WO2024014982A1 - Complexe cuivré de peptide haee pour traiter des maladies neurodégénératives - Google Patents

Complexe cuivré de peptide haee pour traiter des maladies neurodégénératives Download PDF

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WO2024014982A1
WO2024014982A1 PCT/RU2023/000209 RU2023000209W WO2024014982A1 WO 2024014982 A1 WO2024014982 A1 WO 2024014982A1 RU 2023000209 W RU2023000209 W RU 2023000209W WO 2024014982 A1 WO2024014982 A1 WO 2024014982A1
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haee
copper complex
copper
peptide
complex
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PCT/RU2023/000209
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Russian (ru)
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Сергей Александрович КОЗИН
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Общество с ограниченной ответственностью "ЛАЙФМИССИЯ"
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Publication of WO2024014982A1 publication Critical patent/WO2024014982A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic

Definitions

  • the present invention also relates to a method for producing a copper complex of the HAEE peptide, to pharmaceutical compositions based on it, and to the use of a copper complex for the treatment of neurodegenerative diseases.
  • Neurodegenerative diseases have a number of common development factors and a number of other general mechanisms (Litvinenko I.V., Krasakov I.V., Bisaga G.N. et al. Modem conception of the pathogenesis of neurodegenerative diseases and therapeutic strategy. Zhumal Nevrologii i Psikhiatrii imeni S.S. Korsakova. 2017;l 17( 6-2):3-10.
  • Inflammation is one of the typical pathological processes of the human body.
  • a typical pathological process is manifested by a certain set of characteristic signs. It is currently believed that the neurodegenerative process is secondary to the inflammatory one (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 IV, Krasakov IV, Bisaga GN et al. Modem conception of the pathogenesis of neurodegenerative diseases and therapeutic strategy. Zhumal Nevrologii i Psikhiatrii imeni SS Korsakova. 2017;l 17(6-2):3-10. (In Russ.). doi: 10.17116/jnevro2017117623-10).
  • Alzheimer's disease is one of the most common neurodegenerative diseases among older people and is characterized by neuritic plaques, the main component of which is the beta-amyloid (Ap) peptide.
  • the N-terminal 1-16 region of amyloid beta is the metal-binding domain where zinc (Zn) and copper (Cu) bind to peptide A (Guilloreau L., Damian L., Coppel Y., et al. (2006). Structural and thermodynamic properties of Cull amyloid-P 16/28 complexes associated with Alzheimer's disease. JBIC Journal of Biological Inorganic Chemistry, 11(8), 1024-1038, doi: 10.1007/s00775-006-0154-1).
  • Reducing the level of zinc ions in the blood by binding zinc ions with specific chelating agents and/or blocking the interactions of the AR peptide with zinc ions can prevent pathologies associated with these interactions (Ayton S., Lei R., Bush A. Biometals and Their Therapeutic Implications in Alzheimer's Disease. Neurotherapeutics. 2015. 12(1): pp. 109-120).
  • High concentrations of zinc cause precipitation of the Ap peptide and affect the formation in specific parts of the brain (hippocampus, cortex, thalamic nuclei) of extracellular Ap aggregates (amyloid plaques), 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.
  • HAEE Ac-HAEE-NH 2
  • the letters mean: H - histidine, A - alanine, E - glutamic acid.
  • HAEE is capable of inhibiting the interaction of AP peptides with Zn(II) ions, causing a decrease or prevention of aggregation of peptide A in the presence of Zn(II) ions at physiological concentrations of Zn(II) ions of 100-400 ⁇ M.
  • RF patent No. 2709539 From RF patent No. 2709539 it is known that short charged peptides of the HAEE type have a very short lifetime in the blood plasma (from several seconds to several minutes) and hardly pass through the blood-brain barrier (BBB), which significantly weakens the effectiveness of their therapeutic effects.
  • BBB blood-brain barrier
  • the authors of RF patent No. 2709539 have developed a pharmaceutical composition for the delivery of HAEE across the BBB for the treatment of neurodegenerative diseases, including dementia of the Alzheimer's type (Alzheimer's disease), which can improve the pharmacokinetic characteristics and increase 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 a range of neutral carriers and diluents, for example, saline.
  • a pharmaceutically acceptable carrier selected from a range of neutral carriers and diluents, for example, saline.
  • RF patent No. 2709539 The invention according to RF patent No. 2709539 was chosen as the closest analogue, since it is the only complex known for HAEE. From RF patent No. 2709539 it is known that the HAEE peptide forms a specific ternary complex (HAEE-Zn-HSA) with human albumin (HSA) in the presence of zinc ions. However, in the absence of zinc ions, no interactions between HAEE and HSA were observed. This complex is characterized by the fact that with the help of a zinc ion it was possible to obtain intermolecular binding between the HAEE and HSA molecules. The zinc cation is the coordinating cation between these two molecules. This complex was not obtained in solid form. From RF patent No. 2709539 it is known 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.
  • albumin in a neutral environment is -1, in an alkaline environment it is -2, and in a slightly acidic environment it is 0 (isoelectric state) and albumin precipitates (Biochemistry with exercises and tasks. Edited by A.I. Glukhov, E. S. Severina, GEOT AR-Media, Moscow, 2019, p. 19), therefore this complex should not exist in a slightly acidic environment at pH 4-5.5.
  • HAEE-Zn-HSA suitable for preclinical studies, 5 mg of HAEE, 600 mg of human albumin and 0.5 mg of zinc chloride (ZnCh) were dissolved in 20 milliliters of physiological solution. A lyophilized preparation of the synthetic HAEE peptide (purity more than 98%) was used as a source of HAEE. With the 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 moles.
  • the disadvantage of this invention is the high content of human albumin, which is 120 times greater by weight than the content of the active substance HAEE, as well as the high content of zinc, which is 2.6 times greater by weight than the content of the active substance HAEE and can lead to the formation of a complex with a non-stoichiometric ratio of the constituents components into it.
  • Zinc is a significantly more toxic element compared to elements present in significant quantities in the body, such as sodium and calcium.
  • HSA bovine serum albumin
  • HSA can induce an immune response in mice after its administration (Favoretto V. S., Ricardi R., Silva, S. R. et al. (2011). Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin. Toxicon, 57(4), 600-607, doi: 10.1016/j.toxicon.2010.12.023).
  • HSA can be used to improve cognitive function in the treatment of Alzheimer's disease, but only when administered intracerebroventricularly (intracranial). Administration of HSA to blood plasma may reduce its effectiveness (Ezra, A., Rabinovich-Nikitin, L, Rabinovich-Toidman, R., & Solomon, V. (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-l 50694).
  • HAEE and the HAEE-Zn-HSA complex have not previously been characterized in the solid phase and their properties in the solid phase are unknown.
  • the tasks to be solved by the invention are to obtain an effective remedy for the treatment of neurodegenerative diseases in the form of a previously unknown copper complex of the HAEE peptide, including in solid form, to develop pharmaceutical compositions based on it and its use for the treatment of neurodegenerative diseases.
  • the molar content of copper (II) cation and HAEE peptide is equimolar (1:1).
  • the claimed copper complex of the HAEE peptide is described by the formula [Cu(HAEE)]Xr.
  • the claimed copper complex of the HAEE peptide is described by the formula [Cu(HAEE)]X.
  • the claimed copper complex of the HAEE peptide is described by the formula [Cu(HAEE)]3X2.
  • the interaction of the HAEE peptide with a copper ion was also observed at other HAEE:Cu molar ratios, namely, in the range from 1:0.1 to 1:10, which is due to the presence of many donor-acceptor centers (nitrogen, oxygen) in the HAEE molecule and the interaction between HAEE and Cu may be nonstoichiometric.
  • the use of a higher copper content (>1:10) led to the production of a solid mixture of the copper complex of the HAEE peptide and the corresponding copper salt; when using a small amount of copper ( ⁇ 1:0.1), its interaction with the HAEE peptide was not detected physically. chemical methods.
  • the use of HAEE-Zn-HSA improves the pharmacokinetic parameters of the HAEE peptide
  • the use of the copper complex of the HAEE peptide for the treatment of neurodegenerative diseases changes the mechanism of action of the HAEE peptide associated with the conformational structure of the HAEE peptide, which leads to an unexpectedly high therapeutic effect.
  • the EG structure of the copper complex of the HAEE peptide remained unchanged under similar experimental conditions (that is, after extraction of the copper complex of the HAEE peptide from the initial aqueous solution and after extraction of this complex from the blood plasma of wild-type mice after a 2-minute exposure) and corresponded SV structure of the native HAEE peptide extracted from the original aqueous solution.
  • mass spectrometry data the chemical structure of the HAEE peptide was preserved in all experimental samples unchanged both for the native peptide and for the peptide in the copper complex.
  • the change in the release time of the native HAEE peptide or the copper complex of the HAEE peptide from the chromatographic column is mainly due to differences in the hydrophobic interactions of the HAEE peptide with the column material, and these interactions, in turn, are determined by the prevailing 3 D structure of the HAEE peptide in a particular sample.
  • the 3 D structure of the HAEE peptide which corresponds to the “unfolded” conformation of the peptide and is prevalent for the native HAEE peptide in the original aqueous solution, corresponds to the 3 D structure of the copper complex of the HAEE peptide in an aqueous solution and remains unchanged in the case of exposure of the copper complex of the HAEE peptide in the blood plasma, while the native HAEE peptide in the blood plasma loses its “unfolded” conformation.
  • the term 3 D-structure is used as a synonym for spatial structure or the relative arrangement of atoms in a HAEE molecule in three-dimensional space.
  • the HPLC method is based primarily on intermolecular interactions at the interface.
  • the change in the release time of HAEE from the chromatographic column reflects a change in the intermolecular interactions of HAEE with the sorbent, which is a consequence of differences in the spatial structure of HAEE molecules in the native state and in the form of a copper complex.
  • 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 the maintenance biologically significant “unfolded” conformation of this peptide and, as a result, reduces the beneficial therapeutic effects of HAEE.
  • one of the advantages of this invention is the increase in the therapeutic effectiveness of the copper complex of the HAEE peptide due to the preservation of the 3 D structure of this complex in the “unfolded” conformation.
  • the difference between the present invention and its closest analogue, RF patent No. 2709539 is that the claimed copper complex does not contain HSA and zinc, which has a reduced molar concentration of ions compared to the HAEE-Zn-HSA complex with 2.6, as described in the example of RF patent No. 2709539, up to 1 (equimolar amount), as described in the present invention.
  • the invention also relates to the use of a copper complex for the treatment of neurodegenerative diseases, as well as pathologies accompanied by neuroinflammatory processes.
  • the inventive copper complex can effectively influence neurodegenerative diseases, in particular those caused by inflammatory causes, restoring impaired cognitive functions.
  • Copper complex can be used in either solid or liquid form and remains stable for at least 2 years.
  • the technical result of the invention is:
  • the copper complex claimed in the present invention in accordance with the production method used, can be obtained in an amorphous form, as confirmed by x-ray phase analysis (Fig. 1).
  • the copper complex is characterized by a differential scanning calorimetry (DSC) thermograviometry (TG) curve (DSC-TGA).
  • DSC differential scanning calorimetry
  • TG thermograviometry
  • the HAEE molecule on the DSC curve is characterized by: a broad peak with an onset at 62 °C and a maximum at 89 °C; a double melting peak at 203 and 227.5 °C with an onset at 188 °C ( Figure 2).
  • the error for multiple measurements is estimated at ⁇ 3 °C.
  • the claimed copper complex on the DSC curve is characterized by three maxima: the first broad endothermic maximum starts at 70 °C and has a maximum at 88 °C; the second exothermic maximum is observed at a temperature of 174 °C, the third endothermic broad maximum is observed at 250-255 °C (Fig. 3).
  • DSC curves show that the thermal behavior of the copper complex (Figure 3) is significantly different from the behavior of HAEE ( Figure 2), the melting point of the complex is higher compared to the melting point of HAEE, which provides higher stability of the complex, as confirmed by the results of the accelerated storage test .
  • the IR spectrum with Fourier transform of the proposed copper complex is presented in Fig. 4.
  • the inventive copper complex can be obtained in the form of a lyophilisate using standard freeze-drying.
  • the preparation of the copper complex involves mixing aqueous solutions of HAEE and a water-soluble copper salt in equimolar ratios at room temperature, stirring for 10-60 minutes, freezing the solution and freeze-drying.
  • Water-soluble copper salts (up to several wt.%) are nitrate, acetate, chloride, salicylate, lactate, gluconate.
  • anion exchange chromatography or dialysis is performed to remove the primary anion.
  • Another appropriate pharmaceutically acceptable anion is then added to the solution, the solution is frozen and freeze-dried.
  • the anions can be single-, double- or triple-charged and selected from the group of glycinate, tartrate, citrate, etc.
  • the need to remove anions from solution is due to the replacement of one anion with another anion due to the poor solubility of these copper salts.
  • the presence of anions of different nature in the composition of the complex in the liquid or solid phase does not affect the therapeutic effectiveness of the copper complex. It is assumed that only the inner sphere of the copper complex exhibits therapeutic efficacy, regardless of the nature of the anion.
  • the anions for the copper complex are selected from the group, trifluoroacetate, pyruvate, galacturonate, bromide, glutarate, succinate, maleate, fumarate, benzenesulfonate, tosylate and other pharmaceutically acceptable anions.
  • Another object of the invention is pharmaceutical compositions with an effective amount of a copper complex and the presence of excipients.
  • the copper complex can be used without excipients in the form of a lyophilized substance.
  • the effective amount of copper complex depends on the type of neurodegenerative disease, body weight, route of administration, and can therefore vary widely from 0.1 to 100 mg, more preferably from 5 to 50 mg.
  • the copper complex pharmaceutical composition may be in solid form or in the form of an aqueous solution.
  • Solid compositions of the copper complex contain an effective amount thereof and optionally excipients.
  • the solid pharmaceutical composition can be prepared by lyophilizing the copper complex and a set of auxiliary components or adding excipients to the lyophilized copper complex.
  • the lyophilized copper complex can be in an amorphous form, which corresponds to the diffraction pattern in Fig. 1.
  • Excipients are selected from pharmaceutically acceptable additives. Such substances may be mannitol, povidone K 17, trometamol, disodium edetate, sodium chloride, sucrose, histidine, poloxamer 407, and other pharmaceutically acceptable additives.
  • the pharmaceutical composition of the copper complex in the form of an aqueous solution contains an effective amount of the copper complex, water, as well as a set of pharmaceutically acceptable additives and salts.
  • Such substances may be mannitol, povidone K 17, trometamol, disodium edetate, sodium chloride, sucrose, histidine, poloxamer 407, and other pharmaceutically acceptable additives.
  • Another object of the invention is the use of a copper complex for the treatment of neurodegenerative diseases, which consists of administering a copper complex as part of the described pharmaceutical compositions to a patient in an effective amount.
  • the effective amount of copper complex depends on the type of neurodegenerative disease, body weight, route of administration, and can therefore vary widely from 0.1 to 100 mg, preferably from 5 to 50 mg.
  • the administration of the copper complex to the patient can be carried out using all possible types of external, enteral, inhalation and parenteral methods of administration (including intravenous, intraarterial, intraperitoneal, subcutaneous, cutaneous, transdermal, intramuscular, intrathecal, subarachnoid, oral, intranasal, sublingual, buccal, rectal administration ).
  • external, enteral, inhalation and parenteral methods of administration including intravenous, intraarterial, intraperitoneal, subcutaneous, cutaneous, transdermal, intramuscular, intrathecal, subarachnoid, oral, intranasal, sublingual, buccal, rectal administration.
  • the preferred routes of administration are intravenous and intranasal.
  • the copper complex [Cu(HAEE)]C12 was administered six times intravenously at a dose of 0.05 mg/kg, after which a valid test for cognitive abilities “Marble Burying Test” was performed [Santana-Santana M. , Bayascas J. R., Gimenez-Llort L. (2021). Sex-Dependent Signatures, Time Frames and Longitudinal Fine-Tuning of the Marble Burying Test in Normal and AD-Pathological Aging Mice. Biomedicines, 9(8), 994, doi: 10.3390/biomedicines9080994), quantifying more than 2/3 of the buried beads as a percentage of the total number of beads. The greater the number of buried balls (BPS), the higher the cognitive abilities of mice.
  • BPS number of buried balls
  • the value of the CR was 50.6 ⁇ 13.3%. This value was taken as a reference value.
  • the value of the CVS 12.6 ⁇ 4.2%, which indicates a strong deterioration in the behavioral reflexes of transgenic mice of the APP/PS1 line compared to wild-type animals and reflects the disabling effect overexpression of human beta-amyloid, associated with neuroinflammation and the formation of amyloid plaques, on the processes of nervous activity.
  • the claimed copper complex can be effectively used in the treatment of neurodegenerative diseases, in particular those caused by inflammatory complications, restoring cognitive functions to normal.
  • Histochemical analysis of the hippocampal region of the brain of experimental mice showed that the number of plaques (PB) per brain section in the control group of wild mice was zero, in the control group of transgenic APP/PS1 mice the PB value was 31.7 ⁇ 4.9, in the group those receiving HAEE, the BF value was 24.7 ⁇ 3.4; in the group receiving the copper complex, the BF value was 9.5 ⁇ 3.2.
  • One of the mechanisms of action of the copper complex on the possibility of treating neurodegenerative diseases may be its binding to beta-amyloid.
  • the HAEE peptide did not bind to beta-amyloid under similar experimental conditions.
  • This difference between the native HAEE and the copper complex may affect the pharmacological properties, which was confirmed by different tests of cognitive function restoration - the effectiveness of HAEE was significantly lower than the effectiveness of the claimed copper complex.
  • This effect can be related to the above-mentioned HPLC-MS data, which showed changes in the 3D structure of native HAEE after its introduction into the blood plasma, while the copper complex before and after its introduction into the blood plasma has the same retention time in the HPLC chromatogram and, accordingly, a stable 3D structure.
  • Fig. 1 X-ray diffraction pattern of the copper complex [Cu(HAEE)]C12.
  • Fig. 4 IR spectrum with Fourier transform of HAEE (1) and copper complex [Cu(HAEE)]C1 2 (2).
  • Powder X-ray phase analysis was performed on a Rigaku Ultima IV diffractometer (Japan), tube voltage - 40 kV, tube current - 30 mA, tube anode material - Cu.
  • Goniometer 0/0 vertical type, the sample is motionless.
  • the maxima in the diffraction pattern accumulated over 1 hour.
  • the angle 20 ranged from 3 to 70 degrees.
  • Elemental analysis was performed by energy-dispersive X-ray spectroscopy with an EDXA attachment on a TESCAN MIRA3 microscope (Czech Republic). The area of the measured area is 0.04 mm 2 , the number of measurements is 3.
  • Infrared radiation spectra were obtained on a Spectrum Two IR-Fourier spectrometer (Perkin Elmer, USA) with a diffuse reflection attachment in the range of 4000-600 cm'1 with a resolution of 2 cm'1 , the number of scans was 10.
  • the formation of the complex was detected by a biosensor based on the effect of surface plasmon resonance (SPPR) in aqueous buffer systems at physiological pH values.
  • SPPR surface plasmon resonance
  • BPPR experiments were carried out on a BIAcore 3000 instrument (GE Healthcare, USA) using an CM4 optical chip in accordance with the manufacturer’s protocols.
  • the ligand (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAGGGGC) was immobilized on the surface of the optical chip through the disulfide bond of the thiol group of the C-terminal cysteine residue. Copper complex or HAEE were used as analytes.
  • Drying of frozen samples was carried out in penicillin vials on a Heto FD 2.5 sublimator at a pressure of 0.1 -0.2 mbar.
  • the accelerated storage test was carried out in a Memmert HCP50 chamber at 40 °C and 75% humidity. Samples were taken once a month for 6 months and the content of the active substance was determined by HPLC.
  • Example 1 Preparation of the copper complex of the HAEE peptide. To obtain the complex, copper acetate, copper chloride, and other salts were used as a source of copper in different proportions. Table 1 shows various ratios of copper salts to obtain the complex.
  • HAEE a solution of HAEE with a concentration of 10' 5 M was prepared.
  • Mixing was carried out for 5-60 minutes.
  • Equimolar copper complexes were formed at a molar ratio of HAEE and copper salts equal to 1:1.
  • the solution was slowly frozen in a refrigeration unit at -20 or -80 °C and then freeze-dried in a standard manner. Next, the properties of the resulting lyophilized copper complex were studied in detail.
  • a water-soluble copper salt (nitrate, acetate, chloride) was used in the first stage; the anion was removed in a known manner - anion exchange chromatography or dialysis and another compound - tartaric acid, citric acid, etc. - was added to the solution in equimolar quantities. After that, similar operations were performed to freeze and freeze-dry the copper complex.
  • obtaining a copper complex [Cu(HAEE)] m X n where X is benzoate, salicylate, tartrate, citrate, etc. was carried out in three stages.
  • a solution of the copper complex was prepared according to the ratios described in Table 1, then inorganic anions were separated chromatographically and added to obtain benzoate - benzoic acid, salicylate - salicylic acid, tartrate - tartaric acid, citrate - citric acid in equimolar quantities.
  • the resulting solutions were frozen in a freezer (-20 °C) in penicillin vials for 2 hours.
  • the frozen samples were placed in a sublimator with pre-cooled shelves and dried for 16-30 hours. All dried samples were green color.
  • the resulting solid samples of the complex are characterized by an amorphous shape in the diffraction pattern ( Figure 1).
  • the complex characterized by three maxima: the first broad endothermic maximum starts at 70 °C and has a maximum at 88 °C; the second exothermic maximum is observed at a temperature of 174 °C, the third endothermic broad maximum is observed at 250-255 °C (Fig. 3).
  • DSC-TGA it was shown that the copper complex in solid form can contain hydrated or residual water, the content of which for different samples did not exceed 5% (mass loss when heated to 100 °C).
  • samples of the copper complex [Cp(HAEE)]C12 in an aqueous solution with a concentration of 20 ⁇ M were added to a mixture of water and acetonitrile in a 1:1 ratio with the addition of formic acid (to achieve a final concentration of 0.1%).
  • the data obtained indicate that in human or mouse blood samples the conformation of the HAEE peptide differs sharply from the conformation observed under the same conditions for the copper complex. Only 8% of HAEE is in the “unfolded” conformation after its interaction with blood plasma elements. Thus, in blood samples, the copper complex retains its conformation (presumably “unfolded”) unchanged, while HAEE acquires a different conformation, which is characterized by a much more compact structure, presumably “helical”. This means that the copper complex is resistant to changes in conformation in blood plasma.
  • both the copper complex of the HAEE peptide and HAEE require their introduction into the body and presence in the blood plasma, therefore, the fundamental differences in the spatial structure of these molecules in the blood plasma, based on the general concepts of biochemistry and physiology, indicate a different molecular mechanism of action of the copper complex and HAEE, which excludes their bioequivalence.
  • the stability of the copper complex was determined in an accelerated storage test for 6 months, which corresponds to 2 years of storage under normal conditions (Table 3).
  • the copper complex both purified from anions and in a mixture with the indicated anions, retained its color and consistency during storage, did not gain water (the mass of the sample did not increase within 1-2%) and did not lose the active substance.
  • the HAEE sample gained water over time (up to 7% wt.) and, according to HPLC data, degraded to 67.4% of the active substance in 6 months.
  • the sample obtained under RF patent No. 2709539 which is a HAEE-Zn-HSA complex, also took on water (up to 16% by weight) and degraded to a content of 71.9% of the original amount.
  • the stability of the proposed copper complex turned out to be significantly higher compared to HAEE and HAEE-Zn-HSA.
  • Example 2 Pharmaceutical compositions based on the copper complex of the HAEE peptide.
  • compositions based on the HAEE peptide copper complex may contain the active substance in an effective amount (Table 4).
  • the dosage composition is calculated individually and can vary from 0.1 mg to 100 mg per person per day, more preferably from 1 to 50 mg.
  • the compositions of the pharmaceutical compositions are presented in Table 3. The conversion was made to the dosage of the active substance.
  • Other ratios of the active substance in the pharmaceutical composition are possible in the range from 0.1 to 100 mg.
  • the weight of the tablets varies from 100 to 400 mg, the tablets can be coated.
  • Example 3 Effect of the copper complex of the HAEE peptide on behavioral functions and severity of cerebral amyloidosis in experimental animals.
  • Alzheimer's disease To model a neurodegenerative disease, a model of Alzheimer's disease was chosen, which is the most common neurodegenerative disease in older people.
  • mice of the APPswe/PSENldE9 line were used for the experiments.
  • Mice of this strain are also known as APP/PS1: the control group of APP/PS1 mice, starting from 4-6 months of age, exhibit characteristic cognitive signs of Alzheimer's disease-like pathology and have a significant amount of congophilic amyloid plaques in specific parts of the brain, including the hippocampus and cortex (Borchelt D.R., Ratovitski T., Van are J., et al. (1997). Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron, 19(4), 939 -945, doi: 10.1016/S0896-6273(00)80974- 5).
  • control Wild-type mice injected intravenously with saline;
  • control APP/PS1 transgenic mice injected intravenously with saline;
  • HAEE, HAEE-Zn-HSA and the claimed copper complex were injected into the retro-orbital venous plexus in accordance with a known procedure (Yardeni T., Eckhaus M., Morris HD et al. (2011). Retro-orbital injections in mice. Lab animal, 40(5), 155- 160, doi: 10.1038/laban0511-155).
  • Injections at a dose of 0.05 mg/kg were carried out monthly, from 2 to 7 months of age (inclusive), a total of 6 injections were made with HAEE and copper complex preparations, however, only two injections were made with HAEE-Zn-HSA, and then experiments with this drug were stopped due to signs of deterioration in the appearance of experimental animals after the first injection and the death of 5 transgenic mice immediately after the second injection. This is most likely due to the effects of high concentrations of HSA, which is structurally different from endogenous murine albumin (MSA).
  • MSA endogenous murine albumin
  • APP/PS1 transgenic mice and wild-type mice were used as controls.
  • mice were placed in cages with fresh bedding containing eighteen beads arranged in a 3 x 6 matrix. The mice were left in the cage for 30 minutes, after which the percentage of more than two-thirds of beads buried was determined. of the total number of balls.
  • the burying behavior is a sign of obsessive-compulsive behavior. Due to the repetitive and perseverative nature of burying, this behavior may represent neuropsychiatric symptoms such as perseverative behavior and/or stereotypic behavior. Both are neuropsychiatric symptoms commonly present in patients with Alzheimer's disease and other types of dementia.
  • the value of the CR 12.6 ⁇ 4.2%, which indicates a strong deterioration in the behavioral reflexes of APP/PS1 mice compared to wild-type animals and reflects the disabling effect of overproduction of human beta-amyloid on the processes of nervous activity .
  • the process of pouring the sample into paraffin was carried out as follows: poured 75% ethanol and left overnight, then changed to 96% ethanol and kept for 5 minutes, 96% ethanol - 10 minutes, 100% ethanol - 10 minutes (two changes), ethanol-chloroform (1:1) - 30 minutes, and left in pure chloroform overnight. Paraffin embedding was carried out at 60°C for 3 hours (three shifts). Embedding of tissues in paraffin blocks was carried out on a Leica EG 1160 device. Serial sections of the brain (8 ⁇ m) were cut using a Leica RM2265 microtome and placed on glass slides.
  • Example 4 Specific binding of the copper complex of the HAEE peptide to human beta-amyloid (Ap42).
  • the binding of the proposed copper complex to beta-amyloid was assessed as one of the mechanisms of action of this complex in neurodegenerative lesions associated with beta-amyloid.
  • the formation of complexes between the analyte in solution (the claimed copper complex or HAEE) and the immobilized 42-membered human beta-amyloid (ligand) was studied using BPPR. Based on the results of such experiments, the kinetic parameters of interactions and the value of the dissociation constant (KD) of the interaction between copper and the immobilized ligand were calculated. If the K D value ⁇ 10' 4 M, then the interaction between the copper complex and beta-amyloid is biologically significant.
  • Table 1 Volumes of reagents for the preparation of copper complexes of HAEE with various anions.

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Abstract

L'invention concerne un complexe cuivré de peptide HAEE décrit par la formule [Cu(HAEE)]mXn, où Cu est un cation de cuivre à double charge (II), m=1 pour des anions à charge unique ou charge double, m=3 pour des anions à charge triple, HAEE est un peptide synthétique acétylé à l'extrémité N et amidé à l'extrémité C avec une séquence d'acide aminé His-Ala-Glu-Glu, X est un anion pharmaceutiquement acceptable, n=1 pour des anions à charge double, n=2 pour des anions à charge unique ou charge triple. La présente invention concerne également un procédé de production de complexe cuivré de peptide HAEE, des compositions pharmaceutiques à base de complexe cuivré de peptide HAEE, et l'utilisation d'un complexe cuivré de peptide HAEE pour traiter des maladies neurodégénératives.
PCT/RU2023/000209 2022-07-15 2023-07-12 Complexe cuivré de peptide haee pour traiter des maladies neurodégénératives WO2024014982A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012056157A1 (fr) * 2010-10-27 2012-05-03 Kimonella Ventures Ltd Compose peptidique utile pour l'inhibition de la formation de plaques amyloïdes
RU2709539C1 (ru) * 2019-08-15 2019-12-18 Акционерное общество "Опытно-Экспериментальный завод "ВладМиВа" Фармацевтическая композиция на основе пептида HAEE для лечения нейродегенеративных заболеваний

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012056157A1 (fr) * 2010-10-27 2012-05-03 Kimonella Ventures Ltd Compose peptidique utile pour l'inhibition de la formation de plaques amyloïdes
RU2709539C1 (ru) * 2019-08-15 2019-12-18 Акционерное общество "Опытно-Экспериментальный завод "ВладМиВа" Фармацевтическая композиция на основе пептида HAEE для лечения нейродегенеративных заболеваний

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