenous HAEE in the blood compared to the physiological norm.

The use of peptide-amino acid complexes for the treatment of diseases and health disorders in animals associated with neuroinflammation was accompanied by an improvement in the health of experimental animals and a weakening of the clinical manifestations of the corresponding diseases or disorders in these animals.

Claims (13)

1. Peptide-amino acid complex of the formula AA-Zn-HAEE for restoring the deficiency of endogenous HAEE in an animal, where AA is an amino acid selected from Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met or Thr, Zn is a divalent zinc(II) cation, HAEE is a synthetic peptide, acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu. 2. A pharmaceutical composition for the treatment of diseases and health disorders in animals associated with neuroinflammation, containing an effective amount of a peptide-amino acid complex of the formula AA-Zn-HAEE, where AA is an amino acid selected from Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met or Thr, Zn is a divalent zinc(II) cation, HAEE is a synthetic peptide, acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu. 3. The pharmaceutical composition according to item 2, additionally including a set of auxiliary substances. 4. The use of a peptide-amino acid complex of the formula AA-Zn-HAEE for the treatment of diseases and health disorders in animals associated with neuroinflammation, where AA is the amino acid Gly, Leu, Tyr, Ser, Glu, Gln, Asp, Asn, Phe, Ala, Lys, Arg, His, Cys, Val, Pro, Hyp, Trp, Ile, Met, or Thr, Zn is a divalent zinc(II) cation, HAEE is a synthetic peptide, acetylated at the N-terminus and amidated at the C-terminus, with the amino acid sequence His-Ala-Glu-Glu.