WO2024014985A1 - Complexe calcique de peptide haee pour traiter des maladies neurodégénératives - Google Patents

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

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WO2024014985A1
WO2024014985A1 PCT/RU2023/000212 RU2023000212W WO2024014985A1 WO 2024014985 A1 WO2024014985 A1 WO 2024014985A1 RU 2023000212 W RU2023000212 W RU 2023000212W WO 2024014985 A1 WO2024014985 A1 WO 2024014985A1
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haee
calcium complex
calcium
complex
peptide
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PCT/RU2023/000212
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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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Priority claimed from RU2022119475A external-priority patent/RU2785354C1/ru
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Publication of WO2024014985A1 publication Critical patent/WO2024014985A1/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 calcium complex of the HAEE peptide, to pharmaceutical compositions based on it, and to the use of a calcium 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 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 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;117(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 peptide (AR).
  • 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 thermodynamical 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-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 Ap peptide with zinc ions may 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 aggregates of AP (amyloid plaques), which are associated with the development of Alzheimer's disease ((Frederickson C.J., Bush A.I. (2001).
  • HAEE AC-HAEE-NH2
  • 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 Ap peptide aggregation 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 (ZnCE) 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 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, I., 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-150694).
  • 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 and previously unknown calcium complex for the treatment of neurodegenerative diseases, including in solid form, to develop pharmaceutical compositions based on it and its use for the treatment of neurodegenerative diseases.
  • the molar content of calcium (II) cation and HAEE peptide is equimolar (1:1).
  • X is a singly charged anion
  • the claimed calcium complex of the HAEE peptide is described by the formula [Ca(HAEE)]Xr.
  • X is a doubly charged anion
  • the claimed calcium complex of the HAEE peptide is described by the formula [Ca(HAEE)]X.
  • X is a triply charged anion
  • the claimed calcium complex of the HAEE peptide is described by the formula [Ca(HAEE)]3X3.
  • the interaction of the HAEE peptide with calcium was also observed at other HAEE:Ca 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 Ca may be non-stoichiometric.
  • the use of a higher calcium content (>1:10) led to the preparation in solid form of a mixture of the calcium complex of the HAEE peptide and the corresponding calcium salt; when using a small amount of calcium ( ⁇ 1:0.1), its interaction with the HAEE peptide was not recorded physically. chemical methods.
  • the use of HAEE-Zn-HSA improves the pharmacokinetic parameters of the HAEE peptide
  • the use of the calcium 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 3D structure of the native HAEE peptide which in the form of an aqueous solution was placed in the blood plasma of wild-type mice for 2 minutes, differs from the 3D structure of native HAEE in the original aqueous solution, which is confirmed by HPLC by changing the release time with chromatographic column of the HAEE sample extracted from blood plasma, relative to the time of release of the HAEE sample extracted from the original aqueous solution.
  • the 3 D structure of the calcium complex of the HAEE peptide remained unchanged under similar experimental conditions (that is, after extraction of the calcium complex of the HAEE peptide from the original aqueous solution and after extraction of this complex from the blood plasma of wild-type mice after a 2-minute exposure) and corresponded to the 3D structure of the native HAEE peptide extracted from the original aqueous solution.
  • the chemical structure of the HAEE peptide was preserved in all experimental samples unchanged both for the native peptide and for the peptide as part of the calcium complex.
  • the change in the release time of the native HAEE peptide or the calcium 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 3D 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 3D structure of the calcium complex of the HAEE peptide in an aqueous solution and remains unchanged in the case of exposure of the calcium complex of the HAEE peptide in the blood plasma, while the native HAEE peptide in the blood plasma loses its “unfolded” conformation.
  • 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 calcium 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 to increase the therapeutic effectiveness of the calcium 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 calcium complex does not contain HSA and zinc, but contains calcium that is more biocompatible with the body, which has a reduced molar concentration of ions compared to the HAEE-Zn complex -HSA from 2.6, as described in the example of RF patent No. 2709539, to 1 (equimolar amount), as described in the present invention.
  • the invention also relates to the use of a calcium complex for the treatment of neurodegenerative diseases, as well as pathologies accompanied by neuroinflammatory processes.
  • the inventive calcium complex can effectively influence neurodegenerative diseases, in particular those caused by inflammatory causes, restoring impaired cognitive functions.
  • Calcium 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 calcium 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 calcium 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 calcium complex on the DSC curve is characterized by two broad maxima: the first begins at 62 °C and has a maximum at 97 °C; the second endothermic maximum begins at 242 °C and peaks at 277 °C (Fig. 3).
  • DSC curves show that the melting point of the calcium complex is higher compared to the melting point of HAEE, which ensures higher stability of the complex, as confirmed by the results of the accelerated storage test.
  • the IR spectrum with Fourier transform of the claimed calcium complex is presented using the example of [Ca(HAEE)]C12 in Fig. 4.
  • the IR spectrum of the complex repeats all the main lines of the HAEE IR spectrum. There are a number of differences in the vibration bands of the complex compared to HAEE.
  • the maximum shifted by 30 cm' 1 compared to the original HAEE (3045 cm' 1 - > 3075 cm' 1 ).
  • the IR spectrum of the complex contains new maxima at 780, 850 cm' 1 (region of non-planar deformations of the NHz group) and 1150 cm' 1 . Multiple changes occurred in the region 1200-1440 cm' 1 (CN bond). So Thus, after the formation of the complex, changes in the bond vibrations in the HAEE molecule occurred in the IR spectrum, indicating interaction with the calcium ion.
  • the inventive calcium complex can be obtained in the form of a lyophilisate using standard freeze-drying.
  • Preparation of the calcium complex involves mixing aqueous solutions of HAEE and a water-soluble calcium salt in equimolar ratios at room temperature, stirring for 10-60 minutes, freezing the solution and freeze-drying.
  • Water-soluble calcium salts (up to several wt.%) are nitrate, dihydrogen phosphate Ca ⁇ PO b, hydroxide, acetate, chloride, lactate, benzoate, 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 may be single, double or triple charged and selected from the group salicylate, tartrate, citrate and the like.
  • 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 calcium 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 calcium complex. It is assumed that only the inner sphere of the calcium complex exhibits therapeutic efficacy, regardless of the nature of the anion.
  • anions for the calcium complex are selected from the group of trifluoroacetate, pyruvate, galacturonate, bromide, glutarate, succinate, maleate, fumarate, benzenesulfonate, tosylate and other pharmaceutically acceptable anions.
  • each of the above anions to obtain a calcium complex in the solid phase using the freeze-drying method has shown that such a complex may partially retain hydrated or bound water, however, this complex does not accumulate additional water during storage and is stable for a long time.
  • Another object of the invention is pharmaceutical compositions with an effective amount of calcium complex and the presence of excipients. As a medicine, the calcium complex can be used without excipients in the form of a lyophilized substance.
  • the effective amount of calcium complex depends on the type of neurodegenerative disease, body weight, route of administration, and therefore can vary widely from 0.1 to 100 mg, more preferably from 5 to 50 mg.
  • the calcium complex pharmaceutical composition may be in solid form or in the form of an aqueous solution.
  • Solid compositions of the calcium complex contain an effective amount thereof and optionally excipients.
  • the solid pharmaceutical composition can be obtained by lyophilizing the calcium complex and a set of auxiliary components or adding excipients to the lyophilized calcium complex.
  • the lyophilized calcium 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 calcium complex in the form of an aqueous solution contains an effective amount of the calcium 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 calcium complex for the treatment of neurodegenerative diseases, which consists of administering a calcium complex as part of the described pharmaceutical compositions to a patient in an effective amount.
  • the effective amount of calcium 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.
  • Administration of the calcium 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 ).
  • intravenous, intraarterial, intraperitoneal, subcutaneous, cutaneous, transdermal, intramuscular, intrathecal, subarachnoid, oral, intranasal, sublingual, buccal, rectal administration When I When administering a calcium complex in solid form, the preferred route of administration is sublingual. When administering calcium complex in the form of a solution, the preferred routes of administration are intravenous and intranasal.
  • the calcium complex [Ca(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 calcium complex can be effectively used in the treatment of neurodegenerative diseases, in particular those caused inflammatory complications, restoring cognitive functions to normal.
  • PB plaques
  • One of the mechanisms of action of the calcium 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.
  • Fig. 4 IR spectrum with Fourier transform of HAEE (1) and calcium complex [Ca(HAEE)]C1 2 (2), ([Ca(HAEE)]C1 2 (3).
  • 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.
  • 'H-NMR spectra were obtained on a Bruker Avance IIIHD 500 NMR spectrometer, operating frequency 500.13 MHz for 'H' nuclei.
  • Characterization of the calcium complex [Ca(HAEE)]C12 in aqueous solution was carried out by high-resolution mass spectrometry using an LTQ FT Ultra mass spectrometer (Thermo Scientific, Germany), which combines Fourier transform ion cyclotron resonance and ion trap technologies.
  • Electrospray ionization (ESI) was performed using an Ion Max source (Thermo Scientific, Germany) with a metal spray capillary. The following lonMax source parameters were used: flow rate was 3 ⁇ L/min; the temperature of the heated capillary was 200°C; the atomizing gas was turned off; the electrospray capillary voltage was +3.8 kV in the positive mode and -2.5 kV in the negative mode.
  • 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. Calcium complex or HAEE was 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.
  • a HAEE solution with a concentration of 10 s M was prepared.
  • a solution of calcium salt with a concentration of 10 s M was added to it (Table 1).
  • Mixing was carried out for 5-60 minutes.
  • Equimolar calcium complexes were formed at a molar ratio of HAEE and calcium 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 calcium complex were studied in detail.
  • a water-soluble calcium 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 calcium complex.
  • obtaining a calcium complex [Ca(HAEE)] m X n where X is benzoate, salicylate, tartrate, citrate, etc. was carried out in three stages.
  • a solution of the calcium 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 white color.
  • the resulting solid samples of the complex are characterized by an amorphous shape in the diffraction pattern ( Figure 1).
  • the complex in contrast to HAEE (Fig. 2), the complex is characterized by a broad peak with an onset at 62 °C and a maximum at 87 °C; a broad melting peak with an onset at 224 °C and a maximum at 255 °C (Fig. 3).
  • Fig. 3 Using the DSC-TGA method, it was shown that the calcium 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).
  • the IR spectrum with Fourier transform of the complex contains a number of differences from the IR spectrum of HAEE (Fig. 4).
  • the maximum shifted by 30 cm' 1 compared to the original HAEE (3045 cm' 1 - * 3075 cm' 1 ).
  • the IR spectrum of the complex contains new maxima at 780, 850 cm' 1 (region of non-planar deformations of the NH2 group) and 1150 cm' 1 . Multiple changes occurred in the region 1200-1440 cm 1 (CN bond).
  • Characterization of the calcium complex [Ca(HAEE)]C12 in aqueous solution was carried out by high-resolution mass spectrometry using Fourier transform ion cyclotron resonance technology by measuring the exact mass of characteristic molecular ions in both positive and negative ionization modes.
  • samples of the calcium complex [Ca(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%).
  • Mass spectrometric analysis of an aqueous solution of calcium complex [Ca(HAEE)]C12 carried out with a positive mode of an electrospray ionization source, showed the presence of a molecular ion [HAEE+H] +1 with a monoisotopic mass of 526.2314 m/z and a molecular ion [HAEE- H+Ca] +1 with a monoisotopic mass of 564.1811 m/z.
  • 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 calcium complex and HAEE, which excludes their bioequivalence.
  • the stability of the calcium complex was determined in an accelerated storage test for 6 months, which corresponds to 2 years of storage under normal conditions (Table 3).
  • the calcium 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 weight 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 calcium complex turned out to be significantly higher compared to HAEE and HAEE-Zn-HSA.
  • Example 2 Pharmaceutical compositions based on the calcium complex of the HAEE peptide.
  • compositions based on the calcium complex of the HAEE peptide 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 The effect of the calcium complex of the HAEE peptide on behavioral functions and the 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 calcium complex were injected into the retroorbital 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 calcium 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/PS 1 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 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 into paraffin blocks was carried out using a Leica EG1160 device. Serial brain sections (8 ⁇ m) were cut using a Leica RM2265 microtome and mounted on glass slides.
  • the binding of the proposed calcium 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 calcium 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 (K D ) of the interaction between calcium and the immobilized ligand were calculated. If the value of K D ⁇ W 4 M, then the interaction between the calcium complex and beta-amyloid is biologically significant.
  • composition of pharmaceutical compositions with calcium complex of the HAEE peptide IM - intramuscular administration, IV - intravenous administration.

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Abstract

L'invention concerne un complexe calcique de peptide HAEE décrit par la formule [Ca(HAEE)]mXn, où Ca représente un cation de calcium à charge double (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, et n=2 pour des anions à charge unique ou à charge triple. La présente invention concerne également un procédé de production d'un complexe calcique de peptide HAEE, des compositions pharmaceutiques à base de complexe calcique de peptide HAEE, et l'utilisation d'un complexe calcique de peptide HAEE pour traiter des maladies neurodégénératives
PCT/RU2023/000212 2022-07-15 2023-07-12 Complexe calcique de peptide haee pour traiter des maladies neurodégénératives WO2024014985A1 (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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