Rubidium and / or zinc compounds for the treatment of parkinson's disease and other neurodegenerative diseases

Abstract

To provide rubidium and / or zinc compounds for treating Parkinson's disease and other neurodegenerative diseases.SOLUTION: Compositions comprising salts, compounds and complexes of 64 Zn-enriched zinc, such as 64 Zn-enriched zinc aspartate, and optionally a compound of general formula (1) are provided. There is provided a method of treating an NDD such as PD, optionally in combination with any other treatment for the NDD, such as for PD.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field The present disclosure relates to the fields of medicine and pharmacology, and more particularly to methods, compounds, and compositions for treating neurodegenerative disorders (NDDs), particularly Parkinson's disease (PD). [Background technology]

[0002] background Neurodegenerative disorders (NDDs), including Parkinson's disease (PD), are pathological conditions induced by inflammatory processes. No cures have been found, and effective drugs are needed. Summary of the Invention [Means for solving the problem]

[0003] overview In one aspect, the present disclosure provides a method for treating a neurodegenerative disorder (NDD), particularly Parkinson's disease (PD), comprising: 64 Zn-enriched zinc complexes, salts, and compounds, and / or certain 85 The present disclosure provides compositions comprising Rb-enriched rubidium compounds. 64 Zn-enriched zinc-containing compounds (separately or in combination with each other), salts, and complexes, e.g. 64 Zn-enriched zinc aspartate, and 85 Rb-enriched rubidium organic salts (structure shown below) are provided. The disclosed compositions include one or more isotopically enriched compounds, optionally in combination with other active ingredients useful for treating NDD. The disclosed compositions can be used individually and in combination with other anti-NDD treatments. The disclosed compounds have low toxicity to patients.

[0004] In certain embodiments, the present disclosure provides compositions comprising compounds of Formula 1 for treating neurodegenerative disorders (NDDs), particularly Parkinson's disease (PD). [ka]

[0005] The compound of formula 1 is a rubidium salt, and the rubidium 85 Rb-rich, R1 to R 14 are each independently selected from H, OH, F, Cl, Br, I, C1-C6 alkyl, C1-C6 alkoxy, and NO2.

[0006] In certain embodiments, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13 are all H, and

[0007] a) R3 is CH3, and R7, R9, R 12 , and R 14 are all H (compound 1),

[0008] b) R3, R7, R9, R 12 , and R 14 are all H (compound 2),

[0009] c) R3 is CH3 and R 14 is Cl, and R7, R9, and R 12 are all H (compound 3),

[0010] d) R3 is CH3 and R 14 is OH, and R7, R9, and R 12 are all H (compound 4),

[0011] e)R 14 is OH, and R3, R7, R9, and R 12 are all H (compound 5),

[0012] f) R3 is OH, and R7, R9, R 12 , and R 14 are all H (compound 6),

[0013] g)R 14is NO2, and R3, R7, R9, and R 12 are all H (compound 7),

[0014] h)R 12 is Br and R 14 is NO2, and R3, R7, and R9 are all H (compound 8);

[0015] i) R3 and R9 are both OCH3, and R 12 is Br and R 14 is NO2 and R7 is H (compound 9), or

[0016] j) R3 and R9 are both OCH3, and R 14 is NO2, and R7 and R 12 are both H (compound 10).

[0017] In any of the above compounds of Formula 1, in some embodiments, rubidium is at least 75% 85 Rb, at least 85% 85 Rb, or at least 95% 85 Rb, and in some embodiments, at least 99% 85 Rb, e.g. 99.8% 85 Rb. "N% 85 "Rb" is a compound in which N% of the Rb atoms are isotopes. 85 It refers to Rb, which is Rb.

[0018] In certain embodiments, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13 are all H, and the remaining R groups are as defined above. In other embodiments, R, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13 are all H; R3 is selected from H, CH3, OCH3, and NO2; R7 and R9 are each independently selected from H and OCH3; R 12 and R 14are each independently selected from H, Br, I, and NO. In another embodiment, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13 are all H; R3 is selected from H, CH3, OH, OCH3, and NO2; R7 and R9 are each independently selected from H and OCH3; R 12 is selected from H, Br, I, and NO2, and R 14 is selected from H, OH, Cl, Br, I, and NO2.

[0019] Composition types include liquid compositions formulated for intravenous or other parenteral administration, compositions formulated for topical administration, and compositions formulated for oral administration, e.g., tablets. The compositions may include any of those known in the art, including, but not limited to, tablets, pills, capsules, lozenges, granules, etc. In certain embodiments, the compositions contain between 0.4 millimoles and 30 millimoles of the disclosed compounds, for example, between 1 millimoles and 10 millimoles, or for example, 1, 2, 5, 10, 20, 25, or 30 millimoles. The disclosed compositions may further contain one or more excipients suitable for the formulation. Intravenous formulations may include at least one of a suitable solvent, such as water; a salt or ion, such as sodium chloride, potassium chloride, potassium ions, sodium ions, or chloride ions; a sugar, such as glucose or sucrose; a buffer; or other excipients, such as DMSO. Topical formulations may include, but are not limited to, ointments, creams, lotions, and salves, and may include at least one of a suitable vehicle; a penetration enhancer, such as DMSO and related analogs; and an emulsifier. Tablets may contain at least one excipient, such as fillers (e.g., starch, lactose, sucrose, glucose); binders (e.g., carboxymethylcellulose, gelatin, polyvinylpyrrolidone, sucrose); disintegrants (e.g., calcium carbonate, alginic acid, sodium carbonate); wetting agents (e.g., cetyl alcohol, and glycerol monostearate, sodium lauryl sulfate); buffers; lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate); and coatings.

[0020] In certain embodiments, the composition 64 Zn e -asp; in a further embodiment, the composition further comprises a 10% rubidium salt containing Rb-85.

[0021] In another aspect, the present disclosure provides a method for treating NDD, particularly PD, comprising administering a therapeutically effective amount of the disclosed composition to a subject in need thereof.

[0022] In another aspect, the present disclosure provides a method for treating a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the disclosed composition.In certain embodiments, the condition to be treated is a neurodegenerative disease such as Parkinson's disease.In some embodiments, a method for treating or slowing the progression of a neurodegenerative disease such as Parkinson's disease comprises administering a compound of formula 1 with or without conventional NDD or PD treatment. 85 Rb-enriched rubidium compounds, and / or 64 Zn e In certain embodiments, the method comprises administering a therapeutically effective amount of a composition comprising a salt, complex, or compound containing: 64 Zn e a composition comprising a therapeutically effective amount of a compound, salt, or complex containing: 85 a therapeutically effective amount of a composition comprising an Rb-enriched rubidium compound; and 64 Zn e and a compound, salt, or complex containing 85 In a further embodiment, such a composition comprises at least one excipient. 64 Zn e and compounds containing 85 Rb-enriched rubidium compounds exist in a specific ratio to each other, e.g., 90% 64 Zn e and a compound containing 10% of formula 1 85 Rb enriched rubidium compounds, e.g. 90% 64 Zn e -asp and 10% 85 Rb e -E2, where the percentage is based on the mass of the element. 64 Zn e A salt, complex, or compound containing 64 Zn e -aspartate. In some embodiments, the compound is a peptide up to 20 amino acids in length.

[0023] According to a first aspect of the present invention there is provided a method of treating, preventing or delaying the progression of a neurodegenerative disease, comprising: The formula below: [ka] (Wherein, R1 to R 14 are independently H, OH, F, Cl, Br, I, C1-C6 alkyl, C1-C6 alkoxy, and NO2; 85 Rb e At least 75% 85 Rb) of 85 Rb e compounds, and salts, and / or 64 Zn e Compound or complex of to a subject in need thereof.

[0024] 85 Rb e At least 90% 85 is Rb, and / or 64 Zn e At least 90% 64 The method described in

[0023] , wherein the compound is Zn.

[0025] R1, R2, R4 to R6, R8, R 10 , R 11 , and R 13 The method according to

[0023] or

[0024] , wherein all of are H.

[0026] R3 is selected from H, CH3, OCH3, and NO2; R7 and R9 are each independently selected from H and OCH3; R 12 and R 14 are each independently selected from H, Br, I, and NO2.

[0027] a) R3 is CH3, and R7, R9, R12 , and R 14 are all H, b) R3, R7, R9, R 12 , and R 14 are all H, c) R3 is CH3 and R 14 is Cl, and R7, R9, and R 12 are all H, d) R3 is CH3 and R 14 is OH, and R, R, and R 12 are all H, e)R 14 is OH, and R3, R7, R9, and R 12 are all H, f) R3 is OH, and R7, R9, R 12 , and R 14 are all H, g)R 14 is NO2, and R3, R7, R9, and R 12 are all H, h)R 12 is Br and R 14 is NO2, and R3, R7, and R9 are all H; i) R3 and R9 are both OCH3, and R 12 is Br and R 14 is NO2 and R7 is H, or j) R3 and R9 are both OCH3, and R 14 is NO2, and R7 and R 12 The method according to any one of

[0023] to

[0025] , wherein both are H.

[0028] The method according to any one of

[0023] to

[0027] , wherein the composition further comprises at least one excipient.

[0029] R3 is CH3, and R7, R9, R 12 , and R 14 The method according to any one of

[0023] to

[0025] , wherein all of

[0030] A method according to any one of

[0023] to

[0029] , wherein the neurodegenerative disorder is Parkinson's disease.

[0031] A method according to any one of

[0023] to

[0030] , wherein the composition is administered intravenously to the subject.

[0032] A method according to any one of

[0023] to

[0030] , wherein the composition is administered intraperitoneally to the subject.

[0033] A method according to any one of

[0023] to

[0030] , wherein the composition is orally administered to the subject.

[0034] A method according to any one of

[0023] to

[0033] , further comprising administering a formulation containing one or more other therapeutic agents for treating a neurodegenerative disorder before, simultaneously with, or after administering the composition.

[0035] The method described in

[0030] further comprises administering a formulation containing one or more other anti-Parkinson's agents before, simultaneously with, or after administering the composition.

[0036] 64 Zn e The method according to any one of

[0023] to

[0035] , wherein the compound or complex is a part of a zinc finger peptide.

[0037] A method according to any one of

[0023] to

[0036] , wherein the subject is a human subject.

[0038] 85 Rb e The compound was added to 40 mg of 85 Rb e From 2400mg 85 Rb e The method according to any one of

[0023] to

[0037] , wherein the compound is present in an amount equivalent to an amount between

[0023] and

[0037] .

[0039] 64Zn e of compound or complex is administered in an amount of 0.1 to 1.5 mg of pure 64 Zn e is in the range 64 Zn e The method according to any one of

[0023] to

[0038] , wherein the compound is present in an amount equivalent to a dose in the range (by metal).

[0040] 64 Zn e A compound or complex of (metal) is administered in an amount of 1 to 15 mg of pure metal per kg of human body weight. 64 Zn e The method according to any one of

[0023] to

[0038] , wherein the range is: [Brief explanation of the drawings]

[0041] [Figure 1A] Figures 1A and 1B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on microglial phagocytosis in a rat model of experimental parkinsonism. Figure 1A - Relative number of phagocytic cells. Figure 1B - Phagocytosis. Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. sham-operated animals; ## - p ≤ 0.01 vs. sham-operated animals; ^ - p ≤ 0.05 vs. control animals in the parkinsonism model; ^^ - p ≤ 0.01 vs. control animals in the parkinsonism model. [Figure 1B] Figures 1A and 1B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on microglial phagocytosis in a rat model of experimental parkinsonism. Figure 1A - Relative number of phagocytic cells. Figure 1B - Phagocytosis. Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. sham-operated animals; ## - p ≤ 0.01 vs. sham-operated animals; ^ - p ≤ 0.05 vs. control animals in the parkinsonism model; ^^ - p ≤ 0.01 vs. control animals in the parkinsonism model.

[0042] [Figure 2] Figure 2 shows the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on microglial oxidative metabolism in a rat model of experimental parkinsonism. Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. sham-operated animals; ## - p ≤ 0.01 vs. sham-operated animals; ^ - p ≤ 0.05 vs. control animals in the parkinsonism model; ^^ - p ≤ 0.01 vs. control animals in the parkinsonism model; & - p ≤ 0.05 vs. the corresponding index in the unstimulated sample; && - p ≤ 0.01 vs. the corresponding index in the unstimulated sample.

[0043] [Figure 3] Figure 3 shows the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on NO synthesis by microglial cells in a rat model of experimental parkinsonism. Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. animal model of parkinsonism.

[0044] [Figure 4] Figure 4 shows the effect of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on microglial arginase activity in a rat model of experimental parkinsonism. Arginase activity is expressed in mM urea / mg protein / hr.

[0045] [Figure 5A]Figures 5A and 5B show CD14 expression in microglial populations in a rat model of experimental parkinsonism treated with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. The expression is expressed as the CD14 cell fraction (%) (Figure 5A) and CD14 expression (GMean) (Figure 5B). Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. sham-operated animals; ## - p ≤ 0.01 vs. sham-operated animals; ^ - p ≤ 0.05 vs. control animals; ^^ - p ≤ 0.01 vs. control animals. [Figure 5B] Figures 5A and 5B show CD14 expression in microglial populations in a rat model of experimental parkinsonism treated with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. The expression is expressed as the CD14 cell fraction (%) (Figure 5A) and CD14 expression (GMean) (Figure 5B). Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. sham-operated animals; ## - p ≤ 0.01 vs. sham-operated animals; ^ - p ≤ 0.05 vs. control animals; ^^ - p ≤ 0.01 vs. control animals.

[0046] [Figure 6A] Figures 6A and 6B show CD206 expression in microglial populations in a rat model of experimental parkinsonism treated with 64Zne-asp and 64Zne-asp containing 10% E285Rbe, expressed as the CD206+ cell fraction (%) (Figure 6A) and CD206 expression (Gmean) (Figure 6B). Legend: # - p≦0.05 vs. sham-operated animals. [Figure 6B]Figures 6A and 6B show CD206 expression in microglial populations in a rat model of experimental parkinsonism treated with 64Zne-asp and 64Zne-asp containing 10% E285Rbe, expressed as the CD206+ cell fraction (%) (Figure 6A) and CD206 expression (Gmean) (Figure 6B). Legend: # - p≦0.05 vs. sham-operated animals.

[0047] [Figure 7A] Figures 7A and 7B show hemograms from rat models of Parkinson's disease treated with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Results are expressed as cell counts × 103 / μl (or mkl) (Figure 7A) and as cell fractions (%) (Figure 7B). Legend: * - p ≤ 0.05 vs. values ​​for intact animals; # - p ≤ 0.05 vs. values ​​for animal models of Parkinsonism. [Figure 7B] Figures 7A and 7B show hemograms from rat models of Parkinson's disease treated with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Results are expressed as cell counts × 103 / μl (or mkl) (Figure 7A) and as cell fractions (%) (Figure 7B). Legend: * - p ≤ 0.05 vs. values ​​for intact animals; # - p ≤ 0.05 vs. values ​​for animal models of Parkinsonism.

[0048] [Figure 8A] Figures 8A and 8B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on monocyte phagocytosis in a rat model of experimental parkinsonism. Figure 8A - relative number of phagocytic cells (expressed as the percentage of cells emitting fluorescence); Figure 8B - phagocytosis. Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. animal model of parkinsonism. [Figure 8B]Figures 8A and 8B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on monocyte phagocytosis in a rat model of experimental parkinsonism. Figure 8A - relative number of phagocytic cells (expressed as the percentage of cells emitting fluorescence); Figure 8B - phagocytosis. Legend: * - p ≤ 0.05 vs. intact animals; # - p ≤ 0.05 vs. animal model of parkinsonism.

[0049] [Figure 9] Figure 9 shows the effect of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on oxidative metabolism in circulating monocytes in a rat model of experimental parkinsonism. Legend: * - p < 0.05 vs. corresponding index in unstimulated samples; # - p < 0.05 vs. intact animals; & - p < 0.05 vs. control group in the animal model of parkinsonism.

[0050] [Figure 10A] Figures 10A and 10B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on neutrophil phagocytosis in a rat model of experimental parkinsonism. Figure 10A shows the relative number of phagocytic cells (expressed as the percentage of cells emitting fluorescence). Figure 10B shows phagocytosis. Legend: * - p < 0.05 vs. the intact animal group. Legend: # - p < 0.05 vs. the animal model group of parkinsonism. [Figure 10B] Figures 10A and 10B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on neutrophil phagocytosis in a rat model of experimental parkinsonism. Figure 10A shows the relative number of phagocytic cells (expressed as the percentage of cells emitting fluorescence). Figure 10B shows phagocytosis. Legend: * - p < 0.05 vs. the intact animal group. Legend: # - p < 0.05 vs. the animal model group of parkinsonism.

[0051] [Figure 11] Figure 11 shows the effect of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on oxidative metabolism in circulating neutrophils in a rat model of experimental parkinsonism (results expressed as ROS, relative fluorescence). Legend: * - p < 0.05 vs. corresponding index in unstimulated samples; # - p < 0.05 vs. intact animals; & - p < 0.05 vs. control group in the animal model of parkinsonism.

[0052] [Figure 12A] Figures 12A and 12B show CD14 expression in the peripheral blood phagocyte population in a rat model of experimental parkinsonism treated prophylactically with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Legend: * - p < 0.05 vs. the intact animal group; # - p < 0.05 vs. the control group of the animal model of parkinsonism. [Figure 12B] Figures 12A and 12B show CD14 expression in the peripheral blood phagocyte population in a rat model of experimental parkinsonism treated prophylactically with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Legend: * - p < 0.05 vs. the intact animal group; # - p < 0.05 vs. the control group of the animal model of parkinsonism.

[0053] [Figure 13A] Figures 13A and 13B show CD206 expression in the peripheral blood phagocyte population in a rat model of experimental parkinsonism treated prophylactically with 64Zne-asp and 64Zne-asp containing 10% E285Rbe (13A, CD206+ cell fraction, %; 13B, CD206 expression, Gmean). Legend: * - p < 0.05 vs. the intact group; # - p < 0.05 vs. the control group of the animal model of parkinsonism. [Figure 13B]Figures 13A and 13B show CD206 expression in the peripheral blood phagocyte population in a rat model of experimental parkinsonism treated prophylactically with 64Zne-asp and 64Zne-asp containing 10% E285Rbe (13A, CD206+ cell fraction, %; 13B, CD206 expression, Gmean). Legend: * - p < 0.05 vs. the intact group; # - p < 0.05 vs. the control group of the animal model of parkinsonism.

[0054] [Figure 14A] Figures 14A and 14B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on the phagocytosis of peritoneal macrophages in a rat model of experimental parkinsonism. Figure 14A - relative number of phagocytic cells; Figure 14B - phagocytosis. Legend: * - p < 0.05 vs. the intact group. # - p < 0.05 vs. the animal model group of parkinsonism. [Figure 14B] Figures 14A and 14B show the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on the phagocytosis of peritoneal macrophages in a rat model of experimental parkinsonism. Figure 14A - relative number of phagocytic cells; Figure 14B - phagocytosis. Legend: * - p < 0.05 vs. the intact group. # - p < 0.05 vs. the animal model group of parkinsonism.

[0055] [Figure 15] Figure 15 shows the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on the oxidative metabolism of peritoneal macrophages in a rat model of experimental parkinsonism. Legend: * - p≦0.05 vs. the corresponding index in unstimulated samples.

[0056] [Figure 16]Figure 16 shows the effects of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on NO synthesis by microglial cells in a rat model of experimental parkinsonism. * - p < 0.05 vs. the intact group; # - p < 0.05 vs. the animal model of parkinsonism.

[0057] [Figure 17] Figure 17 shows the effect of prophylactic administration of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on arginase activity of peritoneal macrophages in a rat model of experimental parkinsonism. Legend: * - p < 0.05 vs. the intact group; # - p < 0.05 vs. the animal model of parkinsonism.

[0058] [Figure 18A] Figures 18A and 18B show CD14 expression in peripheral macrophage populations in a rat model of experimental parkinsonism treated prophylactically with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Legend: * - p < 0.05 vs. the intact group; # - p < 0.05 vs. the control group of the animal model of parkinsonism. [Figure 18B] Figures 18A and 18B show CD14 expression in peripheral macrophage populations in a rat model of experimental parkinsonism treated prophylactically with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Legend: * - p < 0.05 vs. the intact group; # - p < 0.05 vs. the control group of the animal model of parkinsonism.

[0059] [Figure 19A] Figures 19A and 19B show CD206 expression in a population of CD14+ peritoneal macrophages in a rat model of experimental parkinsonism that received prophylactic treatment with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Legend: * - p < 0.05 vs. the uninjured animal group; # - p < 0.05 vs. the control group of the animal model of parkinsonism. [Figure 19B] Figures 19A and 19B show CD206 expression in a population of CD14+ peritoneal macrophages in a rat model of experimental parkinsonism that received prophylactic treatment with 64Zne-asp and 64Zne-asp containing 10% E285Rbe. Legend: * - p < 0.05 vs. the uninjured animal group; # - p < 0.05 vs. the control group of the animal model of parkinsonism.

[0060] [Figure 20] Figure 20 shows the effect of 64Zne-asp on intracellular ROS production by non-sensitized and bacterial lipopolysaccharide-treated peritoneal macrophages. Legend: * - p≦0.05 vs. unstimulated cells.

[0061] [Figure 21] FIG. 21 shows the effect of 64Zne-asp on nitroblue tetrazolium (NBT test index of non-sensitized and bacterial lipopolysaccharide-treated peritoneal macrophages).

[0062] [Figure 22] FIG. 22 shows the effect of 64Zne-asp on the phagocytosis of non-sensitized peritoneal macrophages.

[0063] [Figure 23] FIG. 23 shows the effect of 64Zne-asp on arginase activity in non-sensitized and bacterial lipopolysaccharide-treated peritoneal macrophages.

[0064] [Figure 24] Figure 24 shows the effect of 64Zne-asp on NO production by unsensitized and bacterial lipopolysaccharide-treated peritoneal macrophages. Legend: * - p < 0.05 vs. unstimulated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0065] [Figure 25]Figure 25 shows the effect of E2+85Rbe on intracellular ROS generation by unsensitized and bacterial lipopolysaccharide-treated peritoneal macrophages. Note: * - p < 0.05 vs. unstimulated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0066] [Figure 26] Figure 26 shows the effect of E2+85Rbe on the NBT test index of unsensitized and bacterial lipopolysaccharide-treated peritoneal macrophages. Legend: * - p < 0.05 vs. unstimulated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0067] [Figure 27] Figure 27 shows the effect of E2+85Rbe on the phagocytosis of unstimulated peritoneal macrophages. Legend: *-p≦0.05 vs. unstimulated cells.

[0068] [Figure 28] Figure 28 shows the effect of E2+85Rbe on arginase activity in unsensitized and bacterial lipopolysaccharide-treated peritoneal macrophages. Legend: * - p < 0.05 vs. unstimulated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0069] [Figure 29] Figure 29 shows the effect of E2+85Rbe on NO synthesis by unsensitized and bacterial lipopolysaccharide-treated peritoneal macrophages. Legend: * - p < 0.05 vs. unstimulated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0070] [Figure 30A] Figures 30A-30C show the stages of collecting brain tissue samples for isolating microglial cells: Figure 30A: decapitation; Figure 30B: opening of the skull; Figure 30C: removal of brain tissue without bleeding. [Figure 30B] Figures 30A-30C show the stages of collecting brain tissue samples for isolating microglial cells: Figure 30A: decapitation; Figure 30B: opening of the skull; Figure 30C: removal of brain tissue without bleeding. [Figure 30C]Figures 30A-30C show the stages of collecting brain tissue samples for isolating microglial cells: Figure 30A: decapitation; Figure 30B: opening of the skull; Figure 30C: removal of brain tissue without bleeding.

[0071] [Figure 31] FIG. 31 shows the production of brain tissue homogenates by mechanical disintegration using a cooling agent.

[0072] [Figure 32] FIG. 32 shows the fractionation of brain tissue homogenates on a Percoll gradient.

[0073] [Figure 33A] Figures 33A and 33B show the separation of circulating phagocytes using a density gradient. [Figure 33B] Figures 33A and 33B show the separation of circulating phagocytes using a density gradient.

[0074] [Figure 34] Figure 34 shows the effect of 64Zne-asp on intracellular ROS generation by non-sensitized rat microglial cells. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0075] [Figure 35] Figure 35 shows the effect of 64Zne-asp on phagocytosis by non-sensitized rat microglial cells. Legend: * - p≦0.05 vs. untreated cells.

[0076] [Figure 36] Figure 36 shows the effect of 64Zne-asp on NO production by non-sensitized rat microglial cells. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0077] [Figure 37]Figure 37 shows the effect of 64Zne-asp on arginase activity in non-sensitized rat microglial cells. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0078] [Figure 38] Figure 38 shows the effect of 64Zne-asp on intracellular ROS generation by non-sensitized rat peripheral blood monocytes. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0079] [Figure 39] Figure 39 shows the effect of 64Zne-asp on the NBT test index of non-sensitized rat peripheral blood monocytes. Legend: *-p≦0.05 vs. non-stimulated cells.

[0080] [Figure 40] Figure 40 shows the effect of 64Zne-asp on the phagocytosis of non-sensitized rat monocytes. Legend: * - p≦0.05 vs. untreated cells.

[0081] [Figure 41] Figure 41 shows the effect of 64Zne-asp on NO production by non-sensitized rat peripheral blood monocytes. Legend: # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0082] [Figure 42] FIG. 42 shows the effect of 64Zne-asp on arginase activity of peripheral blood monocytes from non-sensitized rats.

[0083] [Figure 43] Figure 43 shows the effect of 64Zne-asp on intracellular ROS generation by non-sensitized rat peripheral blood neutrophils. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0084] [Figure 44]Figure 44 shows the effect of 64Zne-asp on the NBT test index of non-sensitized rat peripheral blood neutrophils. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0085] [Figure 45] FIG. 45 shows the effect of 64Zne-asp on the phagocytic activity of peripheral blood neutrophils from non-sensitized rats.

[0086] [Figure 46] Figure 46 shows the effect of 64Zne-asp on NO production by non-sensitized rat peripheral blood neutrophils. Legend: # - p≦0.05 vs. bacterial lipopolysaccharide-stimulated cells.

[0087] [Figure 47] FIG. 47 shows the effect of 64Zne-asp on arginase activity in peripheral blood neutrophils from non-sensitized rats.

[0088] [Figure 48] Figure 48 shows the effect of 64Zne-asp and E2+85Rbe preparations on the spontaneous and PHA-stimulated proliferative activity of human T lymphocytes in vitro. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. PHA-stimulated cells.

[0089] [Figure 49] Figure 49 shows the effect of 64Zne-asp and E2+85Rbe preparations on activation-induced apoptosis of human T lymphocytes in vitro. Legend: * - p < 0.05 vs. untreated cells; # - p < 0.05 vs. PHA-stimulated cells.

[0090] [Figure 50A] FIG. 50A shows a trajectory map, and FIG. 50B shows a map of the cumulative time spent by an animal in a given location in the open field over a 5-minute period. [Figure 50B]FIG. 50A shows a trajectory map, and FIG. 50B shows a map of the cumulative time spent by an animal in a given location in the open field over a 5-minute period.

[0091] [Figure 51A] FIG. 51A shows a trajectory map, and FIG. 51B shows a map of the cumulative time spent by the animal at a given location in the apparatus over a 5-minute period. [Figure 51B] FIG. 51A shows a trajectory map, and FIG. 51B shows a map of the cumulative time spent by the animal at a given location in the apparatus over a 5-minute period.

[0092] [Figure 52] FIG. 52 is a graphical representation of the experimental scheme in studying the therapeutic effects of 64Zn-asp on behavioral and motor function in experimental parkinsonism and apomorphine-induced rotational behavior (Example 7).

[0093] [Figure 53] Figures 53A and 53B show the results of the apomorphine test in the LPS rat model of parkinsonism before and after administration of 64Zn-asp. Figure 53A: Mean number of rotations / 30 min in the first and second apomorphine test, M±SD; Figure 53B: Percentage ratio of rats with increased and decreased number of rotations per 30 min between the first and second apomorphine tests.

[0094] [Figure 54-1]Figures 54A-O show immunohistochemical identification of neurons with tyrosine hydroxylase activity in the midbrain of an LPS rat model of parkinsonism after administration of 64Zn-asp. TH-positive staining (brown). Oc. 40, ob. 10. Figure 54A (animal 1), Figure 54B (animal 2), Figure 54C (animal 3), Group I - intact rats (n=3); Figure 54D (animal 1), Figure 54E (animal 2), Figure 54F (animal 3), Group II - sham-operated rats + HO (n=3); Figure 54G (animal 1), Figure 54H (animal 2), Figure 54I (animal 3), Group III - sham-operated rats + 64Zn-asp (n=3); Figure 54J (animal 1), Figure 54K (animal 2), Figure 54L (animal 3), Group V - LPS + HO (n=3); Figure 54M (animal 1), Figure 54N (animal 2), Figure 54O (animal 3), Group VI - LPS + 64Zn-asp (n=3). [Figure 54-2] Figures 54A-O show immunohistochemical identification of neurons with tyrosine hydroxylase activity in the midbrain of an LPS rat model of parkinsonism after administration of 64Zn-asp. TH-positive staining (brown). Oc. 40, ob. 10. Figure 54A (animal 1), Figure 54B (animal 2), Figure 54C (animal 3), Group I - intact rats (n=3); Figure 54D (animal 1), Figure 54E (animal 2), Figure 54F (animal 3), Group II - sham-operated rats + HO (n=3); Figure 54G (animal 1), Figure 54H (animal 2), Figure 54I (animal 3), Group III - sham-operated rats + 64Zn-asp (n=3); Figure 54J (animal 1), Figure 54K (animal 2), Figure 54L (animal 3), Group V - LPS + HO (n=3); Figure 54M (animal 1), Figure 54N (animal 2), Figure 54O (animal 3), Group VI - LPS + 64Zn-asp (n=3).

[0095] [Figure 55] Figure 55 is a graph showing the therapeutic effect of 64Zn-asp on the number of TH-positive neurons in the midbrain of experimental parkinsonism. M±SD, P<0.001.

[0096] [Figure 56A]Figure 56A is a graph of the therapeutic effect of 64Zn-asp on body weight in the LPS rat model of parkinsonism. *p<0.05, **p<0.01, ***p<0.001 vs. values ​​on day 1 of the experiment. Figure 56B is a graph showing the change in body weight over the course of treatment with 64Zn-asp in the LPS rat model, M±SD, *p<0.05, **p<0.01, ***p<0.001 vs. values ​​in the LPS group. [Figure 56B] Figure 56A is a graph of the therapeutic effect of 64Zn-asp on body weight in the LPS rat model of parkinsonism. *p<0.05, **p<0.01, ***p<0.001 vs. values ​​on day 1 of the experiment. Figure 56B is a graph showing the change in body weight over the course of treatment with 64Zn-asp in the LPS rat model, M±SD, *p<0.05, **p<0.01, ***p<0.001 vs. values ​​in the LPS group.

[0097] [Figure 57] FIG. 57 shows fecal water content in the LPS rat model of Parkinson's disease, M+SD.

[0098] [Figure 58] FIG. 58 displays the therapeutic effect of 64Zn-asp on fecal water content in the LPS rat model of Parkinson's disease, M±SD.

[0099] [Figure 59A] Figures 59A-59E show the effects of 64Zn-asp on behavioral response parameters measured in the open field test: Figure 59A: total distance traveled by animals per group; time spent in the outer perimeter; Figure 59B: and time spent in the inner perimeter; Figures 59C and 59D: total number of feedings; Figure 59E: total number of defecations. [Figure 59B] Figures 59A-59E show the effects of 64Zn-asp on behavioral response parameters measured in the open field test: Figure 59A: total distance traveled by animals per group; time spent in the outer perimeter; Figure 59B: and time spent in the inner perimeter; Figures 59C and 59D: total number of feedings; Figure 59E: total number of defecations. [Figure 59C] Figures 59A-59E show the effects of 64Zn-asp on behavioral response parameters measured in the open field test: Figure 59A: total distance traveled by animals per group; time spent in the outer perimeter; Figure 59B: and time spent in the inner perimeter; Figures 59C and 59D: total number of feedings; Figure 59E: total number of defecations. [Figure 59D] Figures 59A-59E show the effects of 64Zn-asp on behavioral response parameters measured in the open field test: Figure 59A: total distance traveled by animals per group; time spent in the outer perimeter; Figure 59B: and time spent in the inner perimeter; Figures 59C and 59D: total number of feedings; Figure 59E: total number of defecations. [Figure 59E] Figures 59A-59E show the effects of 64Zn-asp on behavioral response parameters measured in the open field test: Figure 59A: total distance traveled by animals per group; time spent in the outer perimeter; Figure 59B: and time spent in the inner perimeter; Figures 59C and 59D: total number of feedings; Figure 59E: total number of defecations.

[0100] [Figure 60A] Figures 60A-60D show the effects of 64Zn-asp on behavioral response parameters measured in the elevated plus maze: (Figure 60A) total distance traveled by the animal; (Figure 60B) time spent in the open arms; (Figure 60C) time spent in the closed arms; (Figure 60D) total number of entries. [Figure 60B] Figures 60A-60D show the effects of 64Zn-asp on behavioral response parameters measured in the elevated plus maze: (Figure 60A) total distance traveled by the animal; (Figure 60B) time spent in the open arms; (Figure 60C) time spent in the closed arms; (Figure 60D) total number of entries. [Figure 60C] Figures 60A-60D show the effects of 64Zn-asp on behavioral response parameters measured in the elevated plus maze: (Figure 60A) total distance traveled by the animal; (Figure 60B) time spent in the open arms; (Figure 60C) time spent in the closed arms; (Figure 60D) total number of entries. [Figure 60D] Figures 60A-60D show the effects of 64Zn-asp on behavioral response parameters measured in the elevated plus maze: (Figure 60A) total distance traveled by the animal; (Figure 60B) time spent in the open arms; (Figure 60C) time spent in the closed arms; (Figure 60D) total number of entries.

[0101] [Figure 61] FIG. 61 displays the effect of 64Zn-asp on behavioral response parameters measured in the pick-up test.

[0102] [Figure 62] Figure 62 displays the effect of 64Zn-asp on the levels of circulating leukocytes in the LPS rat model of parkinsonism, M±SD. Note: #p<0.05 vs. uninjured animals, $p<0.05 vs. sham-operated animals.

[0103] [Figure 63] Figure 63 shows the effect of 64Zn-asp on the absolute numbers of various populations of circulating leukocytes in the LPS rat model of Parkinsonism, M±SD. Note: #p<0.05 vs. intact animals, $p<0.05 vs. sham-operated animals, *p<0.05 vs. Parkinson's disease animal model.

[0104] [Figure 64] Figure 64 shows the effect of 64Zn-asp on the relative numbers of various populations of circulating leukocytes in an LPS rat model of Parkinsonism, M±SD. Note: #p<0.05 vs. intact animals, $p<0.05 vs. sham-operated animals, *p<0.05 vs. Parkinson's disease animal model.

[0105] [Figure 65A]Figures 65A and 65B show the therapeutic effect of 64Zn-asp on microglial phagocytosis in an LPS rat model of Parkinson's disease. Figure 65A - relative number of phagocytes, Figure 65B - phagocytosis. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals, * - p≦0.05 vs. control animal model of Parkinson's disease. [Figure 65B] Figures 65A and 65B show the therapeutic effect of 64Zn-asp on microglial phagocytosis in an LPS rat model of Parkinson's disease. Figure 65A - relative number of phagocytes, Figure 65B - phagocytosis. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals, * - p≦0.05 vs. control animal model of Parkinson's disease.

[0106] [Figure 66] Figure 66 shows the therapeutic effect of 64Zn-asp on microglial oxidative metabolism in an LPS rat model of Parkinson's disease. #-p<0.05 vs. intact animals; $p≦0.05 vs. sham-operated animals, *p≦0.05 vs. control animal model of Parkinson's disease, @-p≦0.05 vs. unstimulated samples.

[0107] [Figure 67A] Figures 67A and 67B show the expression of phenotypic markers in microglial populations in an LPS rat model of experimental Parkinsonism treated with 64Zn-asp. Figure 67A - Number of expressing cells in the analyzed population, Figure 67B - Expression level. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease. [Figure 67B] Figures 67A and 67B show the expression of phenotypic markers in microglial populations in an LPS rat model of experimental Parkinsonism treated with 64Zn-asp. Figure 67A - Number of expressing cells in the analyzed population, Figure 67B - Expression level. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease.

[0108] [Figure 68A] Figures 68A and 68B show the therapeutic effect of 64Zn-asp on the phagocytosis of circulating monocytes and granulocytes in an LPS rat model of Parkinson's disease. Figure 68A - Relative number of phagocytes, Figure 68B - Phagocytosis. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease. [Figure 68B] Figures 68A and 68B show the therapeutic effect of 64Zn-asp on the phagocytosis of circulating monocytes and granulocytes in an LPS rat model of Parkinson's disease. Figure 68A - Relative number of phagocytes, Figure 68B - Phagocytosis. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease.

[0109] [Figure 69A] Figures 69A and 69B show the therapeutic effect of 64Zn-asp on the oxidative metabolism of circulating monocytes (Figure 69A) and granulocytes (Figure 69B) in an LPS rat model of Parkinson's disease. # - p<0.05 vs. intact animals; $ - p≦0.05 vs. sham-operated animals; * - ≦0.05 vs. control animal model of Parkinson's disease; @ - p≦0.05 vs. unstimulated samples. [Figure 69B] Figures 69A and 69B show the therapeutic effect of 64Zn-asp on the oxidative metabolism of circulating monocytes (Figure 69A) and granulocytes (Figure 69B) in an LPS rat model of Parkinson's disease. # - p<0.05 vs. intact animals; $ - p≦0.05 vs. sham-operated animals; * - ≦0.05 vs. control animal model of Parkinson's disease; @ - p≦0.05 vs. unstimulated samples.

[0110] [Figure 70A] Figures 70A and 70B show the expression of phenotypic markers by circulating phagocytes in an LPS rat model of experimental Parkinsonism treated with 64Zn-asp. Figure 70A - Number of expressing cells in the analyzed population, Figure 70B - Expression level. # - p<0.05 vs. intact animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease. [Figure 70B] Figures 70A and 70B show the expression of phenotypic markers by circulating phagocytes in an LPS rat model of experimental Parkinsonism treated with 64Zn-asp. Figure 70A - Number of expressing cells in the analyzed population, Figure 70B - Expression level. # - p<0.05 vs. intact animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease.

[0111] [Figure 71A] Figures 71A and 71B show the therapeutic effect of 64Zn-asp on phagocytosis of peritoneal macrophages in an LPS rat model of Parkinson's disease. Figure 71A - relative number of phagocytes, Figure 71B - phagocytosis. # - p<0.05 vs. uninjured animals; $ - p<0.05 vs. sham-operated animals; * - p<0.05 vs. control animal model of Parkinson's disease. [Figure 71B] Figures 71A and 71B show the therapeutic effect of 64Zn-asp on phagocytosis of peritoneal macrophages in an LPS rat model of Parkinson's disease. Figure 71A - relative number of phagocytes, Figure 71B - phagocytosis. # - p<0.05 vs. uninjured animals; $ - p<0.05 vs. sham-operated animals; * - p<0.05 vs. control animal model of Parkinson's disease.

[0112] [Figure 72] Figure 72 shows the therapeutic effect of 64Zn-asp on the oxidative metabolism of peritoneal macrophages in an LPS rat model of Parkinson's disease. #-p<0.05 vs. intact animals, $-p≦0.05 vs. sham-operated animals, *-≦0.05 vs. control animal model of Parkinson's disease, @-p≦0.05 vs. unstimulated samples.

[0113] [Figure 73A]Figures 73A and 73B show the expression of phenotypic markers by peritoneal macrophages in an LPS rat model of experimental Parkinsonism treated with 64Zn-asp. Figure 73A - Number of expressing cells in the analyzed population. Figure 73A - Expression level. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease. [Figure 73B] Figures 73A and 73B show the expression of phenotypic markers by peritoneal macrophages in an LPS rat model of experimental Parkinsonism treated with 64Zn-asp. Figure 73A - Number of expressing cells in the analyzed population. Figure 73A - Expression level. # - p<0.05 vs. uninjured animals; $ - p≦0.05 vs. sham-operated animals; * - p≦0.05 vs. control animal model of Parkinson's disease.

[0114] [Figure 74] Figure 74 shows neuronal nitric oxide synthase expression in astrocytes in an LPS rat model of Parkinsonism treated with 64Zn-asp. @-p≦0.05 vs. control animal model of Parkinson's disease. 1 is control; 2 is sham-operated + HO; 3 is sham-operated + Zn-asp 1.5 mg / kg; 4 is LPS + HO; 5 is LPS + Zn-asp 1.5 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0115] Detailed Description As used herein, the word "a" or "plurality" before a noun refers to one or more than one of the particular noun.

[0116] Regarding the terms "for example" and "such as" and their grammatical equivalents, Unless expressly stated, it is understood to be followed by the phrase "and without limitation." As used herein, the term "about" is intended to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about," unless expressly stated otherwise, whether or not this term is explicitly used. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0117] As used herein, the suffix "e" attached to an isotope symbol refers to that isotopically enriched element. For example, 64 Zn e "teeth, 64 Zn refers to concentrated zinc. 85 Rb e "teeth, 85 Rb refers to enriched rubidium.

[0118] "Compound 1" as used herein refers to N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine. 85 It is called Rb-enriched rubidium organic salt and is called "E2+ 85 Rb e "or" 85 Rb e -E2." "E2" refers to N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine.

[0119] As used herein, 64 Zn e is at least 50% 64 Zn. Preferably, the zinc compounds, complexes, and salts of the compositions used in the disclosed methods are at least 75% 64 Zn, e.g., at least 80%, 85%, 90%, 95%, or 99% 64 Zn, 64 Zn e Includes.

[0120] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent that produces a beneficial effect on a patient. The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent that produces a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, amelioration, alleviation, reduction, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease or disorder in a patient, or any other desired alteration of a biological system. An effective amount can be administered in one or more administrations.

[0121] As used herein, "patient" and "subject" are synonymous terms and may refer to a human patient or an animal subject.

[0122] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, databases, entries, and other references mentioned herein are incorporated by reference in their entirety.In the event of any discrepancy, the present specification, including definitions, will control.

[0123] Neurodegenerative disorders

[0124] Neurodegenerative disorders (NDDs), including Parkinson's disease (PD), are pathological conditions induced by inflammatory processes. Calabrese V, et al. Free Radic Biol Med. 2017;115:80-91. doi: 10.1016 / j.freeradbiomed.2017.10.379.1; Franceschi C, et al., Trends Endocrinol Metab. 2017;28(3):199-212. doi: 10.1016 / j.tem.2016.09.005; Monti D, et al.. Mech Ageing Dev. 2017;165(Pt B):129-138. doi: 10.1016 / j.mad.2016.12.008. The development of NDD is characteristic of the elderly. The pathophysiological basis of this phenomenon is called "inflammaging," which is characterized by the elevated expression of inflammatory mediators in tissues, organs, and body fluids, as well as the metabolic profile of immune system cells, particularly effectors of innate immunity, such as mononuclear and polymorphonuclear phagocytes (monocytes, macrophages, and neutrophils). It is a chronic, low-grade systemic inflammatory process accompanied by a pro-inflammatory shift in the immune system. Franceschi C, Campisi J. J Gerontol A Biol Sci Med Sci. 2014 Jun;69 Suppl 1:S4-9. doi: 10.1093 / gerona / glu057. One of the most important inflammatory mediators is Reactive oxygen species (ROS) exert several important biological functions in both the development and pathological conditions. Myeloid cells of the immune system (monocytes, neutrophils, and macrophages) are the primary source of ROS. Monocytes and neutrophils are myeloid cells that constantly circulate in peripheral blood. Macrophages are resident cells present in all tissues without exception and act as sentinels of the immune system. They are formed through the differentiation of monocytes. Under normal conditions, ROS function as signaling molecules that control cellular metabolism. This ROS-mediated signaling is involved in tissue repair processes related to the stimulation of angiogenesis and the differentiation of stem tissue elements necessary for the reconstruction of tissue lesions. ROS have bactericidal functions and are effector molecules for the killing of pathogenic microorganisms both intracellularly and extracellularly. However, enhanced ROS generation leads to abnormal cell signaling and has significant effects on protein properties, causing problems in protein folding (the formation of their functional tertiary structure), the formation of protein aggregates, and consequently the loss / loss of their physiological functions and / or the acquisition of pathological functions. Among all proteins in the body, those incorporating sulfur-containing amino acids, namely cysteine ​​and methionine, are the most important targets of ROS. Ahmad S, et al. Front Biosci (Schol Ed). 2017;9:71-87. Luo S, Levine RL. et al., FASEB J. 2009;23(2):464-72. doi: 10.1096 / fj.08-118414. Cysteine ​​and methionine act as ROS scavengers, thus preventing the development of oxidative stress. The mechanism of the antioxidant effect of these amino acids is the immediate repair of their oxidized forms by reductases present in all cells of the body. For example, the oxidized form of methionine is repaired by methionine sulfoxide reductase (MSR). However, with aging, the synthesis of reductases, including MSR, significantly decreases, which is one of the reasons for the realization of the pathogenic effects of ROS and the development of inflammation.

[0125] ROS are also a pathogenic factor in PD. Targets of the pathogenic action of ROS in parkinsonism are several methionine-containing proteins in the brain, such as α-synuclein, tyrosine hydroxylase, mitochondrial complex I, PINK1 (PTEN-induced kinase 1), and the antioxidant DJ-1 [Herrero MT, et al., Front Neuroanat. 2015;9:32. doi: 10.3389 / fnana.2015.00032; Glaser CB, et al., Biochimica et Biophysica Acta 2005;1703;157-169; Danielson SR, Andersen JK. Free Radic Biol Med. 2008 May 15;44(10):1787-94. doi: 10.1016 / j.freeradbiomed.2008.03.005]. For example, ROS-mediated oxidation of methionine as part of α-synuclein This causes a decrease in its ability to exist in the protofibrillar form, promoting the aggregation of monomers along with the formation of pathological forms of this protein. Initially, the inflammatory process accompanying the development of PD was thought to be localized solely within the brain, where activated microglial cells (the brain's resident macrophages) are the source of ROS. However, recent studies have convincingly demonstrated the systemic nature of the inflammatory process in PD, involving the involvement of phagocytes, localizing the disease outside the brain in pathogenesis. The development of PD in both humans and experimental animals has been shown to involve proinflammatory activation of myeloid cells circulating in the peripheral blood (monocytes and neutrophils). These cells exhibit two types of pathological changes, quantitative and functional changes: monocytosis with neutropenia, accompanied by a proinflammatory shift in monocyte function, and a sudden increase in neutrophil oxidative metabolism. Furthermore, quantitative and functional characteristics of circulating myeloid cells are considered informative markers for monitoring the PD process and assessing the effectiveness of treatments for this condition. Funk N, et al., Mov Disord. 2013;28(3):392-5. doi: 10.1002 / mds.25300. Grozdanov V, et al., Acta Neuropathol. 2014;128(5):651-63. doi: 10.1007 / s00401-014-1345-4. The onset of PD is also , closely associated with phagocytes localized within the gastrointestinal tract, including peritoneal macrophages. Research conducted over several years has convincingly demonstrated a relationship between the development of parkinsonism and dysbiosis of the intestinal microbiota. A simplified scheme of dysbiosis in the development of PD appears as follows: Dysbiosis in the intestine is accompanied by proinflammatory activation of myeloid cells in the intestinal wall and peritoneal phagocytes, which become a source of local ROS generation. Increased ROS concentrations lead to a pathological shift in the aerobic / anaerobic ratio in the intestinal microbiota, increasing the relative number of aerobic forms of microorganisms compared to those without immune tolerance mechanisms. Increased numbers of pathogenic aerobic microorganisms further enhance proinflammatory activation of myeloid cells. Accelerated ROS synthesis leads to the formation of polymeric forms of α-synuclein in the intestine. α-synuclein aggregation causes pathological changes in the enteric nervous system and spreads throughout the body, reaching the brain via a prion-like mechanism. Felice VD, et al., Parkinsonism Relat Disord. 2016;27:1-8. doi: 10.1016 / j.parkreldis.2016.03.012. Obata Y, Pachnis V. Gastroenterology. 2016 v;151(5):836-844. doi: 10.1053 / j.gastro.2016.07.044. Yoo BB, Mazmanian SK. T Immunity. 2017;46(6):910-926. doi: 10.1016 / j.immuni.2017.05.011. Mukherjee A, Biswas A, Das SK. World J Gastroenterol. 2016 ;22(25):5742-52. doi: 10.3748 / wjg.v22.i25.5742. The crucial role of dysbiosis in the pathogenesis of PD is evidenced by the fact that the only way to prevent this disease is the so-called Mediterranean diet, characterized by a high content of complex carbohydrates, which stabilize the metabolism of normal gut microbiota, and polyunsaturated plant fatty acids, which function as powerful antioxidants. Cassani E., et al. Parkinsonism Relat Disord. 2017;42:40-46. doi: 10.1016 / j.parkreldis.2017.06.007.

[0126] See also U.S. Patent Publication Nos. 2019 / 0105345 and 2016 / 0153957; and U.S. Patent Nos. 10,226,484, 9,861,659, and 10,183,041.

[0127] Disclosed compounds, compositions, and methods

[0128] The present disclosure relates to methods, compounds, and compositions for treating neurodegenerative diseases in a subject, such as Parkinson's disease.

[0129] The present disclosure provides: 64 Zn-enriched zinc-containing compounds (separately or in combination with each other), salts, and complexes, e.g. 64 Zn Enriched Zinc Aspartate, or any 64 Zn-enriched zinc amino acids, and 85 Rb-enriched rubidium organic salts (structure shown below) are provided for use in treating neurodegenerative diseases in a subject, such as Parkinson's disease.

[0130] In another aspect, the present disclosure provides a method for treating neurodegenerative disorders (NDD), particularly Parkinson's disease (PD), comprising: 64 Zn-enriched zinc complexes, salts, and compounds, and / or certain 85 Compositions containing Rb-enriched rubidium compounds are provided. The disclosed compositions include one or more of the isotope-enriched compounds, optionally in combination with other active ingredients useful for treating NDD. The disclosed compositions can be used individually or in combination with other anti-NDD treatments. The disclosed compounds have low toxicity to patients.

[0131] In certain embodiments, the present disclosure provides compositions comprising compounds of Formula 1 for treating neurodegenerative disorders (NDDs), particularly Parkinson's disease (PD). [ka]

[0132] The compounds of formula 1 are rubidium salts, and rubidium is 85 Rb-rich, R1 to R 14 are each independently selected from H, OH, F, Cl, Br, I, C1-C6 alkyl, C1-C6 alkoxy, and NO2.

[0133] In certain embodiments, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13 are all H, and

[0134] a) R3 is CH3, and R7, R9, R 12 , and R 14 are all H (compound 1),

[0135] b) R3, R7, R9, R 12 , and R 14 are all H (compound 2),

[0136] c) R3 is CH3 and R 14 is Cl, and R7, R9, and R 12 are all H (compound 3),

[0137] d) R3 is CH3 and R 14 is OH, and R, R, and R 12 are all H (compound 4),

[0138] e)R 14 is OH, and R3, R7, R9, and R 12 are all H (compound 5),

[0139] f) R3 is OH, and R7, R9, R 12 , and R 14 are all H (compound 6),

[0140] g)R 14 is NO2, and R3, R7, R9, and R 12 are all H (compound 7),

[0141] h)R 12 is Br and R 14 is NO2, and R3, R7, and R9 are all H (compound 8);

[0142] i) R3 and R9 are both OCH3, and R 12 is Br and R 14 is NO2 and R7 is H (compound 9), or

[0143] j) R3 and R9 are both OCH3, and R 14 is NO2, and R7 and R 12 are both H (compound 10).

[0144] In any of the above compounds of formula 1, rubidium is preferably at least 75% 85 Rb, more preferably at least 85% 85 Rb, and even more preferably at least 95% 85 Rb, and in some embodiments, at least 99% 85 Rb, e.g. 99.8% 85 Rb. "N% 85 "Rb" is a compound in which N% of the Rb atoms are isotopes. 85 It refers to Rb, which is Rb.

[0145] In certain embodiments, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13 are all H, and the remaining R groups are as defined above. In other embodiments, R, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13are all H; R3 is selected from H, CH3, OCH3, and NO2; R7 and R9 are each independently selected from H and OCH3; R 12 and R 14 are each independently selected from H, Br, I, and NO. In another embodiment, R, R, R, R, R, R, R of Formula 1 10 , R 11 , and R 13 are all H; R3 is selected from H, CH3, OH, OCH3, and NO2; R7 and R9 are each independently selected from H and OCH3; R 12 is selected from H, Br, I, and NO2, and R 14 is selected from H, OH, Cl, Br, I, and NO2.

[0146] In certain embodiments, the composition 64 Zn e -asp; in a further embodiment, the composition further comprises 10% rubidium salt containing Rb-85.

[0147] In an exemplary embodiment, compound 1 of the invention has R3 as CH3 and the remaining R groups as H. This compound is referred to herein as N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine. 85 Rb-enriched organic rubidium salts and 85 Rb e -E2" and "E2+" 85 Rb e "E2" refers to N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine (in the figure, "E2+ 85 The terms "Rb," "E2Rb85," and "85RbE2" are 85 Rb e -E2).

[0148] The disclosed compounds are prepared as disclosed herein and / or using chemical synthesis methods known in the art.

[0149] Rubidium is a chemical element in the main subgroup of group 1, period 5 of the periodic table, with atomic number 37. 85 Rb is a stable isotope with a natural abundance of 72.2%. That is, naturally occurring rubidium consists of a mixture of rubidium isotopes. In a naturally occurring sample of rubidium, 72.2% of the rubidium atoms are isotopes. 85 It is Rb.

[0150] As used herein, " 85 "Rb enriched rubidium" is more than 72.2% 85 Rubidium is composed of Rb. Thus, in the disclosed compounds, rubidium is greater than 72.2%. 85 Rb, e.g., at least about 75% (e.g., at least 75%), at least about 80% (e.g., at least 80%), at least about 85% (e.g., at least 85%), at least about 90% (e.g., at least 90%), or at least about 95% 85 Rb (e.g., at least 95% 85 Rb).

[0151] In certain embodiments of the disclosed compounds, compositions, and methods, rubidium is at least about 90% 85 Rb (e.g., at least 90% 85 Rb), and more than 99% 85 Rb, for example, about 99.8% 85 Rb (e.g., 99.8% 85 Rb). The disclosed compositions may be such 85 The disclosed methods involve administering such compositions, including Rb-enriching compounds.

[0152] As used herein, the term "about" refers to plus or minus 3% of the amount of interest (e.g., "about 80%" refers to a range of 77.6% to 82.4%).

[0153] In another aspect, the present disclosure provides a method of treating or slowing the progression of a neurodegenerative disease, such as Parkinson's disease, comprising administering to a patient a compound of formula 1 85 Rb-enriched rubidium compounds and / or 64 Zn e In certain embodiments, the method comprises administering a therapeutically effective amount of a composition comprising a compound containing a salt, complex, or compound alone or in combination with conventional forms of NDD or PD treatment. 64 Zn e a therapeutically effective amount of a composition comprising a compound, salt, or complex of formula 1; 85 a therapeutically effective amount of a composition comprising an Rb-enriched rubidium compound; or 64 Zn e a compound, salt, or complex containing 85 In a further embodiment, such a composition comprises at least one excipient. 64 Zn e containing compounds and 85 Rb-enriched rubidium compounds exist in a specific ratio to each other, e.g., 90% 64 Zn e containing compounds and 10% of the 85Rb enriched rubidium compound of formula 1, e.g., 90% 64 Zn e -asp and 10% 85 Rbe-E2, where the percentages are based on the mass of the element. 64 Zn e The containing salt, complex, or compound is 64 Zn e -aspartate. In some embodiments, the compound is a peptide up to 20 amino acids in length.

[0154] In certain embodiments, the disclosed compositions comprise: 64 Zn eIn some embodiments, the compound is a zinc finger-shaped protein or peptide, containing one or two zinc ions and one or about 20 amino acids. In such structures, zinc may act as a structural stabilizer.

[0155] 64 Zn e and 85 Rb e In certain embodiments, in compositions comprising both compounds: 64 Zn e and 85 Rb e The ratio of the components is 64 Zn e is 9 parts (by weight) 85 Rb e is 1 part; in other embodiments, the ratio is 64 Zn e 7 parts (by weight) and 85 Rb e In some embodiments, the ratio is: 64 Zn e and 85 Rb e In a composition comprising both compounds 64 Zn e is 1 to 7 parts (by weight) 85 Rb e is 3 to 9 parts (by weight).

[0156] Instead of one composition containing both, 64 Zn e or 85 Rb e Also provided are two compositions each comprising any of the compounds of: 64 Zn e and 85 Rb e The ratio of the components is 64 Zn e is 9 parts (by weight) 85 Rb e is 1 part; in other embodiments, the ratio is 64 Zn e7 parts (by weight) and 85 Rb e In some embodiments, the ratio is: 64 Zn e and 85 Rb e In a composition comprising both compounds 64 Zn e is 1 to 7 parts (by weight) 85 Rb e is 3 to 9 parts (by weight).

[0157] In certain embodiments, compounds such as any of compounds 1-10 (e.g., N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine) are used. 85 Compositions are provided that include one or more compounds of Formula 1, optionally also containing one or more other anti-NDD agents, e.g., one or more anti-PD agents, such as Rb-enriched rubidium salts.

[0158] In some embodiments, the disclosed compositions comprise, in addition to the compound of Formula 1: 64 Zn e Containing compounds, salts, or complexes, e.g. 64 Zn e Aspartate (referred to herein as " 64 Zn e -asp), 64 Zn e of glutamate, or another amino acid 64 Zn e Salts, such as those containing any of the 18 other most common naturally occurring amino acids.

[0159] Disclosed are methods that include administering to a human or veterinary animal a disclosed compound or composition, and optionally at least one other form of anti-NDD therapeutic agent. The methods may be used to treat NDD (e.g., PD) or slow the progression of the disease in patients with the disease.

[0160] In certain embodiments, N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine 85 A compound containing Rb-enriched rubidium salt (Compound 1) is disclosed, and the enriched rubidium is 99.8% 85 It is Rb.

[0161] Combination therapy

[0162] In various embodiments, the disclosed compositions are administered prior to, concurrently with, or after administration of another NDD or PD therapy or treatment. In certain embodiments, if not administered simultaneously, the interval between administrations is about 48 hours or less, e.g., 48 hours or less, 36 hours or less, or 24 hours or less.

[0163] In other embodiments, the disclosed compositions comprise: 64 Zn e Containing compounds, salts, or complexes, e.g. 64 Zn e Aspartate, 64 Zn e of glutamate, or another amino acid 64 Zn e Salts, such as any of the 18 other most common naturally occurring amino acids, do not contain the compound of Formula 1. In some embodiments, such compositions contain one or more additional anti-NDD agents, such as an anti-PD agent.

[0164] In certain embodiments, the compound of formula I 85 In a composition containing both an Rb-enriched rubidium salt and an anti-PD drug, an appropriate dose 85 Rb e The salt and the previously prior art anti-NDD drug, e.g., previously prior art anti-PD drug, are present, for example, at a dose between 0.05 and 2.5 times the approved dosage (or at the dosage at which the anti-NDD drug would have been prescribed if not administered in conjunction with the disclosed compound).

[0165] The anti-PD drug may be any anti-PD drug, for example, an anti-PD agent such as L-dopa (optionally in combination with carbidopa), melevodopa, or etilevodopa; a dopamine agonist such as ropinirole, pramipexole, rotigotine, bromocriptine, pergolide, dihydroergocriptine mesylate, cabergoline, piribedil, and apomorphine; a monoamine oxidase B inhibitor such as rasagiline, selegiline, opicapone, and safinamide; The anti-PD drug composition may include one or more drugs selected from the group consisting of tetracholoro-O-methyltransferase inhibitors, such as entacapone and tolcapone; anticholinergic agents, such as trihexyphenidyl, benztropine biperiden, methixene, procyclidine, profenamine, dexetimide, fenglutarimide, mazaticol, bornaprine, tropatepine, ethanautine, orphenadrine (chloride), ethibenztropine, and budipine; and amantadine. The anti-PD drug composition optionally contains one or more adjuvant agents.

[0166] Formulation and Administration of Compositions

[0167] The disclosed compositions include any conventional form known in the art for administration to humans or animals.

[0168] In some embodiments, the disclosed compositions comprise at least one pharmaceutically acceptable vehicle or excipient, including, as needed, for example, diluents, fillers, disintegrants, solubilizers, dispersants, preservatives, wetting agents, stabilizers, buffers (e.g., phosphates, citrates, acetates, tartrates), suspending agents, emulsifiers, and penetration enhancers, e.g., D MSO included.

[0169] In certain embodiments, the composition may further contain suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers, or release aids, and other pharmaceutically acceptable excipients.

[0170] In some embodiments, the disclosed compositions are solutions for injection, such as intravenous injection.Water is preferably used in the injection composition as a dosage vehicle and diluent.Other pharmaceutically acceptable solvents and diluents may be used in addition to or instead of water, such as saline, glycerol, and ethanol.

[0171] A complete description of pharmaceutically acceptable excipients can be found, for example, in Remington's Pharmaceutical Sciences (Mack Pub., Co., NJ 1991) or other standard pharmaceutical textbooks, such as Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed. 2017).

[0172] Composition types include liquid compositions formulated for intravenous or other parenteral administration, compositions formulated for topical administration, and compositions formulated for oral administration, such as any type known in the art, including but not limited to tablets, pills, capsules, lozenges, granules, etc.

[0173] In certain embodiments, the composition comprises between 0.4 millimoles and 30 millimoles of a disclosed compound, such as between 1 millimoles and 10 millimoles, or for example, 1, 2, 5, 10, 20, 25, or 30 millimoles. The composition further comprises one or more excipients suitable for formulation.

[0174] The intravenous formulation comprises at least one of a suitable solvent, such as water; a salt or ion, such as sodium chloride, potassium chloride, potassium ions, sodium ions, or chloride ions; a sugar, such as glucose and sucrose; a buffer; or other excipient, such as DMSO.

[0175] Topical formulations include, but are not limited to, ointments, creams, lotions, and salves, and contain at least one of a suitable vehicle; a penetration enhancer, such as DMSO and related analogues; and an emulsifier.

[0176] Tablets contain at least one excipient, such as a filler (e.g., starch, lactose, sucrose, glucose); a binder (e.g., carboxymethylcellulose, gelatin, polyvinylpyrrolidone, sucrose); a disintegrant (e.g., calcium carbonate, alginic acid, sodium carbonate); a wetting agent (e.g., cetyl alcohol, and glycerol monostearate, sodium lauryl sulfate); a buffer; a lubricant (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate); and a coating.

[0177] Large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and copolymers of amino acids can also be used as vehicles for the disclosed agents and compositions.

[0178] For any disclosed compound, the therapeutically effective dose can be initially estimated from cell culture or animal model assays. Mice, rats, guinea pigs, rabbits, dogs, or pigs are commonly used as animal test models. Animal models can be used to determine the appropriate range of concentrations and routes of administration. Such information can then be used to determine the therapeutically effective dose in humans. The dosage conversion table provided in the Guidance for Industry and the Reviewers document (2002, US Food and Drug Administration, Rockville, MD, USA) can be used to determine the appropriate dose and route of administration for a patient. To estimate the human equivalent dose, it is recommended to use the dosage conversion table provided in the Guidance for Industry and the Reviewers document (2002, US Food and Drug Administration, Rockville, MD, USA). The exact effective dose for a patient will depend on various considerations, including the severity of the disease, the patient's general health, the patient's age, weight, and sex, nutrition, time and frequency of administration, route of administration, drug combinations, reaction sensitivities, and tolerability / response to treatment. The exact dose can be determined by routine experimentation and according to the professional judgment and discretion of the attending physician. In one embodiment, the total dose of the compound of Formula 1 of the present invention is about 1.25 mg 85 Rb e Approximately 12.5 mg / kg body weight 85 Rb e / kg body weight. 64 Zn e The prophylactic dose of (metallic) is 0.1 to 1.5 mg of pure metal per kg of human body weight. 64 Zn e The range is. 64 Zn e The therapeutic dose (of the metal) is 1 to 15 mg of pure metal per kg of human body weight. 64 Zn e The range is.

[0179] In certain embodiments, 85 Rb e The prophylactic dose of (metallic) is 0.1 to 1.25 mg of pure metal per kg of human body weight. 85 Rb e In certain embodiments, the therapeutic dose of 85Rbe (metallic) ranges from 1.25 to 12.5 mg pure Rbe per kg of human body weight. 85 Rb e The range is.

[0180] In some embodiments, the disclosed compositions are administered intravenously, intraperitoneally, orally, or intramuscularly. For IV, the dosage form may be a solution. Other conventional routes of administration may also be used, including, but not limited to, other injection routes, as well as via oral and topical administration.

[0181] Exemplary Methods for Making the Disclosed Compounds

[0182] The disclosed compositions may be prepared in accordance with common practices applied in the pharmaceutical industry, such as those exemplified in the latest edition of Remington's Pharmaceutical Science Handbook, Mack Pub. NY, USA. Prepared using the methods described in the lines.

[0183] Light isotopes may be purchased commercially: Zn-64 oxide and Rb-85Cl at the required enrichments may be purchased, for example, from Oak Ridge National laboratory, Oak Ridge, TN, USA.

[0184] A compound of formula 1, wherein R 9 Compounds where is H can be prepared as schematized below. Step 1. Aryl sulfonation: [ka] Step 2. Acylation: [ka]

[0185] Step 3: Obtain the rubidium complex. 85 To prepare Rb-enriched compounds, 85 Rb e Cl (e.g., 85 Rb e is 99% 85 Rb) is used in the final step shown below. [ka]

[0186] The compound of formula 1 can be prepared using intermediate (v) above, or a salt thereof, as a starting material or intermediate in the synthesis of the compound of formula 1.

[0187] In certain embodiments, the R groups of the compounds of intermediate (v) and salts thereof are the same as the corresponding R groups of the compounds of formula 1. In certain embodiments, the synthetic method proceeds as outlined below: [ka]

[0188] This synthesis produces a potassium salt as an intermediate and a rubidium salt as a product. 85 To obtain a product enriched with respect to Rb, 85 Rb e Cl can be used in place of RbCl.

[0189] R 9 Synthetic schemes for preparing certain disclosed compounds where is H are set forth below. Step 1. Arylsulfonation of o-phenylenediamine: [ka] Step 2. Acylation of N-R-phenylsulfonyl-o-phenylenediamine: [ka]

[0190] Step 3: Obtaining N-(R12,R14-benzoyl)-N'-(R3-phenylsulfonyl)-o-phenylenediamine rubidium complex. 85 To prepare Rb-enriched compounds, 85 Rb e Cl (e.g.,85 Rb e is 99% 85 Rb) is used in the final step shown below. [ka]

[0191] Compounds 1-10 may be prepared by the synthesis described above. [Example]

[0192] Example

[0193] In order that the present invention may be better understood, the following examples are set forth, which are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any manner.

[0194] Example 1 64 Zn e -asp and 64 Zn e -asp+10%E2 85 Rb e Effect of prophylactic administration of the preparation on the functional profile of phagocytes of different localizations in a rat model of Parkinson's disease.

[0195] Methods: Adult, mature rodents were used in the study. Before the start of the experiment, they were randomized by weight and divided into seven groups of eight animals each: Group 1 - intact animals; Group 2 - sham-operated animals; Group 3 - 64 Zn e Group 4 - Sham-operated animals administered with -asp; Group 5 - Animal model of Parkinson's disease induced by stereotactic intracerebral administration of 6-hydroxydopamine (6-OHDA); 64 Zn e -Animals with parkinsonism treated with prophylactic injections of asp; Group 6 - 64 Zn e -asp+10%E2 85 Rb eGroup 7 - sham-operated animals, which received injections of 64 Zn e -asp+10%E2 85 Rb e Parkinsonism after prophylactic injection Animal model. Before the prophylactic administration of experimental drugs, animals were subjected to the open field test to assess their overall locomotor activity level and anxiety. The prophylactic course consisted of 10 daily intraperitoneal injections of the drug (in the case of combined drugs, the components were administered at 2-hour intervals). After the prophylactic course, Parkinson's disease was modeled in animals. The degree of neuronal destruction in the substantia nigra was studied dynamically during apomorphine tests 7 and 14 days after the onset of the disease. Ten days after the final (second) apomorphine test, the behavioral responses of the animals were assessed in the open field test, after which they were euthanized and the functional (metabolic and phenotypic) profiles of differently localized phagocytes were analyzed. Thus, a study of the functional profile of phagocytes in a rat model of Parkinson's disease was performed 28 days after the end of the course of prophylactic treatment with experimental drugs. Resident peritoneal macrophages were isolated from a sterile cell suspension of peritoneal exudate; the suspension was concentrated by 2-hour adhesion. Nonadherent and dead cells were separated by centrifugation. Functional characteristics of circulating phagocytes (monocytes and neutrophils) were evaluated in whole blood collected in tubes containing EDTA, used as an anticoagulant. Mononuclear and polymorphonuclear phagocytes were analyzed by flow cytometry using a gating method. Microglial phagocytes were isolated using a Percoll density gradient. To assess the phenotypic profile of phagocytes, expression indices of the pan-phagocytic marker CD14 (a coreceptor for bacterial lipopolysaccharide) and the alternative polarization marker CD206 (mannose receptor 1) of phagocytes were used. To characterize the metabolic profile, microglial oxidative metabolism and phagocytic activity, as well as pathways of arginine metabolism, were studied using flow cytometry and spectroscopy, respectively. FITC-labeled Staphylococcus aureus wood 46 was used as a target for phagocytosis. Hemogram parameters were analyzed with a hematological analyzer, particle counter model PCE 210 (Elma Sales Co., Ltd., Japan), adapted for the study of blood cells in rats and mice.

[0196] Results: At the end of the experiment (14 days after the end of the prophylactic drug course), a reduction in the degree of neuronal destruction in the substantia nigra was recorded in both experimental groups. The number of animals in the control group with 100% unilateral destruction of neurons in the substantia nigra reached 75%, whereas animals 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn e In the -asp group, only 35% (more than 50% less than controls) and 30% (60% less than controls) of animals, respectively, showed complete loss of neurons in the substantia nigra. Furthermore, 86% of dopaminergic neurons were destroyed. 64 Zn e -asp+10%E2 85 Rb e It was recorded in 14% of the animals in the group (Table 1).

[0197] [Table 1]

[0198] Rotational speed during the apomorphine test in animals with 100% neuronal destruction of the substantia nigra compared with controls was 64 Zn e It should also be noted that the rate of mitochondrial death was 2.2 times faster than in a rat model of experimental parkinsonism after prophylactic administration of -asp.

[0199] The documented effects of drugs that reduce the degree of damage to dopaminergic neurons in the substantia nigra are accompanied by the following changes in the functional profile of phagocytes of different localizations.

[0200] Microglia

[0201] Increased microglial phagocytic activity is an indicator of their activation, both in repair and in inflammation processes. The final stage of inflammation is accompanied by a complete loss of the microglial capacity for phagocytosis, associated with the acquisition by microglial cells of the properties of antigen-presenting cells, and the activation of an adaptive pro-inflammatory immune response. See, e.g., Janda E, Boi L, Carta AR. Front Mol Neurosci. 2018;11:144. doi: 10.3389 / fnmol.2018.00144;Fu R, Shen Q, Xu P, Luo JJ, Tang Y. Mol Neurobiol. 2014;49(3):1422-34. doi: 10.1007 / s12035-013-8620-6. The development of parkinsonism is related to 6-OHDA toxicity. The number of phagocytic cells in microglia of animals receiving prophylactic drug treatment was significantly increased, while their endocytic activity was reduced. This change in metabolic processes was more pronounced when the drug was used in combination (Figure 1A & B).

[0202] for the phagocytic activity of microglial cells in intact animals 64 Zn e The modulatory effect of -asp in vitro was the opposite of that observed in vivo. It can be assumed that the modulatory effect of this drug on the phagocytic activity of microglial cells depends on its basal level, and the nature of the effect is homeostatic (normal).

[0203] Enhanced microglial oxidative metabolism is also characteristic of their activated state, which occurs during both repair and destructive inflammatory processes. The development of Parkinsonism (and surgery without 6-OHDA administration) was accompanied by a decrease in microglial oxidative metabolism in laboratory animals. In animal models of Parkinson's disease receiving preventive drug treatment, indicators of microglial oxidative metabolism were significantly higher than in control rat models of Parkinson's disease (2-fold for animals receiving zinc-based drugs and 4.5-fold for animals receiving combination drugs).

[0204] at the same time, 64 Zn e In the group receiving -asp, this index was at the level of control intact animals, and 10% of E2 85 Rb e Contains 64 Zn e In the group receiving -asp, the index of reactive oxygen species (ROS) generation was two times higher than in intact animals (Figure 1.1.2). Considering the nature of the clinical preventive effect, it can be assumed that the enhancement of microglial oxidative metabolism after the action of the drug is due to the activation of repair processes mediated by microglial cells.

[0205] In addition to the basal level of oxidative metabolism, we also analyzed the metabolic reserve of this function. For this purpose, cells were treated in vitro with phorbol myristate acetate (PMA). The metabolic reserve of microglial oxidative metabolism was absent in all animals. (Figure 2)

[0206] Increased synthesis of reactive nitrogen species, especially NO, characterizes the arginine metabolic pathway in phagocytes through the use of NO synthase and is a criterion for pro-inflammatory (M1) activation of microglia, which is accompanied by destructive inflammatory processes. demonstrates an anti-inflammatory metabolic shift in phagocytes, including in phagocytes.

[0207] The development of parkinsonism is accompanied by increased production of reactive nitrogen species by microglial phagocytes, indicating proinflammatory activation of these cells (Figure 3).

[0208] In the drug-treated animal model of Parkinson's disease, the level of NO synthesis did not differ from that in intact animals. This is evidence of the drug's anti-inflammatory and immunomodulatory effects on microglial cells. This assumption is supported by the fact that NO synthesis is inversely related to ROS synthesis. Singh AK, et al., Nitric Oxide. 2016;58:28-41. doi: 10.1016 / j.niox.2016.06.002. Given the fact that increased ROS synthesis was recorded in the drug-treated animal model of Parkinsonism, it is logical to assume that this situation leads to a decrease in the synthesis of apoptotic reactive nitrogen species by microglial cells and is associated with reduced neuronal destruction in the substantia nigra.

[0209] It should be noted that in in vitro studies, both drugs had an inhibitory effect on the synthesis of reactive nitrogen species by non-sensitized microglial cells in intact animals, which supports the above assumption.

[0210] Arginase activity is a second or alternative pathway for arginine utilization by phagocytes. The arginine metabolic pathway is consistently characterized by changes in both indices (NO synthesis level and arginase activity). Arginase activity was not statistically significant in the various groups of animals. Figure 4.

[0211] In addition to the nature of the changes in NO synthesis levels by microglial cells in the different groups of animals, this further confirms the assumption regarding an anti-inflammatory metabolic shift of microglial phagocytes in a rat model of experimental parkinsonism after prophylactic administration of the drug.

[0212] The drug did not exert any statistically significant effect on arginase activity in unsensitized microglial cells from intact animals, indicating that the drug's effect on arginine metabolism is mediated and dependent on alterations in phagocyte oxidative metabolism.

[0213] CD14 is a pan-phagocytic marker (expressed by all phagocytes, including microglia). Its expression level increases with functional maturation of the cells associated with their activation (both pro- and anti-inflammatory). The onset of parkinsonism is accompanied by an increase in the number of CD14+ cells in microglia, which is thought to be the result of migration of circulating phagocytes to the brain as a result of the breakdown of the blood-retinal barrier, which is related to the development of neurodegenerative diseases (Figures 5A and 5B).

[0214] a. 64 Zn e Prophylactic administration of -asp was accompanied by a decrease in the relative number of CD14+ cells, along with an increase in their expression level (Figure 5B), possibly indicating the drug's ability to reduce the occurrence of blood-retinal barrier breakdown and / or phagocytic migration, while simultaneously stimulating their functional maturation, which may be both pro- and anti-inflammatory. Based on our own experience, we believe that the increase in the number of CD206+ cells in microglia is evidence of the activation of this population as a whole. Such an increase in expression level is evidence of an alternative (anti-inflammatory) metabolic polarity of brain phagocytes. According to our findings, the onset of parkinsonism was accompanied by an increase in the fraction of CD206+ cells (Figure 6A), without a significant increase in the mean level of their expression (Figure 6B), which, considering the pathogenesis of the disease, is evidence of pro-inflammatory microglial activation. 64 Zn e Prophylactic administration of -asp, along with an increase in its expression level, caused a significant increase in the relative number of CD206+ cells in microglia, indicating an anti-inflammatory shift in the functional profile of these cells. It should be noted that there was significant individual variation in both indices. b. Microglial phenotypic profile in animal models of parkinsonism 64 Zn e -asp+10%E2 85 Rb e The effects of this drug had slightly different characteristics. At the end of the experiment, the relative number of CD14+ cells in microglia of animals that underwent the preventive course was not significantly different from that of the control group of Parkinson's disease animal models (Figure 5B). However, the expression level of this marker was significantly increased (Figure 6B). Both facts indicate that the drug probably does not have an effect on the migration of peripheral phagocytes to the brain, but stimulates the functional maturation of resident microglial cells. The relative number of CD206+ cells in microglia after administration of this drug was not significantly different from that of the control animal model of Parkinsonism (Figure 6A). At the same time, its expression level was significantly increased (Figure 6B), convincingly demonstrating the drug's ability to induce anti-inflammatory activation of brain phagocytes.

[0215] circulating phagocytes

[0216] Parkinson's disease is a multisystem neurodegenerative disorder based on the irreversible destruction of dopaminergic neurons against a background of an inflammation-induced shift in metabolism in resident brain phagocytes called microglia. However, a growing number of recent experimental studies and clinical observations indicate that the inflammatory process in Parkinson's disease is systemic in nature and involves an inflammation-induced functional shift in phagocytes localized in the brain periphery, including circulating mononuclear and polymorphonuclear phagocytes. Smith AM, et al., Mov Disord. 2018;33(10):1580-1590. doi: 10.1002 / mds.104. The development of parkinsonism is associated with monocytosis and an increased risk of developing monocyte precursors in the bone marrow. It is associated with an increase in monocyte counts. Raj T, Rothamel K et al. Science. 2014;344(6183):519-23. doi: 10.1126 / science.1249547. Monocytosis in Parkinson's disease is associated with a decrease in the number of circulating granulocytes (neutrophils in the first place). Wijeyekoon RS, et al. Front Neurol. 2018 Oct 16;9:870. doi: 10.3389 / fneur.2018.00870. Circulating monocytes in Parkinson's disease are characterized by an activated state, as evidenced by increased expression of apoptotic markers (activation-induced apoptosis). Wijeyekoon RS, et al., Front Neurol. 2018 Oct 16;9:870. doi: 10.3389 / fneur.2018.00870; Lin WC, et al., Biomed Res Int. 2014;2014:635923. doi: 10.1155 / 2014 / 635923). There are also data on the decreased phagocytic activity of peripheral blood monocytes in patients with parkinsonism. Grozdanov V, et al., Acta Neuropathol. 2014;128(5):651-63. doi: 10.1007 / s00401-014-1345-4. Disruption of monocyte function is associated with the expression of LRRK2 kinase ( Associated with genetically determined defects in the expression of leucine-rich repeat kinase 2. Bliederhaeuser C, et al., Acta Neuropathol Commun. 2016;4(1):123.

[0217] The onset of parkinsonism was accompanied by a statistically significant leukocytosis (Figures 7A and 7B). Analysis of the percentages of major leukocyte populations (lymphocytes, monocytes, and granulocytes (neutrophils)) indicated that monocytosis is likely the cause of leukocytosis in parkinsonism, as the fraction of these phagocytes was statistically significantly increased in the rat model of Parkinson's disease (Figure 7B). Leukocytosis was also observed in sham-operated animals. However, in this case, given that the study was conducted long after sham surgery, it was considered to be caused by an increase in the lymphocyte fraction, a natural sign of resolution of the inflammatory process. In the animal model of Parkinson's disease treated with drugs containing trace elements, absolute circulating leukocyte counts were not significantly different from the same values ​​in the control group of the rat model of Parkinson's disease. However, after administration of the drugs, no significant changes were recorded in the population composition of peripheral blood leukocytes, and there was a decrease in monocytosis and normalization of relative circulating lymphocyte counts, indicating their anti-inflammatory systemic effect.

[0218] At the end of the experiment, the phagocytic activity of circulating monocytes in the rat model of parkinsonism was reduced compared to intact animals (FIG. 8), which correlates with the clinical observations described above.

[0219] In animals receiving zinc as a drug, monocyte phagocytosis did not differ from that in intact animals. In rats receiving the combined drug, monocyte phagocytosis was significantly reduced. The number of phagocytic cells in animals receiving prophylactic treatment with trace elements was also reduced (Figure 8B), which correlates with the data on the reduction in monocyte counts after prophylactic drug treatment (Figures 7A and 7B).

[0220] Monocyte oxidative metabolism in animal models of Parkinson's disease was statistically significantly lower than in intact animals (Figure 9). In rats receiving a prophylactic course of trace elements, oxidative metabolism in circulating monocytes was within the normal range.

[0221] Functional conservation of oxidative metabolism was present in all animal cell groups. It should be noted that in in vitro studies, drugs containing trace elements slightly inhibited intracellular oxidative metabolism in non-synthetic monocytes from peripheral blood of intact rats. In this case, as can be seen in Figure 9, the drugs normalized the oxidative metabolism that was weakened in in vivo studies. The nature of the effect of zinc and rubidium drugs on the intracellular production of reactive oxygen species by immature forms of mononuclear phagocytes appears to depend on the initial value of this index within the cells.

[0222] The modulatory effects of trace element-containing drugs on the function of peripheral blood granulocytes (neutrophils) in rat models of Parkinsonism were also documented. The relative number of circulating neutrophils in all animals in the experimental groups was lower than in intact animals (Figure 7B). At the same time, the phagocytic function of these cells in the rat models of Parkinson's disease was the same as in intact animals (Figure 1.1.10). In the groups of animal models of Parkinsonism that underwent preventive treatment with trace elements, the phagocytic activity of peripheral blood neutrophils was significantly reduced compared to control animal models of Parkinson's disease and to intact animals. The obtained results are consistent with the characteristics of the in vitro modulatory effects of trace element-containing drugs on the phagocytic function of unsensitized neutrophils in the peripheral blood of intact rats (Figures 10A and 10B).

[0223] In the development of Parkinson's disease, intracellular oxidative metabolism of peripheral blood neutrophils was reduced compared to intact animals (Fig. 11).

[0224] 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn e Prophylactic administration of -asp was associated with the absence of any disturbance in oxidative metabolism in a rat model of experimental parkinsonism. A functional reserve of neutrophil oxidative metabolism was present in all groups of animals.

[0225] Data on the evaluation of the phenotypic profile of peripheral blood phagocytes in animals confirm the results of the evaluation of their metabolic characteristics. The fraction of CD14+ peripheral blood phagocytes in control animal models of Parkinsonism was statistically significantly lower than that in intact animals, indicating circulating immature cell forms, possibly associated with monocytosis (Figures 12A and 12B). The expression level of this marker in rat models of Parkinson's disease was also lower than that in intact animals, which is confirmed by the presence of immature circulating phagocyte forms (Figures 12A and 12B).

[0226] The relative number of circulating phagocytes expressing CD206 and the expression level of this phenotypic marker in the rat model of parkinsonism were higher than in intact animals, which should be considered as a sign of activation of immature phagocytes recruited from the bone marrow (Figures 13A and 13B).

[0227] At the end of the experiment, CD206 expression in the circulating phagocyte population of animals that had undergone a preventive course with a drug containing trace elements was significantly higher than that of control and intact animals in the animal model of Parkinson's disease. This is further evidence of the drug's anti-inflammatory effect on circulating phagocytes. In general, analysis of the metabolic and phenotypic characteristics of circulating phagocytes demonstrates the drug's ability to reduce the appearance of the systemic inflammatory response associated with monocytosis, a hallmark sign of parkinsonism.

[0228] Peritoneal macrophages

[0229] 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn eAnalysis of the functional and phenotypic profiles of peritoneal macrophages in an animal model of Parkinson's disease that underwent a preventive course with -asp stemmed from three circumstances. First, the drug was administered intraperitoneally, and peritoneal macrophages were the first cells of the immune system to be exposed to the action of the preparation containing trace elements. Second, it is known that the blood-brain barrier is not an absolute barrier to the interaction between the brain and the peripheral immune system, and that brain antigens are transported via lymphatic vessels in the central nervous system to regional lymph nodes, the nose, and the deep neck, which are part of the mucosa-associated lymphoid tissue (MALT) (Louveau A, et al., Trends Immunol. 2015 Oct;36(10):569-577. doi: 10.1016 / j.it.2015.08.006; Raper D, et al., Trends Neurosci. 2016 Sep;39(9):581-586. doi: 10.1016 / j.tins.2016.07.001). MALT also includes the peritoneal cavity. According to the scheme of immune response activation in MALT, the presence of antigenic stimuli in any of its compartments may be accompanied by activation of MALT as a whole. Third, a correlation has been established between neurodegenerative diseases and disorders of the normal intestinal microbiota, i.e., dysbiosis, the onset of which involves activation of phagocytes in the peritoneal cavity adjacent to the intestine. Kishimoto Y, et al., Neuromolecular Med. 2019 May 11. doi: 10.1007 / s12017-019-08539-5.

[0230] The development of Parkinson's disease was accompanied by activation of rat peritoneal macrophages. Accordingly, peritoneal phagocytes in animal models of Parkinsonism were characterized by statistically significantly increased endocytic activity compared with intact animals (Figures 14A and 14B).

[0231] 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn eProphylactic administration of -asp prevented the increase in phagocytosis by peritoneal macrophages in parkinsonism.

[0232] Oxidative metabolism of peritoneal macrophages in a rat model of Parkinson's disease was also higher than in intact animals. Figure 15.

[0233] Prophylactic administration of drugs containing trace elements did not affect this index in animal models of parkinsonism.

[0234] Significantly enhanced reactive nitrogen species production also indicates pro-inflammatory activation of peritoneal macrophages in parkinsonism.

[0235] 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn e Prophylactic administration of -asp prevented the increase in NO synthesis by peritoneal macrophages in an animal model of parkinsonism.

[0236] Further evidence for the ability of trace element-containing drugs to prevent the pro-inflammatory shift in arginine metabolism in peritoneal macrophages in Parkinson's disease is provided by data on the arginase activity of these cells. As mentioned above, arginase and NO synthase are the two enzymes that metabolize arginine by phagocytes. The arginine metabolic pathway is the main metabolic criterion for the functional polarization of phagocytes. Increased arginase activity is a sign of an anti-inflammatory shift in metabolism in these cells, while increased production of reactive nitrogen species is a sign of their pro-inflammatory shift. Our results show that arginase activity in peritoneal macrophages in a rat model of parkinsonism was not different from that in intact animals, indicating a proinflammatory shift in the metabolism of these cells along with increased NO production (Figure 17).

[0237] 10% E285 Rb e Contains 64 Zn e Zinc-asp significantly increased arginase activity in peritoneal phagocytes, which, together with reduced production of reactive nitrogen species, indicates an anti-inflammatory metabolic profile of these cells. Zinc-based drugs also contributed to an anti-inflammatory shift in arginine metabolism by peritoneal phagocytes, as evidenced by reduced NO synthesis and the absence of any change in arginase activity.

[0238] Analysis of the phenotypic profile of peritoneal macrophages confirmed their involvement in the pathogenesis of the disease. To evaluate the phenotypic profile of peritoneal macrophages, we used the same phenotypic markers analyzed in microglial phagocytes and circulating phagocytes. Our findings showed that CD14 expression by peritoneal phagocytes did not change statistically significantly under disease conditions or under the influence of drugs containing trace elements, which is typical of resident tissue phagocytes, since CD14 expression on the membrane is physiologically maximal (Figures 18A and 18B).

[0239] 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn e CD14 expression in a population of peripheral macrophages in a rat model of experimental parkinsonism treated with prophylactic -asp. * -p ≤ 0.05 vs. the group of intact animals; #-p ≤ 0.05 vs. the control group of the animal model of parkinsonism.

[0240] The fraction of CD206+ cells in the peritoneal cavity is very low; therefore, we analyzed the number of cells expressing this marker in the population of CD14+ peritoneal phagocytes. The results of the analysis showed that the fraction of peritoneal CD14+ phagocytes expressing CD206 did not change with the onset of Parkinsonism (Figure 19A). However, the expression level of this marker was reduced in the animal model of Parkinson's disease compared with that in intact animals.

[0241] 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn e Prophylactic administration of -asp was associated with a further decrease in the expression levels of this marker in the peritoneal phagocyte population, which is inconsistent with an altered metabolic profile of the cells.

[0242] Findings from Example 1:

[0243] 1. The development of experimental parkinsonism is accompanied by changes in the functional and phenotypic profile of differently localized phagocytes along with signs of their inflammatory activation.

[0244] 2. 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn e Prophylactic systemic administration of -asp modulated the metabolic and phenotypic profiles of differently localized phagocytes in an animal model of Parkinsonism, reducing the cellular inflammatory metabolic shift that occurred against the background of reduced levels of dopaminergic neuron destruction in the animals.

[0245] 3. The nature of the modulatory effect of drugs containing trace elements on phagocytes varied depending on their localization and maturity. Microglial cells in animals receiving the drugs were characterized by the activation of anti-inflammatory (repair) pathways. Preventive drug administration prevented metabolic and phenotypic shifts in circulating phagocytes that are normally recruited to the brain during neurodegenerative disorders, which was also one of the reasons for the reduction of neuronal destruction in the substantia nigra. Drug administration was also associated with a reduction in the peritoneal The drug prevented proinflammatory activation of phagocytes within the intestinal cavity. The mechanism of this phenomenon may be complex and involve drug effects on cells of the immune system, as well as on the microbiota and intestinal epithelial cells, leading to the prevention of the pathological gut-brain axis. Analysis of such complex effects requires studies of the drug's effects on intestinal barrier integrity, the translocation of intestinal bacteria, and their metabolism.

[0246] 4. Functional status and quantitative parameters of peripheral blood phagocytes in neurodegenerative disorders 64 Zn e -asp and 10% E2 85 Rb e Contains 64 Zn e -asp can be considered a sensitive criterion for monitoring its effectiveness. In these calculations, mass properties of pure elements are used ( 64 Zn e and 85 Rb e ). For example, in a mixture 64 Zn e 9 parts and 85 Rb e Part 1 means that the calculation is based on the mass of the pure element ( 64 Zn e of pure elements in 9 parts and 85 Rb e 1 part of pure element).

[0247] Example 2 Metabolic profile of non-sensitized mouse peritoneal macrophages 64 Zn e -asp and E2+ 85 Rb e Influence of preparation

[0248] Materials and Methods: Peritoneal macrophages (PM) were isolated from the peritoneal cavity of male mice of the C57 / B6 strain weighing an average of 22 g without prior sensitization. The metabolic profile of phagocytes was characterized by the arginine metabolic pathway (by the level of arginase activity and NO synthesis measured using a photocolorimetric method), the formation of intracellular reactive oxygen species (ROS) by esterase, and the level of phagocytic activity measured using flow cytometry, as well as the level of NADH and NADPH oxidase activation as determined by the NBT test. The duration of treatment of standardized suspensions of non-sensitized macrophages from the peritoneal cavity varied depending on the methodological characteristics of the metabolic response. Kim DK, Pfeifer J. Surg Forum. 1977;28:85-7. A 90-minute exposure to drugs did not affect their oxidative metabolism and phagocytic activity. An 18-hour exposure to the drug was used to analyze the effect on the arginine metabolic pathway. Lastra MD, et al., J Trace Elem Med Biol. 2001;15(1):5-10. To analyze the effect of the drug under study on stimulated metabolic activity of phagocytes, cells were treated with lipopolysaccharide (LPS) from E. coli (Sigma, USA) according to Haddad JJ. Mol Immunol. 2009;47(2-3):205-14. doi: 10.1016 / j.molimm.2009.09.034.

[0249] result:

[0250] 90 minutes 64 Zn e PM treatment with -asp had a mild stimulatory effect on spontaneous and LPS-stimulated ROS generation by macrophages (Fig. 20). Treatment with the drug at a concentration of 10 μg / ml caused a greater stimulation of expressive ROS generation than after treatment with LPS. There was no clear dose-response relationship.

[0251] Treatment of cells with drugs for 90 min had no statistically significant effect on either the spontaneous or induced state of hexose monophosphate shunt (activation of NADH and NADPH oxidase) in peritoneal macrophages (Figure 21).

[0252] It should be noted that after 90 min of PM exposure, the action of LPS did not alter the levels of NADH and NADPH oxidase activation by peritoneal phagocytes, which may be due to the insufficient duration of treatment.

[0253] Drugs containing stable isotopes of zinc had no significant effect on the phagocytosis of non-sensitized PMs (FIG. 22).

[0254] A trend towards a slight decrease in the intensity of endocytosis in treated cells was observed only after using the lowest drug concentrations.

[0255] The drug's ability to exert a weak, multidirectional effect on arginase activity at the trend level was observed 18 hours after treatment of PM with the drug (Figure 23). It should be noted that there was significant variability in optical density values ​​within each experimental option. A non-significant dose-dependent decrease in arginase activity of treated cells was observed in the concentration range of 5 to 20 μg / ml. Further reductions in concentration were accompanied by a loss of the dose-response relationship. There was no statistical significance to the effect.

[0256] 64 Zn e -asp had the most pronounced modulating effect on the production of reactive nitrogen species by peritoneal phagocytes (Figure 24).

[0257] Exposure of cells to drugs in the concentration range of 1.25 to 10 μg / ml for 18 hours resulted in a statistically significant inhibition of spontaneous production of reactive nitrogen species by PM. There was no dose-response relationship. Treatment of bacterial LPS-stimulated cells with preparations containing stable isotopes of zinc also caused a statistically significant reduction in NO synthesis. A trend toward an inverse dose-dependence of the inhibitory effect of zinc-based drugs on phagocyte-induced production of reactive nitrogen species was noted.

[0258] E2+ on the metabolic activity of peritoneal phagocytes 85 Rb e The modulatory effect of the compound is 64 Zn e This was even more pronounced than in the case of -asp. Exposure of non-sensitized PMs to the drug at concentrations ranging from 62 to 500 μg / ml for 90 min caused a rapid inhibition of their spontaneous ROS production as a result of esterase activation (Figure 25). No dose-response relationship was observed. The drug (E2+) at concentrations of 8, 15.5, and 31 μg / ml significantly inhibited the ROS production. 85 Rb e ) did not cause any statistically significant changes in the spontaneous realization of metabolic functions by the cells.

[0259] E2+ used in the same concentration range 85 Rb e caused a rapid inhibition of induced (bacterial LPS-stimulated) intracellular ROS generation by peritoneal phagocytes. No dose-response relationship was observed. Figure 25.

[0260] E2+ for the activation of NADH and NADPH oxidase in PM 85 Rb eThe nature of the effect of the drug on the reactive metabolic production of oxygen by esterases (Figure 26) was different from that on the reactive metabolic production of oxygen by esterases (Figure 26). When used at a concentration of 500 μg / ml, the drug caused a slight increase in phagocyte oxidase activity. All drug concentrations below 500 μg / ml did not exert a statistically significant effect on this index of PM metabolic activity. The nature of the drug's effect on induced (LPS-stimulated) oxidase activity was similar to that of the spontaneous realization of this function by the cells. When used at lower concentrations, the drug demonstrated a tendency toward the ability to reduce the formation of reactive oxygen species by oxidases. However, given the non-significant level of the effect and the disproportionate variation in its direction depending on the concentration, it should be considered nonspecific and due to variations in optical density (Figure 26).

[0261] Drugs containing stable isotopes of rubidium exerted similar effects on the phagocytosis of non-sensitized peritoneal phagocytes (Figure 27). When used at concentrations of 500 and 250 μg / ml, they modestly enhanced endocytosis in peritoneal phagocytes. Decreasing drug concentrations were accompanied by its lack of effect on this index of PM metabolism.

[0262] E2+ at concentrations of 500 and 250 μg / ml with 18 h exposure 85 Rb e The use of E2+ caused a statistically significant increase in PM arginase activity (Figure 28). The stimulatory effect of the drug on this metabolic indicator of phagocytes was independent of additional treatment of the cells with bacterial LPS and was more pronounced compared to its effect on the phagocyte function of the cells. 85 Rb e The dose-dependence of the modulatory effect of was not statistically significant.

[0263] E2+ 85 Rb e The most visible effect of 64 Zn e As in the case of -asp, the generation of reactive nitrogen species by PM was recorded after 18 hours of exposure (Figure 29).

[0264] When used at all of the above concentrations except 8 μg / ml, the drug inhibited the spontaneous production of reactive nitrogen species by PM. No dose-response relationship was observed. The drug also tended to attenuate the induced production of NO by cells treated with bacterial LPS, again without a dose-response relationship.

[0265] Findings regarding Example 2.

[0266] 1. Both drugs under study have an anti-inflammatory modulatory effect on the metabolic profile of unsensitized and bacterial LPS-stimulated peritoneal phagocytes. This conclusion is justified by the altered effect of the drugs on the arginine metabolic pathway in phagocytes, toward a sharp decrease in the generation of reactive nitrogen species, which are products of arginine metabolism by inducible NO synthase (iNOS). Reactive oxygen species are potent mediators of the inflammatory response and signaling molecules involved in the activation of apoptosis. Reduced iNOS activity characterizes an alternative phenotype (M2) of phagocytes. There is no clear dose-response relationship.

[0267] 2. More pronounced effects were noted with drugs containing stable isotopes of rubidium, which also exhibit strong antioxidant activity.

[0268] 3. The high level of variability in some of the variables under study could be a result of the unstable solubility of both preparations, especially the zinc-based preparation, in the polar solvent (saline solution).

[0269] The lack of immunomodulatory effects of zinc-based preparations may be due to insufficient exposure of cells to the drug. In this regard, since in vitro studies are limited by methodological constraints, it is recommended to conduct additional in vivo studies of the drug's effect on phagocyte oxidative metabolism using intact animals, which would allow for a longer-term effect of the agent on this cellular function.

[0270] Example 3 on the metabolic profiles of non-sensitized microglial cells, circulating monocytes, and neutrophils in healthy rats 64 Zn e -asp and E2+ 85 Rb e Effect of the preparation

[0271] Materials and Methods: Rat microglial cells were isolated by mechanical (cell sieve) disaggregation of brain tissue (Figures 30A, 30B, and 30C), followed by fractionation of tissue homogenates on a Percoll density gradient (Microglia: Methods and Protocols, Edited by Bertrand Josef and Jose Luis Venero, Springer protocol. Humana Press, 2013, 350 p.) (Figure 31). Before collecting the brain tissue, decapitation was performed to isolate the brain during sampling. Bleeding was avoided (Figure 32). Decapitation was performed immediately after anesthesia to minimize the death of brain tissue, including microglial cells. All procedures for the isolation of microglial cells were performed using a cooling agent. Circulating neutrophils were isolated from venous blood collected in tubes containing EDTA on a two-step Percoll gradient (Alsharif KF et al. Vascul Pharmacol. 2015 Aug;71:201-7. doi: 10.1016 / j.vph.2015.02.006) (Figures 33A and 33B). Circulating monocytes were isolated from venous blood collected in heparin-containing tubes using a two-step Hypaque gradient (Macrophages and dendritic cells: Methods and Protocols, Edited by Neil E. Reiner, NY, Humana Press, 2009: 368 p). The metabolic profile of phagocytes was characterized based on the pathway of arginine metabolism (arginase activity and NO synthesis levels measured using a photocolorimetric method), the level of intracellular reactive oxygen species (ROS) formation by esterase, and an index of phagocytic activity determined by flow cytometry, as well as the activation levels of NADH and NADPH oxidases determined by the NBT test. The duration of treatment of all the above-mentioned populations of phagocytes varied depending on the methodological characteristics of the metabolic response. Kim et al., 1977 (cited above) A 90-minute exposure to drugs according to the method described above was used to analyze their effects on oxidative metabolism and phagocytic activity. An 18-hour exposure according to Lastra et al., 2001 (cited above) was used to analyze drug effects on the arginine metabolic pathway. To analyze the effect of the drugs under study on the induced (stimulated) metabolic activity of phagocytes, cells were treated with lipopolysaccharide (LPS) from E. coli (Sigma, USA) according to Haddad et al., 2009 (cited above).

[0272] result:

[0273] Microglia

[0274] 64 Zn eTreatment of non-sensitized microglial cells with -asp had multidirectional effects on the spontaneous and LPS-stimulated generation of intracellular ROS without a clear dose-response relationship (Figure 34). At concentrations of 20 and 10 μg / ml, the drug stimulated esterase-dependent oxidative metabolism in microglial cells, whereas concentrations of ≦5 μg / ml had no statistically significant effect on this index. At concentrations of 20 and 5 μg / ml, the drug significantly inhibited LPS-stimulated ROS generation. When used at other concentrations, it had no effect on esterase-dependent oxidative metabolism induced in the cells under study.

[0275] At all concentrations studied, the drug mildly increased the phagocytic activity of microglial cells without affecting the number of phagocytes in the population (Figure 35). No clear dose-response relationship was observed.

[0276] Under normal conditions, resident microglial phagocytes are characterized by a low level of phagocytic activity, the increase of which is a sign of functional activation of these cells.

[0277] Regardless of concentration, the drug reduced spontaneous nitric oxide production by glial cells (Figure 36). There was a weakly expressed inverse dose-response relationship. At a concentration of 10 μg / ml, the drug also statistically significantly reduced the stimulatory activity of NO synthase, as evidenced by a reduction in nitrite synthesis.

[0278] Regardless of the concentration, the drug had virtually no effect on spontaneous arginase activity in microglial cells (Figure 37). In the background of LPS treatment, at a concentration of 2.5 μg / ml, the drug modestly enhanced this metabolic function of brain phagocytes.

[0279] The absence of an effect on arginase activity in combination with an inhibitory effect on nitrite synthesis indicates an anti-inflammatory modulatory effect of the drug on this population of phagocytes.

[0280] Peripheral blood monocytes

[0281] 64 Zn e Treatment of peripheral blood monocytes with -asp resulted in a decrease in spontaneous esterase-dependent intracellular ROS production by the cells. A weakly expressed inverse dose-response relationship was observed (Figure 38).

[0282] At a concentration of 10 μg / ml, the drug statistically significantly reduced this metabolic function in cells treated with bacterial LPS.

[0283] At the same concentrations, the drug slightly increased spontaneous oxidative metabolism in peripheral blood monocytes depending on NADH and NADPH oxidase (FIG. 39).

[0284] The drug had no effect on the LPS-stimulated oxidase-dependent respiratory burst of monocytes.

[0285] At concentrations of ≦5 μg / ml, the drug reduced the phagocytic activity of monocytes (FIG. 40). It should be noted that there was a significant variability in the indices studied, which was due to the 64 Zn e This may be due to incomplete solubility of -asp or insufficient functional maturity of monocytes. To confirm these hypotheses, parallel studies of the phagocytic function of cells and the expression of one or more than one endocytic receptor on the membrane of these cells are recommended.

[0286] Regardless of the concentration used, there was no significant change in spontaneous nitrite production by sensitized circulating monocytes after treatment with these drugs. However, the drugs modestly increased nitric oxide production after treatment with LPS (Figure 41). The mechanism of this action is mediated by the drug's effect on intracellular ROS production. The synthesis of reactive oxygen species is known to negatively affect the synthesis of reactive nitrogen species by mononuclear phagocytes, especially in functionally immature cells, including monocytes. Therefore, the slight increase in nitric oxide synthesis may be due to the drug's inhibitory effect on ROS production.

[0287] On monocyte arginase activity 64 Zn e There was no statistically significant effect of -asp, both after treatment of resting cells and after addition of the drug to a background of bacterial LPS cell treatment (Figure 42). The results of studies on monocyte arginase and NO synthase activity indicate that there is no significant effect of the drug on the arginine metabolic pathway in these cells. Microglial phagocytes differ from circulating monocytes in terms of maturity and functional plasticity. Resident microglial cells are functionally mature tissue phagocytes derived from the yolk sac, capable of self-renewal, and characterized in the literature as cells with a very conservative metabolism. Circulating monocytes are functionally immature cells (other than a subpopulation of monocytes patrolling along the endothelial wall, which are not separated by the methods used in this study).

[0288] Many metabolic responses (including signaling pathways) reside in these cells in an inactive and / or semi-active state, significantly increasing the metabolic plasticity of monocytes.

[0289] peripheral blood neutrophils

[0290] 64 Zn e Treatment of circulating unsensitized neutrophils with -asp resulted in a sharp decrease in intracellular ROS production by these cells (Figure 43). The modulatory effect had an inverse dose-dependence. Addition of the drug to cells against the background of their treatment with bacterial LPS also caused a sharp decrease in esterase-dependent oxidative metabolism in neutrophils. Neutrophils 64 Zn e It should be noted that neutrophils were the cells most sensitive to the antioxidant effects of -asp. Neutrophils differ from all previous phagocytes in their functional properties. Neutrophils are functionally mature cells with a highly reactive metabolism, and their short life span is important. These are exclusively circulating cells and are among the first of the main effectors of the inflammatory response (associated with both infectious processes and sterile inflammation). The metabolic plasticity of neutrophils is the least studied.

[0291] The drug also had a strong negative effect on NADH and NADPH oxidase-dependent ROS generation, independent of treatment of cells with bacterial LPS (FIG. 44).

[0292] However, there was no dose-response relationship in this case. The low variability of the studied parameters should also be noted.

[0293] No statistically significant effect of the drug on neutrophil phagocytosis was observed (FIG. 45).

[0294] It should be noted that the index of neutrophil phagocytic activity was low. A specific feature of rat blood is its low number of circulating neutrophils compared to other laboratory rodents. No literature data were found regarding the specific characteristics of the intensity of neutrophil endocytosis in these animals.

[0295] 64 Zne-asp had no significant effect on the synthesis of reactive nitrogen species by resting neutrophils and produced a moderate negative effect on this metabolic response after treatment of the cells with LPS (FIG. 46).

[0296] The arginase activity of neutrophils treated with both resting and bacterial LPS remained virtually unchanged under the action of the drugs (FIG. 47).

[0297] Figure 47 shows the effect of arginase on peripheral blood neutrophils from non-sensitized rats. 64 Zn e -shows the effect of asp.

[0298] All these data indicate that the drug has only a modest effect on the pathway of arginine metabolism in circulating neutrophils.

[0299] Findings from Example 3:

[0300] 1. Metabolic Profiles of Four Phagocyte Populations 64 Zn e Comparative analysis of the effects of -asp revealed that microglial cells were the most sensitive to the drug's action (treatment with the drug caused changes in almost all of the functions studied), while peritoneal macrophages were the least sensitive.

[0301] 2. Of all the metabolic reactions studied, the drug had the greatest effect on the oxidative metabolism of all cells in the population studied; it had no effect on arginase activity and virtually no effect on the endocytic activity of the cells.

[0302] 3. The drug inhibited induced oxidative metabolism in all phagocyte populations studied and inhibited the induced production of reactive nitrogen species in virtually all cells, demonstrating the anti-inflammatory properties of its immunomodulatory effect. The greatest antioxidant effect was recorded in neutrophils.

[0303] The response of microglial resident phagocytes to drugs is largely similar to that of peripherally localized (peritoneal macrophages) tissue phagocytes.

[0304] Example 4 On the spontaneous and phytohemagglutinin-induced proliferation of human peripheral blood mononuclear cells 64 Zn e -asp and E2+ 85 Rb e Effects of preparations.

[0305] Introduction: It is known from the relevant literature that mitogen-stimulated T lymphocytes activate the expression of transferrin receptors, which promotes zinc influx into the cells (Mathe G, et al., Med Oncol Tumor Pharmacother. 1985;2(3):203-10; F Flynn A. Nutrition Research. 1985;5(5):487-495.). The zinc-transferrin complex is a marker of zinc transport in activated T lymphocytes. It stimulates DNA synthesis in lymphocytes and has virtually no effect on this process in resting lymphocytes (Rosenkranz E, et al. Eur J Nutr. 2017 Mar;56(2):557-567. doi: 10.1007 / s00394-015-1100-1; Barnett JB, et al., Am J Clin Nutr. 2016 Mar;103(3):942-51. doi: 10.3945 / ajcn.115.115188). Published data show that rubidium salts (chloride) stimulate the process of differentiation of lymphoid and myeloid lineages of blood cells in the bone marrow and therefore have a negative effect on their proliferation (Hammarstroem L, Smith CI. Exp Cell Res. 1979 Mar 15;119(2):343-8;Petrini M, et al., Haematologica. 1990 Jan-Feb;75(1):27-31). The literature also includes the effect of leukemia on the proliferation activity of differentiated T lymphocytes. There are no data on the effects of bidium salts.

[0306] Materials and Methods: Peripheral blood mononuclear cells were isolated from healthy donors by density gradient centrifugation on Histopaque-1077 (Sigma, USA). Phytohemagglutinin (PHA) (Sigma, USA) was used to activate the proliferative activity of T lymphocytes. Cells were exposed to the action of both preparations for 48 hours. Stimulatory (drugs added as single components) and costimulatory (drugs added in combination with PHA) effects were evaluated. The CD3 phenotypic marker was used to selectively measure the proliferative activity of T lymphocytes. T cell proliferative activity was assessed by analyzing the DNA status of CD3+ cells using flow cytometry techniques. The results are presented in the form of a proliferation index calculated by the following formula:

[0307] PI=((S+G2M) / (G0G1+G2M))×100(Peng X, et al., Biol Trace Elem Res. 2011 Dec;144(1-3):688-94. doi: 10.1007 / s12011-011-9077-y).

[0308] result:

[0309] When used in vitro separately from PHA, 64 Zn e -asp had a weak stimulatory effect on CD3+ T lymphocytes in a dose-dependent manner, in contrast to zinc chloride, which according to published data has no effect on the proliferation of unstimulated T cells (Figure 48).

[0310] When used in combination with PHA, a classical mitogen for T lymphocytes, the preparation increased the cell proliferation activity compared to samples treated with the mitogen alone and to untreated samples. A dose-response relationship was not observed. The obtained data are consistent with published data on the costimulatory effect of zinc chloride on T cell proliferation.

[0311] E2+ 85 Rb edid not have any in vitro effect on the spontaneous proliferation activity of T lymphocytes, but significantly inhibited the mitogen-induced proliferation of the cells. Consequently, the effect of the drug differs from that of rubidium chloride, which, according to published data, has an inhibitory effect on the proliferation of both resting and activated lymphocytes.

[0312] T cell apoptosis (activation-induced apoptosis) (Brenner D, et al., Crit Rev Oncol Hematol. 2008 Apr;66(1):52-64. doi: 10.1016 / j.critrevonc.2008.01.002) is an additional criterion for T cell activation. In this context, apart from PHA, Activation-induced apoptosis of T lymphocytes treated with test drugs in combination with phenylalanine and PHA As can be seen from Figure 49, when used alone, 64 Zn e -asp had no effect on activation-induced T cell apoptosis. However, when this drug was used in combination with a mitogen (10 μg / ml), the level of activation-induced T cell apoptosis was significantly reduced, indicating its ability to inhibit apoptosis of actively proliferating cells. This effect should be taken into consideration when investigating the antitumor properties of the drug.

[0313] E2+ at a concentration of 500 μg / ml 85 Rb e The preparation had a significant proapoptotic effect both when used separately and in combination with mitogens. This suggests that the drug has anti-neoplastic properties, at least with respect to malignant T lymphocytes. Furthermore, a positive effect of the drug on inflammatory pathological processes accompanied by the activation of T cell proliferation (autoimmune diseases, allergic pathologies) is possible.

[0314] Findings from Example 4:

[0315] 1. 64 Zn e-asp has stimulatory and costimulatory effects on T lymphocyte proliferation, as well as inhibiting apoptosis of actively proliferating T cells.

[0316] 2.E2+ 85 Rb e has no effect on the spontaneous proliferative activity of T lymphocytes and inhibits mitogen-induced cell proliferation as well as stimulating apoptosis of actively proliferating T cells.

[0317] Overall conclusion

[0318] Thus, the metabolic profiles of differently localized phagocytes 64 Zn e -asp and E2+ 85 Rb e The results of in vitro studies on the effects of have demonstrated that microglial cells, circulating phagocytes (monocytes and neutrophils), and peritoneal macrophages are sensitive to both drugs. Microglial cells showed the greatest sensitivity to zinc-based preparations. Microglial phagocytic activity is enhanced under its action, a sign of its activation. 64 Zn e -asp increased ROS production by resting (unstimulated) microglial cells, indicating the drug's ability to activate the repair activity of brain phagocytes. At the same time, ROS production by activated microglial cells (a situation simulating an inflammatory process) decreased under the drug's action. The arginine metabolic pathway in microglial cells shifted toward increased arginase activity under the drug's action, again confirming the anti-inflammatory nature of its immunomodulatory effect (arginine metabolism via arginase leads to the formation of molecules that are precursors of extracellular matrix components required for repair processes, whereas arginine metabolism via NO synthase leads to the formation of NO, a reactive nitrogen species with cytotoxic functions). 64 Zn eThe nature of the metabolic changes in circulating phagocytes under the action of -asp differed from those in microglia: the drug caused a decrease in ROS production by both resting and stimulated monocytes and neutrophils, and had a weak effect on phagocytosis and arginine metabolism in these cells. 64 Zn e The response of peritoneal macrophages to -asp is similar to that of microglial cells, which is entirely logical given that both cell populations represent resident tissue-derived mature phagocytes of ontogeny origin, as opposed to circulating phagocytes derived from the bone marrow. 64 Zn e Under the action of -asp, ROS production was enhanced and simultaneously the synthesis of reactive nitrogen species was sharply reduced, indicating an anti-inflammatory (repair) shift in their metabolism.

[0319] Different localization of phagocytes and 64 Zn e -asp and E2+ 85 Rb e Combined treatment with the preparations caused slightly different changes in their metabolism: the phagocytic activity of microglia was reduced by such treatment, and ROS synthesis by both resting and stimulated cells was also reduced, along with a decrease in the production of reactive nitrogen species, but all these together indicated that the combination of the preparations containing trace elements Together, anti-inflammatory regulation of the function of these cells is demonstrated. 64 Zn e -asp and E2+ 85 Rb e The nature of the effect of combined treatment of circulating phagocytes with zinc-based drugs was essentially no different from that of the use of zinc-based drugs as monotherapy. The drugs reduced ROS production by both monocytes and neutrophils, and also reduced the production of reactive nitrogen species by circulating phagocytes, indicating their anti-inflammatory effect.

[0320] E2+ 85 Rb eTreatment of peritoneal macrophages with zinc-based drugs resulted in a more pronounced anti-inflammatory shift in metabolism in these cells than treatment of them with zinc-based drugs when used as the sole treatment.

[0321] These in vitro results suggest that preventive anti-inflammatory modulation of phagocyte function in different localizations prior to the onset of PD in in vivo studies would be advantageous. In our opinion, such advanced preparations of brain, peripheral blood, and peritoneal phagocytes could inhibit their inflammatory activation that accompanies the onset of the disease and attenuate / reduce the extent of dopaminergic neuronal destruction in animals within the substantia nigra.

[0322] This hypothesis was confirmed by the results of prophylactic drug administration before the induction of PD in rats. Both zinc-based drugs, used as monotherapy and in combination with a rubidium-containing preparation, caused a reduction in the degree of dopaminergic neuron destruction in animals during the development of PD. This reduction in the degree of dopaminergic neuron destruction was accompanied by positive changes in the localization of phagocytes in animals receiving the drug under investigation as prophylactic medication, in contrast to control PD animal models, which were characterized by proinflammatory activation of phagocytes in different localizations, which was largely abolished by prophylactic administration of a trace element-containing preparation. Microglia in control PD animal models differed from those in intact animals by reduced ROS production, while at the same time, the synthesis of reactive nitrogen species, which have a profound destructive effect on neurons in the substantia nigra, was enhanced. Prophylactic drug administration was accompanied by increased ROS production by microglial cells, concomitant with a reduction in the synthesis of reactive nitrogen species. As mentioned above, increased ROS synthesis is a necessary condition for the activation of repair processes. Thus, the enhanced oxidative metabolism in microglial cells combined with its anti-inflammatory polarization (increased scavenger receptor expression and reduced synthesis of reactive nitrogen forms) demonstrates the ability of trace element-based preparations to activate repair processes in the brain of animal models of PD subjected to their systemic intraperitoneal administration. Circulating phagocytes in rat models of parkinsonism were characterized by a pronounced proinflammatory polarization, confirming literature data regarding the involvement of these cells in the systemic inflammation that accompanies the development of PD. Prophylactic administration of trace element-based preparations prevented the inflammatory activation of these cells. The same is true for the metabolic changes in peritoneal macrophages in rat models of PD after prophylactic administration of trace element-based preparations.

[0323] The preventive effect of zinc-containing drugs was more pronounced but less stable over time, whereas the effect of combined use of zinc and rubidium-based drugs was less pronounced but more stable over time.

[0324] Example 5 Reduced Toxicity of the Disclosed Compounds

[0325] E2+ on the metabolic activity of normal human fibroblasts 85 Rb e In vitro evaluation of the effect (MTT assay).

[0326] E2 series E2+Rb reference drug E2+ 85 Rb e Concentration of the compound

[0327] Cellular metabolic activity, % *

[0328] 150μg / ml 99.3±3.2 72.8±2.1

[0329] 75μg / ml 97.7±7.5 67.4±4.7

[0330] 38μg / ml 98.8±7.1 68.5±1.8

[0331] 20μg / ml 96.8±9.8 74±1.3

[0332] 10μg / ml 104.6±14.2 82.5±1.3

[0333] 5μg / ml 103.4±9.3 82.3±3

[0334] 2.5μg / ml 128.2±8.3 84.4±4.6

[0335] 1.25μg / ml - 105.1±2.3

[0336] * Control group - compared to 100% live cells

[0337] Cell proliferation and metabolic activity were determined by colorimetric methods.

[0338] The metabolic activity of the cells (ie, the number of viable cells) was assessed by MTT staining.

[0339] Twenty-four hours after the last reagent, cells were seeded into wells of a 96-well plate at a concentration of 1 x 10 cells / well in complete DMEM nutrient medium containing 10% FBS and 40 μg / ml gentamicin. The cells were cultured for 24 hours in a humidified atmosphere at 5% CO2 and 37°C. After 24 hours, various doses of experimental substances were added to each well. The cells were incubated for an additional 72 hours at 37°C and 5% CO2.

[0340] After completion of the incubation with the preparations, the proliferation and metabolic activity of the experimental cells was evaluated by colorimetric method by staining the MTT cells:

[0341] 10 μl of MTT solution (5 mg / ml dye in phosphate-buffered saline) was added to each well of the plate; the plate was incubated for 3 hours at 37° C. in a CO incubator. Afterwards, the medium was removed from the wells, and the formed tetraformazan crystals were dissolved in 100 μl of dimethyl sulfoxide.

[0342] When MTT was used, the results were evaluated using a multiwell spectrophotometer at an excitation wavelength of 540 nm. The percentage of viable cells was calculated by the formula:

[0343] IR = (A 540 (experimental) / A 540 (control)) × 100%.

[0344] Example 6

[0345] Compound 7 was prepared as follows: To obtain the desired product, 85 Rb e The same procedure can be used to prepare other compounds of Formula 1 by using Cl as needed, along with starting materials and intermediates containing appropriate substituents. An alternative synthesis is provided below for when R is OCH.

[0346] Phase 1

[0347] 10.8 g of o-phenylenediamine base was dissolved in 100 ml of isopropyl alcohol, and while maintaining the mixture at room temperature, 200 ml of an alcoholic solution of benzenesulfonyl chloride (17.65 g) was added. The resulting mixture was stirred at room temperature for 1 hour and at 65-70 °C for 0.5 hour. After cooling, the resulting light-colored precipitate was filtered by thoroughly washing with cold water to remove any unreacted o-phenylenediamine residues. The precipitate from the filter was boiled in 10% hydrochloric acid and filtered while hot to remove the by-product bis-dibenzenesulfonyl-o-phenylenediamine. The filtrate was clarified with activated carbon, and after cooling, N-benzenesulfonyl-o-phenylenediamine hydrochloride was filtered off in the form of fine needle-like crystals (weight 7.11 g per dry substance, 25% yield). [ka]

[0348] Phase 2

[0349] The N-benzenesulfonyl-o-phenylenediamine hydrochloride (7.11 g) obtained in Phase 1 was suspended in 50 ml of toluene, and 5.1 g of o-nitrobenzoyl chloride and 5.32 g (7.1 ml) of triethylamine were added. The reaction mixture was boiled under reflux in an oil bath for 3 hours, and after cooling, the precipitate formed was filtered off. The resulting material was recrystallized from isopropyl alcohol and purified with activated carbon. The yield of dry N-benzenesulfonyl-N'-2-nitrobenzoyl-o-phenylenediamine was 6.45 g (65%). [ka]

[0350] Phase 3

[0351] 85 Rb ePreparation of an aqueous solution of the rubidium salt of N-benzenesulfonyl-N'-2-nitrobenzoyl-o-phenylenediamine at a concentration of 0.001 g / ml: 0.073 g of KOH and 0.467 g of N-benzenesulfonyl-N'-2-nitrobenzoyl-o-phenylenediamine were dissolved in 90 ml of deionized water. The solution was heated and stirred to completely dissolve the precipitate. After cooling to room temperature, the solution was filtered and 0.1417 g of 85 Rb e Cl( 85 Rb e is 99% 85 Rb) was added and the mixture was quickly The reaction mixture was stirred at room temperature for 30 minutes and filtered again. The filtrate was made up to 100 ml with deionized water and used for further studies. [ka]

[0352] The following methods, among others, can be used to identify newly synthesized compounds: measurement of PMR spectra on a Varian VXR 200 spectrometer for TMS in DMSO-d; IR spectra (4000–600 cm) on a Bruker ALPHA FT-IR spectrometer using an ATR accessory. -1 ) measurement; silica gel 60 F 254 TLC on plates (eluent: chloroform); mass spectra determination using a Kratos MS 890 mass spectrometer with direct injection of the sample into the ion source at an ionization chamber temperature of 180-250 °C and an ionization electron energy of 70 eV.

[0353] When R9 is OCH3, the following alternative synthesis may be used: In the last step, 85 Rb e Cl is 85 It can be used in place of RbCl to prepare Rb-enriched products. [ka]

[0354] Example 7 On local and systemic immune reactivity, behavior, and motor function in rats with LPS-induced experimental parkinsonism 64 Effect of Zn-asp

[0355] Zinc is recognized as an essential trace metal required for human health, and its deficiency is strongly linked to neuronal and immune system defects. Zn also acts as a potent modulator of the immune response.

[0356] Bacterial endotoxin (lipopolysaccharide, LPS) is a TLR4 agonist and a potent pro-inflammatory activator of cells of the innate immune system, including microglia, the brain's resident mononuclear phagocytes. Subramanian Vignesh K, Deepe GS Jr. Arch Biochem Biophys. 2016;611:66-78. doi: 10.1016 / j.abb.2016.02.020;Liu M, Bing G. Parkinsons Dis. 2011;2011:327089. doi: 10.4061 / 2011 / 327089.

[0357] TLR4 is expressed by all cells of the innate and adaptive immune system, as well as adipocytes, epithelial cells, and many other cells. Interaction of TLR4 with LPS activates the NFκB-dependent signaling pathway, resulting in, among other things, the synthesis of proinflammatory mediators, including cytokines, chemokines, and eicosanoids. Hersoug LG, et al. Obes Rev. 2016;17(4):297-312. doi: 10.1111 / obr.12370; Vergadi E, et al. Front Immunol. 2018;9:2705. doi: 10.3389 / fimmu.2018.02705. Increased numbers of circulating LPS molecules are one of the most important factors in activating neurodegenerative processes and are considered an essential component of the pathogenesis of Parkinson's disease (PD), Alzheimer's disease (AD), and other conditions. Sfera A, et al., Front Neurol. 2018;9:1062. doi: 10.3389 / fneur.2018.01062;Perez-Pardo P, et al., Gut. 2018. pii: gutjnl -2018-316844. doi: 10.1136 / gutjnl-2018-316844.

[0358] Based on the above, the LPS model of PD is of particular interest in the study of α-synucleinopathy, as it allows the assessment of the inflammatory component in the pathogenesis of the disease and, with it, provides the most appropriate assessment of the therapeutic efficacy of anti-inflammatory drugs for the treatment of this pathological condition.

[0359] The conditions for the generation of an LPS-induced model of experimental parkinsonism were optimized: among two stereotaxic coordinates for endotoxin injection described in the literature, those ensuring maximal neuroinflammation and subsequent high damage to the neurons of the substantia nigra were selected.

[0360] material and method

[0361] experimental animals

[0362] Male Wistar rats (220-250 g) were used. Animals were maintained in an animal facility under standard conditions with unlimited access to food and water.

[0363] Modeling hemiparkinsonism in rats

[0364] Chronic dopamine deficiency in the left hemisphere was modeled by unilateral destruction of dopaminergic neurons in the substantia nigra pars compacta of the brain. This was induced by stereotaxic microinjection of 10 μg of endotoxin (lipopolysaccharide) (Escherichia coli O111:B4, cat. L2630, Sigma) dissolved in 2 μl of sterile 0.9% NaCl (Infuzia CJSC, Ukraine). The solution was prepared on the day of surgery. The flask containing the resulting solution was sealed with a drop of silicone oil (STEP-ELECTRONICA LLC, Ukraine) and placed in a refrigerator.

[0365] Animals were anesthetized using a mixture of ketamine (75 mg / kg, diluted with sterile water for injection, Sigma, USA) and 2% xylazine (100 μl / rat, Alfasan International BV, Netherlands) administered intraperitoneally in a total volume of 1 ml. The animals were then placed in a modified rat stereotaxic apparatus (SEZH-4). The animals were then scalped, and a trephine opening was made directly into the substantia nigra with an injection needle (AP = -5.3; ML = ±2.0; DV = -7.2). Dissolved LPS was collected in a homemade microinjector, and its tip was dropped into the trephine opening.

[0366] Endotoxin was injected into the brain tissue at a rate of 1 μl / min (every 15 seconds) at a dose of 10 μg in a volume of 2 μl. After endotoxin administration, the tip of the microinjector was left in the brain tissue for 5 minutes. The microinjector was then removed, and several stitches were placed in the scalp soft tissue of the animals. Control animals received 2 μl of sterile 0.9% NaCl instead of LPS (sham-operated animals).

[0367] Apomorphine test

[0368] The percentage of destroyed dopaminergic neurons was calculated using the apomorphine test. The intensity of the animals' rotational movement toward the opposite hemisphere to the one into which endotoxin was injected was assessed. Similar motor activity was induced by systemic intraperitoneal administration of apomorphine, a dopamine receptor agonist (0.5 mg / kg, Sigma, USA). The intensity of such rotational movement over a 30-minute period indicated the degree of degradation of the nigrostriatal dopamine system (SA Ta Ilanov et al., Neurophysiology 38 (2), 128-133). Apomorphine Test I and I Between I and I, the number of animals whose rotation rate increased, decreased, or remained unchanged within 30 minutes was quantified.

[0369] Immunohistochemical identification of dopamine neurons

[0370] LPS-induced degeneration was assessed using immunohistochemical tyrosine hydroxylase (TH) antibody staining [Walsh, S., Finn, DP, Dowd, E., 2011. Neuroscience 175, Immunohistochemical staining of 5 μm paraffin-embedded midbrain sections was performed at 1:2 Immunofluorescence was performed using primary TH antibody at a dilution of 0.00 (Millipore, AB152). Endogenous peroxidase activity was quenched using endogenous peroxidase blocking solution (Dako, EnVision Flex, DM821). Nonspecific antibody binding was blocked with a 4% solution of dried milk in Tris-buffered saline (TBS) containing 0.2% Triton X-100.

[0371] The primary antibody was diluted in TBS containing 0.2% Triton X-100, applied to the sections, and incubated overnight (+4°C). The secondary antibody (biotinylated anti-rabbit, 1:200) was incubated for 60 minutes. The immunoreaction was developed by applying diaminobenzidine (Dako, EnVision) for 5 minutes. Evaluation of TH staining was performed at low light level using a Primo Star microscope, Zeizz. The intensity of the peroxidase mark (within the area of ​​maximum expression) was evaluated. Receptor expression was assessed using a scoring system. Staining intensity was scored from 0 to 3: 0 - no staining; 1 - weak staining; 2 - moderate staining; 3-Strong staining intensity It was.

[0372] Measurement of fecal moisture content

[0373] Feces were collected on days 8, 21, and 28 of the experiment. They were weighed (wet weight m ww ), dried in a thermostat at t = 60 °C for 24 h, and then weighed again (dry weight m dw ). The water content (M) of feces can be calculated using the following formula: M=100-(m dw ×100% / m ww ) and calculated as the percentage difference between the wet and dry weight of feces.

[0374] Behavioral testing

[0375] Open field test

[0376] Methods. The open field test is a commonly used technique to measure the locomotor activity of rodents and their exploratory activity in an unfamiliar environment, as well as to assess the level of neophobia, anxiety, or increased orientation response in rodents (Denenberg VH, et al., Physiology and Behavior, 1969;4:403-406.; Pellow S et al., J Neurosci Methods. 1985;14(3):149-67. The behavioral patterns measured in the open field test were similar between the outer and inner periphery. The statistics include distance traveled, duration spent on the outer and inner perimeters, total distance traveled, and the percentage of the number of inner quadrangles traversed relative to the total number of quadrangles.

[0377] The open field was a square chamber measuring 100 × 100 cm with walls 60 cm high. The chamber was enclosed in two 60 watt batteries each suspended at a height of 2 m. The open field was illuminated with 1000 LED bulbs. An IP camera was used to record the rodent's movements. Parameters were analyzed using MATLAB®-based software. Statistical analysis was performed using GraphPad Prism. Significance was set at <0.005. Data collected from the open field were analyzed using one-way ANOVA (Tukey's test or Dunnett's test) and unpaired t-test or Mann-Whitney.

[0378] Data Analysis and Interpretation. A novel environment that an animal finds itself in provokes a conflict between fear and the desire to explore it. These two behavioral tendencies are characterized by different temporal variations and different spatial advantages. For the analysis, the arena was divided into two zones: inner and outer. Total locomotor activity and time spent in the inner and outer zones are the most significant parameters for assessing the rat's level of anxiety. During a 5-minute test, an animal is considered normal if it spends most of its time near the walls and on the periphery, rarely entering the inner "open" zone. Increased entry into the inner zone is interpreted as a decrease in the animal's anxiety. Animal emotional and exploratory behavior are affected by various disorders in brain regions such as the hippocampus and amygdala. Furthermore, various substances with anxiolytic, stimulating, and muscle-relaxing effects can affect the outcome of the experiment. Figures 50A and 50B show an example of the analysis of a rat's trajectory in the experimental arena.

[0379] elevated cross maze

[0380] Methods. The elevated plus maze is a widely used behavioral assay in rodents that allows for the assessment of sedative or anxiogenic effects of drugs, the definition of mechanisms underlying anxiety-related behavior, and the assessment of rats' locomotor activity and their exploratory activity levels, which can be correlated with data collected in the open field test. (Campos AC, et al., Braz J Psychiatry. 2013;35 Suppl 2:S101-11. doi: 10.1590 / 1516-4446-2013-1139; Braun AA, et al., Neurobiol Learn Mem. 2012;97(4):402-8. doi: 10.1016 / j.nlm.2012.03.004). Behavior was assessed according to the following main parameters: total distance traveled, number of entries into each arm, time spent in each arm, total number of entries, and percentage of entries. Each animal was placed at the junction of the four arms of the maze, and the above parameters were observed for 5 minutes. Throughout the experiment, the animals had free access to food and water. Before the start of the measurements, the animals were given time to adapt to the new environment to reduce stress.

[0381] The apparatus used in the elevated plus maze had a cross-shaped configuration, including two open arms (50 × 10 cm) that crossed each other and intersected perpendicularly to two closed arms (50 × 10 × 30 cm). The entire apparatus was elevated 50 cm above the floor. At the center of the junction was a central area of ​​10 square cm, where the animal began facing the open arms. The apparatus was illuminated by two 60-watt LED bulbs suspended at a height of 2 m. The rat's location was recorded using an IP camera, and scoring was later completed using MATLAB®-based software. Statistical analysis was performed using GraphPad Prism. Significance was set at <0.005. Data collected from the open field were analyzed using one-way ANOVA (Tukey's test or Dunnett's test) and unpaired t-tests or Mann-Whitney.

[0382] Data Analysis and Interpretation. The test is based on the rat's natural aversion to open areas and its preference for the closed area of ​​the apparatus. The animal's anxiety level is assessed as the total time spent in the open and closed arms. Animals that spend more time in the open arms have lower levels of anxiety compared to animals that prefer the closed arms. Furthermore, as in the open field test, the novel environment that the animal finds itself in creates a conflict between fear and the desire to explore this environment. Therefore, the data collected from these two tests The collected data can be correlated with the distance traveled and the time spent in the outer and inner perimeters. During a 5-minute test, an animal is considered normal if it spends most of its time in the closed arms but also makes a few exploration entries into the open arms. Therefore, an increase in entries into the open arms is interpreted as a decrease in the animal's anxiety. Figures 51A and 51B show an example of an analysis of a rat's trajectory in the experimental arena.

[0383] Pickup Test

[0384] The pick-up test is a test for increasing irritability. The animal is picked up by grabbing its body. Responses are graded as follows: 1, very easy; 2, easy with vocalization; 3, somewhat difficult, the rat rears up on its hind legs and faces the hand; 4, the rat remains motionless (with or without vocalization); 5, difficult, the rat moves far away and avoids the hand; and 6, very difficult, the rat behaves defensively and may attack the hand (Brandt C et al., Neuropharmacology. 2006 Sep;51(4):789-804).

[0385] Experimental scheme

[0386] The animals were assigned to seven groups: I (n = 12) - intact animals maintained under standard animal facility conditions and not subjected to any manipulation; II (n = 12) - rats administered 0.1 ml of ultra-light water intravenously (iv) daily for 10 days after surgery, rats were sham-operated; III (n = 12) - rats after surgery. 64 Rats were sham-operated after receiving Zn-asp (1.5 mg / kg, iv, 10 days) solution daily; IV (n=15) - rats were sham-operated after surgery after receiving 0.1 ml of ultra-light water (iv, 10 days) daily, which resulted in the generation of LPS-induced Parkinson's disease model; V (n=15) - after surgery, 64 Rats were given Zn-asp (1.5 mg / kg, iv, for 10 days) solution daily, and then an LPS-induced Parkinson's disease model was generated; VI (n=15). - After surgery, 64 Zn-asp+10%E2+ 85 Rats were administered Rb (1.5 mg / kg, iv, 10 days) solution daily, and rats were sham-operated. VII (n = 15) - After surgery, 64 Zn-asp+10%E2+ 85 Rats were administered Rb (1.5 mg / kg, iv, for 10 days) solution daily, and then an LPS-induced Parkinson's disease model was generated (Figure 52).

[0387] 1. On day 1 of the experiment, rats were operated on to model hemiparkinsonism or sham-operated. From day 9 to day 18 of the experiment, animals were injected with either ultra-light water or test substance for 10 days according to their group assignment. On day 8 of the experiment (before the start of treatment), - Apomorphine Test I) and on the 21st day of the experiment (after the administration of the test substance was completed) (Apomorphine Test II) Animals were subjected to the apomorphine test to analyze the onset of hemiparkinsonism, which correlates with the number of destroyed dopaminergic neurons. Furthermore, follow-up analyses of fecal water content were performed on postoperative day 8 before treatment began, as well as on days 21 (after treatment ended) and 28 (before autopsy). On day 24 of the experiment, behavioral tests were performed in all groups. All animals were weighed before autopsy. During autopsy, two-thirds of the animals from each group were sacrificed by cervical dislocation, and brain and blood samples were collected for further biochemical and immunological studies. One-third of the animals were used for further immunohistochemical studies of the brain; for this purpose, a rapid, controlled, and uniform fixation procedure of the animals' whole bodies under anesthesia was performed using 4% paraformaldehyde perfused through the rat heart to obtain the best possible preservation of brain structure for immunohistochemistry. Gage GJ, et al., J Vis Exp. 2012 Jul 30;(65) .

[0388] Hematological evaluation

[0389] Blood counts were analyzed at the end of the experiment (day 28). Absolute white blood cell counts, as well as absolute and relative numbers of lymphocytes, monocytes, and neutrophil granulocytes were calculated.

[0390] Isolation of phagocytes of various localizations

[0391] To evaluate the anti-inflammatory and immunomodulatory effects of test substances, microglia and peritoneal macrophages were isolated. The phenotypic and functional characteristics of peripheral blood phagocytes were evaluated without fractionation (using whole blood). To isolate microglia, brain tissue homogenates were obtained by manual homogenization, followed by removal of undissociated clumps using a cell strainer (No. 70). Microglial cells were isolated from the resulting homogenates by centrifugation through a two-step Percoll gradient. Peritoneal macrophages were isolated without prior sensitization by perfusion of ice-cold growth medium into the peritoneal cavity of rats. The adherent fraction of peritoneal exudate cells was used in the experiments.

[0392] Assessment of phagocytic activity of phagocytes of various localizations

[0393] Phagocytic activity of microglia, peritoneal macrophages, and peripheral blood phagocytes was analyzed by flow cytometry using FITC-labeled S. aureus Wood 46 cells as phagocytic targets. S. aureus cells were obtained from the Microbiology Collection of the Department of Microbiology and Immunology, ERC Institute of Biology and Medicine, Taras Shevchenko National University.

[0394] Oxidative metabolism of phagocytes at various locations

[0395] The oxidative metabolism of phagocytes of various localizations was analyzed by flow cytometry using cell-permeable 2′7′-dichlorodihydrofluorescein-diacetate (DHP) (carboxy-H2DCFDA, Invitrogen, USA), which is converted by intracellular esterases to the non-fluorescent membrane-impermeable carboxy-H2DCF form.

[0396] Assessment of the phenotypic profiles of phagocytes of different localizations

[0397] The phenotypic profile of phagocytes of different localizations was characterized by the expression of markers of functional maturity and metabolic polarization (CD206, CD80, and CD86), which was determined by flow cytometry and by the use of monoclonal antibodies of appropriate specificity marked with fluorescent dyes (Abcam, Becton Dickinson).

[0398] Statistical Data Analysis Methods

[0399] Numerical results other than those collected from behavioral tests were processed using statistical data analysis methods using the Statistica 8.0 software package. Student's t-test was used to determine the statistical significance of reliable differences between the results shown by each group. Significance was set at p<0.05.

[0400] Statistical data processing of behavioral tests was performed using the GraphPad Prism 7 statistical software package. Nonparametric tests were used for statistical data processing. The median value of each group is indicated by a horizontal line. Significant differences between groups were determined using the Kruskel-Wallis one-factor test and post-hoc Dunn's test. Significance was set at p<0.05.

[0401] result

[0402] On apomorphine-induced rotational behavior in a rat model of Parkinson's disease 64 Therapeutic effect of Zn-asp

[0403] In a preliminary study, we observed that the LPS animal model of Parkinson's disease had significantly less pronounced rotational behavior than the 6OHDA-induced PD model, and this fact was confirmed in this study with a larger number of animals (groups IV, V, and VII). In this example, 38 rats (86%) of 44 LPS models of Parkinson's disease (one rat did not recover from anesthesia) rotated less than 100 times per 30 minutes.

[0404] A physiological decrease in the number of rotations between the first and second apomorphine trials was observed in 33.3% (5 of 15 animals) of rats in the control group with LPS-induced parkinsonism and injected with HO (Group IV), whereas an increase in the number of rotations was observed in 66.6% (10 of 15 animals) of rats in this group, although the mean number of rotations did not differ significantly between the first (35.3 ± 25.5 rotations / 30 min) and second (43.4 ± 36.1 rotations / 30 min) apomorphine trials (Figures 53A and 53B).

[0405] 64 Effect of Zn-asp

[0406] 64 Considering the fact that Zn-asp administration began the day after the first apomorphine test and that the second apomorphine test was performed 3 days after the last injection of the test substance, the number of animals that produced fewer rotations in the second apomorphine test than in the first apomorphine test is a relevant marker of the efficacy of the test substance in preventing the destruction of dopaminergic neurons. There was no significant difference in the mean rotations between the first (91.2±107.7 rotations / 30 min) and second apomorphine tests (86.7±106.3 rotations / min) (Figure 53A). However, in contrast to rats injected with ultra-light water, 64 85.7% of rats (12 of 14 animals) treated with Zn-asp produced fewer rotations between the first and second apomorphine trials, and 14.3% of rats (2 of 14 animals) produced an increased rotation (Fig. 53B), which may be a role for Zn-asp in preventing the intensification of the degenerative process of dopaminergic neurons in the substantia nigra of the left hemisphere. 64 This shows the significant efficacy of Zn-asp.

[0407] on the number of TH-positive neurons in the midbrain in a rat model of Parkinson's disease 64 Therapeutic effect of Zn-asp

[0408] To further confirm the positive therapeutic effect of the test substance in preventing the degeneration of dopamine neurons, we performed an immunohistochemical study of the number of TH-positive neurons in the midbrain. Tyrosine hydroxylase (TH) is an enzyme that limits the synthesis of dopamine and is a classic marker of dopamine neurons. The study results showed that the color of the substantia nigra in animals from the intact group (Group I) and the sham-operated group (Group II) was significant and corresponded to a score of 3. At the same time, administration of LPS to generate the PD model resulted in a significant decrease in the number of TH-positive neurons in the midbrain (score 1) compared with the intact group and the sham-operated group (Figures 54A-54O, Table 2).

[0409] to sham-operated animals 64 Administration of Zn-asp did not cause any changes in the intensity of TH-positive staining compared to intact animals and sham-operated animals injected with water. 64 The LPS rat model treated with Zn-asp (group VI) showed an increased number of TH-positive neurons in the midbrain compared with the LPS animal model administered with water (group V) ( Figure 5 These data correlate with the apomorphine study data (Figs. 53A and 53B) and demonstrate the role of apomorphine in preventing the severe degeneration of dopamine neurons in the substantia nigra of the left hemisphere. 64 The remarkable effectiveness of Zn-asp is confirmed.

[0410] Effect of LPS on body weight changes in the LPS rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0411] The body weight of animals is a classic clinical index that allows the assessment of their physiological state. Extensive research has reported that weight loss can precede the diagnosis of Parkinson's disease by years and is also associated with the severity and duration of the disease

[26] . Furthermore, PD-related motor and non-motor symptoms can affect the patient's body mass index. Weight loss is not an independent pathological factor, but is associated with the pathogenesis of Parkinson's disease and Alzheimer's disease [Joly-Amado A et al., (2016). Neurobiol. Aging 44, 62-73]. Alzheimer's disease In Parkinson's disease, weight loss is one of the criteria for the clinical diagnosis of dementia. Similarly, in the context of Parkinson's disease, weight loss may precede motor symptoms and be considered an indicator of the onset of the disease. Kai Ma, et al., Current Knowledge and Future Prospects / / Front Aging Neurosci. 2018; 10: 1.

[0412] In these experiments, intact animals gained approximately 39% body mass between days 1 and 28 of the experiment (within 4 weeks). Sham-operated rats also gained an average of 30% weight.

[0413] However, it should be noted that the LPS rat model of Parkinsonism only gained 2% weight between day 1 (day of surgery) and day 28 (day of necropsy) of the experiment, indicating a relationship between the weight of the animals to assess their overall clinical condition and the effect of the test substance on their condition in Parkinson's disease (Figure 57).

[0414] to the LPS rat model of Parkinson's disease 64 Administration of Zn-asp caused a significant increase in their body weight (the difference in values ​​in this group on days 1 and 28 of the experiment (Figure 56A) and in relation to the value in the group of LPS rat models given ultra-light water on day 28 of the experiment (Figure 56B)).

[0415] [Table 2]

[0416] Effect of LPS on fecal water content in the LPS rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0417] Despite the main clinical signs of Parkinson's disease, such as tremor, motor weakness, muscle rigidity, and postural instability, it is the early stages of Parkinson's disease that clearly affect patients' quality of life. Karaban IN Non-motor symptoms in the clinical picture of Parkinson's disease / IN Karaban., OV Shalenko, SA Krizhanovsky / / International Neurological Journal. - 2017. - No.1(87). - P. - 58-63. The main discomforts among non-motor symptoms include gastric motility disorders, constipation, and anorectal insufficiency. Delayed gastric emptying, which progresses to gastroparesis, is reported in up to 100% of PD patients and occurs at all stages of the disease with serious consequences for the patient's quality of life [Gastric motor dysfunctions in Parkinson's disease: Current pre-clinical evidence / C. Pellegrini [et al.]. / / Parkinsonism Relat Disord.- 2015. - Vol. 21(12). - P.1407-14]. The water content of feces is It depends on digestive function, not on food intake, and this accurately reflects it. Intestinal peristalsis and water absorption are related, and water absorption is further related to intestinal blood circulation. In all cases, fecal hyperhydration is one of the first signs of colonic mucosal irritation. Jensen R, et al. Clin Chem. 1976 ;22(8):135.

[0418] In this study, increased fecal water content was recorded in rats in the sham-operated (SO) group compared to intact animals. These deficits were stable over time and remained significant even 28 days after surgery (Figure 57). In the LPS rat model of Parkinsonism, significantly lower fecal water content was observed 8 days after surgery compared to sham-operated animals. However, it should be noted that by the 28th day of the experiment, a trend toward increased fecal water content was observed in the LPS rat model (Figure 57). This fact confirms the relevance of this model to the pathological picture, i.e., GI dysfunction in Parkinson's disease.

[0419] In this study, animals were injected with LPS to induce parkinsonism, followed by a 10-day follow-up period. 64 Administration of Zn-asp (FIG. 58) did not result in any significant change in the water content of the feces.

[0420] on behavioral responses in the LPS rat model of Parkinson's disease in the open field test 64 Effect of Zn-asp

[0421] on CNS function 64 To evaluate the effects of Zn-asp, a commonly accepted open-field test was used. Observation of the rats' behavioral responses in this test allows us to determine the extent of the test substance's influence on the animals' locomotor and exploratory activity, orientation response, and emotional reactivity. The test results are shown in Table 3 and Figures 59A-59E.

[0422] [Table 3]

[0423] The results of the open field test showed that none of the behavioral parameters of animals from the SO group (group II) were significantly different from those of animals from intact controls (group I).

[0424] Significant groups, namely group II (SO) and group III (SO+ 64The Kruskal-Wallis test used to make comparisons between the IV (Zn-asp) and IV (LPS) groups revealed the following: - a slight decrease in the total distance covered by the animals (P = 0.0505); - Increased time spent in the outer periphery and decreased time spent in the inner periphery (P=0.0020), significantly different between groups II (SO) and III (SO+ 64 Zn-asp) (Dunn's multiple comparison test P = 0.0193); - Reduction in total rearing times by group (P=0.0160), and Group II (SO) and Group III (SO+ 64 There is a significant difference between the two (Zn-asp) (Dunn's multiple comparison test P=0.0053).

[0425] Furthermore, the effects of the open field test on behavioral parameters 64 To analyze the effect of Zn-asp, significant groups, i.e., group IV (LPS) and group V (LPS + 64 Comparisons were made between the Zn-asp and Zn-asp groups and showed that: test substance administration affected the total distance traveled (Kruskal-Wallis test - P value = 0.7031), the time spent in the outer (left chart) and inner (right chart) perimeters (Kruskal-Wallis test - P value = 0.2319), the total number of rearing episodes (Kruskal-Wallis test - P value = 0.7001); the number of grooming episodes (Kruskal-Wallis test - P value = 0.3022).

[0426] 64 It should be noted that the frequency of defecation in the LPS rat model of PD treated with Zn-asp was increased compared with the LPS rat model of PD injected with ultra-light water (Dunn's multiple comparison P = 0.0030).

[0427] on behavioral responses in the LPS rat model of Parkinson's disease in the elevated plus maze 64 Effect of Zn-asp

[0428] Using the Elevated Plus Maze to Evaluate CNS Function in Laboratory Animals 64 The effect of Zn-asp was analyzed. This test allowed us to evaluate the degree of effect of the test substance on the anxiety level of rats by observing the behavioral response. The test results are presented in Table 4 and Figures 60A to 60D.

[0429] [Table 4]

[0430] The elevated plus maze results showed that none of the behavioral parameters of animals from the SO group (Group II) were significantly different from those of animals from intact controls (Group I).

[0431] Significant groups, namely group II (SO) and group III (SO+ 64 Kruskal-Wallis and ANOVA tests used to compare between Group IV (Zn-asp) and Group IV (LPS) revealed no significant changes in any of the parameters analyzed. However, quantitative data (Table 4) show that the total distance traveled, time spent in the open arms, and number of entries in Group IV (LPS) were reduced by 1.3-, 1.4-, and 1.6-fold, respectively, when compared with Group II (SO).

[0432] on behavioral parameters of rats in the elevated plus maze 64 To further analyze the effect of Zn-asp, significant groups, i.e., group IV (LPS) and group V (LPS + 64 Comparisons were made between the Zn-asp and the test substance, revealing that the administration of the test substance did not have any effect on the behavioral parameters of the experimental animals. 64 It is shown that Zn-asp increased the total distance traveled and the number of entries in group V by 1.2 and 1.3 times, respectively, when compared with group IV (LPS).

[0433] Behavioral responses of the LPS rat model of Parkinson's disease in the pick-up test64 Effect of Zn-asp

[0434] The pick-up test is a test for increased irritability. The animal was picked up by grasping it around its body. The response was scored from 1 to 6. The test results are presented in Figure 61.

[0435] The results of the pick-up test showed that none of the behavioral parameters of animals from the SO group (group II) were significantly different from those of animals from intact controls (group I).

[0436] Significant groups, namely group II (SO) and group III (SO+ 64 Kruskal-Wallis test and ANOVA test used to compare between IV (Zn-asp) and IV (LPS) groups revealed no significant changes in the behavioral responses of the animals.

[0437] for behavioral parameters observed in the pick-up test 64 To further analyze the effect of Zn-asp, comparison was made between the significant groups, i.e., group IV (LPS) and group V (LPS + 64 Zn-asp) and compared with IV group (LPS), V group (LPS + 64 A statistically significant increase in stimulation with Zn-asp was revealed (Dunn's multiple comparison test P=0.0136).

[0438] Effect of LPS on blood cell counts in the LPS rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0439] Parkinson's disease is now considered a disease involving systemic meta-inflammation. Causes of systemic inflammation include disruption of the blood-brain barrier through the secretion of proinflammatory brain mediators into the periphery, transport of inflammatory mediators via the glymphatic system, and peripheral disorders accompanied by neurodegeneration, including gastrointestinal dysfunction (e.g., peptic ulcers, impaired gut motility) and dysbiosis. Grozdanov V, et al. Acta Neuropathol. 2014 Nov;128(5):651-63. doi: 10.1007 / s00401-014-1345-4; Akil E, et al., Neurol Sci. 2015 Mar;36(3):423-8. doi: 10.1007 / s10072-014-1976-1. It should be noted that the analysis of blood cell counts was carried out after the end of the experiment, i.e., 10 days after the end of treatment with the test substances.

[0440] Analysis of blood samples from the LPS animal model of Parkinson's disease showed no statistically significant changes in the absolute numbers of circulating leukocytes in these rats compared to intact and sham-operated animals (Figure 62).

[0441] Therapeutic administration of the test substances did not affect this hemogram parameter. Analysis of the absolute number of leukocytes in the main populations showed a slightly different picture (Fig. 63). Sham-operation was accompanied by a mild monocytosis. Administration of the zinc preparation eliminated this inflammatory phenomenon.

[0442] Induction of parkinsonism was accompanied by neutrophilia, an increased neutrophil-to-lymphocyte ratio, a typical feature of blood counts resulting from idiopathic parkinsonism, an early marker of disease progression, and an important prognostic criterion for its severity. Wijeyekoon RS, et al., Front Neurol. 2018;9:870. doi: 10.3389 / fneur.2018.00870. Therapeutic administration of zinc preparations was associated with a normalization of the neutrophil-to-lymphocyte ratio, primarily due to increased lymphocyte levels, a criterion for resolution of inflammation involving regulatory cells.

[0443] A somewhat different picture was obtained in the analysis of the relative numbers of leukocytes in the various populations (Figure 66).

[0444] Sham surgery caused an increase in the relative number of monocytes, confirming the presence of systemic inflammation in response to surgery. The use of test substances eliminated monocytosis. In addition to an increase in the ratio of neutrophils to lymphocytes, the onset of parkinsonism was also accompanied by monocytosis, as demonstrated by the results of the analysis of the relative numbers of major populations of leukocytes. Therapeutic administration of test substances reduced peripheral inflammation.

[0445] To characterize the function and phenotype of microglia in the LPS rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0446] Microglial phagocytic activity is an indicator of its activation state, and any changes in it should be viewed in the context of changes in other functional and phenotypic characteristics. The results of this study showed that the number of phagocytic microglial cells and their endocytic activity in sham-operated animals were significantly higher than in intact animals, which, given the satisfactory physiological state of this group of animals, should be considered a sign of activation of the reparative inflammatory process after surgical intervention (Figures 65A and 65B). Administration of zinc preparations to sham-operated animals was accompanied by a decrease in the relative number of phagocytic microglia and their endocytic activity, indicating an anti-inflammatory effect. PD progression was accompanied by a decrease in microglial phagocytic activity, which, given the emergence of disease symptoms, could be considered a sign of suppression of the scavenging or homeostatic patrol function of tissue-resident macrophages in the brain. Therapeutic administration of zinc preparations had various effects on this functional indicator of microglia in the LPS animal model of PD. We observed a sharp increase in microglial phagocytic activity in zinc-fed animals, which, considering the physiological state of this group of animals, can be considered as a sign of the activation of repair processes in the brain.

[0447] The results of this study of microglial oxidative metabolism are supported by data on the phagocytic activity of these cells (Figure 66). Microglia in intact animals were characterized by the lack of significant response to in vitro stimulation, indicating a quiescent state of metabolically conservative phagocytes, i.e., resident mononuclear phagocytes.

[0448] The oxidative metabolism of sham-operated animals was enhanced during the experiment, confirming our assumption of a persistent reparative inflammatory process. An additional criterion for such a process is the presence of functional preservation of microglia in this group of animals, as evidenced by a statistically significant positive response to in vitro PMA treatment. The presence of functional preservation can be considered as an indication of the presence of recruited circulating mononuclear phagocytes in the analyzed population.

[0449] Administration of zinc preparations to sham-operated animals was accompanied by a decrease in microglial oxidative metabolism, confirming our conclusions regarding its anti-inflammatory effect. Oxidative metabolism in PD animal models was sharply reduced compared to that in sham-operated animals. Considering the physiological state of the animals and the fact that this group of rats also underwent surgery, the lack of activated generation of reactive oxygen species can be considered a sign of functional defects in microglia. Administration of the test substance slightly but statistically significantly increased microglial oxidative metabolism, confirming our earlier suggestion that this preparation activates repair processes.

[0450] The results of the analysis of the microglial phenotypic profile are presented in Figures 67A and 67B.

[0451] To characterize the phenotypic profile, the following markers were selected: CD206 (a scavenger receptor, a marker of the alternative polarization of extracerebral phagocytes and also a marker of activated resident microglia (see previous project report)) and CD80 / 86 (costimulatory molecules involved in the process of antigen presentation, which are a marker of the proinflammatory activation of extracerebral phagocytes and are also overexpressed by myeloid-derived suppressor cells, negative regulators of the proinflammatory response of innate and adaptive immunity). The results of this study showed that the expression of all analyzed markers was increased in sham-operated animals, which indicates an activated state of microglia. The administration of zinc preparations significantly reduced the expression of all phenotypes under study. This was accompanied by a decrease in the expression of phenotypic markers. However, the numbers of CD206+ and CD80+ cells in this group of animals increased dramatically. Clearly, this is also a marker of an enhanced repair process. Microglia originate from the yolk sac, and the microglial pool colonizes the brain early during embryonic development. This unique population of resident phagocytes maintains their numbers by self-repair (proliferation and differentiation of stem tissue elements) and does not require the recruitment of immature myeloid precursors from the circulation. Increased expression of any phenotypic marker is a sign of a cell differentiation process, allowing us to conclude that the expansion of the CD206+ and CD80+ fractions occurred as a result of differentiation of immature resident tissue elements.

[0452] The phenotypic profile of the LPS rat model of PD differed from that of rats with 6-OHDA-induced disease. Microglia in the LPS animal model of PD were characterized by significantly reduced expression levels of all studied markers (compared to sham-operated animals), confirming our assumption of a functional defect in these cells caused by a prolonged inflammatory process. However, the fraction of CD206+ cells was very large, which may indicate any healing or repair inflammation. (It should be noted that analysis of all phagocyte immunoreactivity indicators was performed at the end of the experiment, i.e., 28 days after disease induction.) Administration of the zinc preparation was accompanied by increased expression of CD206, a marker of alternative (anti-inflammatory, repair) activation of microglia phagocytosis, and a significant increase in the expression of CD86, a marker specific for myeloid-derived suppressor cells. It can be hypothesized that the zinc preparation promotes the differentiation and / or recruitment of regulatory inflammatory cells. However, the suggested hypothesis requires additional experimental confirmation, since the study protocol did not allow us to differentiate the myeloid-derived suppressor cells phenotypically.

[0453] Therefore, the development of LPS-induced Parkinson's disease is most likely accompanied by a functional defect of microglia caused by a long-term local inflammatory process. The use of zinc preparations with a positive therapeutic effect is associated with an increase in the metabolic activity of microglia, whose significant activation of phagocytic function is involved in the repair process, which may indicate a repair-homeostatic effect of the test substance.

[0454] Functional and phenotypic characteristics of circulating phagocytes in the LPS rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0455] As mentioned above, the development of PD is accompanied by the formation of systemic inflammation, which increases and maintains the persistence of the neuroinflammatory process. This situation makes effector cells of the systemic inflammatory process less attractive targets for anti-inflammatory treatment in PD than resident brain inflammatory effectors. This was one of the reasons for analyzing the functional and phenotypic characteristics of circulating phagocytes. Another reason for this analysis was the fact that zinc preparations were administered intravenously, which makes circulating phagocytes the frontline responding cells. As mentioned above, blood count results indicated the presence of a systemic inflammatory process in the LPS animal model of PD, with significant neutrophilia and an increased neutrophil-lymphocyte ratio, valid indicators of a systemic inflammatory response. Analysis of the functional and phenotypic characteristics of circulating phagocytes confirmed these observations (Figures 68A and 68B). The number of phagocytes in the peripheral blood of sham-operated animals exceeded that of uninjured animals, likely indicating hematopoietic activation associated with the inflammatory repair process. Zinc had no effect on this parameter.

[0456] The phagocytic activity of monocytes and granulocytes in the peripheral blood of sham-operated animals was not significantly different from that of intact rats. Administration of the test substance had no effect on this parameter. The oxidative metabolism of circulating phagocytes in sham-operated animals was significantly reduced compared to that of intact animals (Figures 69A and 69B).

[0457] The zinc preparation did not have any significant effect on this parameter. In general, it can be concluded that the metabolic profile of circulating phagocytes in this group of animals had anti-inflammatory characteristics (which may be evidence of a permanent repair process after surgical intervention) and was not significantly affected by the administration of the test substances.

[0458] The levels of phagocytes in the blood of PD animal models were lower than in sham-operated rats. Given the surgical procedure, which requires activation of repair, the results obtained from these animals may indicate a deficiency / inhibition of hematopoiesis (myelopoiesis) caused by disease progression. Zinc treatment was accompanied by a slight decrease in the relative number of circulating monocytes and did not have any effect on the aforementioned granulocyte indices.

[0459] The phagocytic activity of circulating monocytes and granulocytes in the PD rat model was significantly lower than that in sham-operated and intact rats. At the same time, the oxidative metabolism of these cells significantly exceeded that of the control group. This is evidence of a proinflammatory metabolic shift in the cells and a marker of the systemic inflammatory process. Circulating phagocytes in both populations of the PD animal model treated with zinc preparations were characterized by a sharply reduced oxidative metabolism, demonstrating the anti-inflammatory nature of the systemic regulatory effect exhibited by the test substance. It should be noted that zinc preparations have the ability to restore the functional integrity of the oxidative metabolism of circulating phagocytes.

[0460] Analysis of the expression of phenotypic markers by circulating phagocytes in animals of all groups revealed the following (Figures 70A and 70B): expression of CD206 (a marker of an alternative anti-inflammatory polarization of extracerebral phagocytes) was significantly higher in sham-operated animals compared to intact rats. The number of positive cells did not differ from that in healthy animals. The revealed phenomenon supports the above-mentioned assumption regarding the reparative nature of the inflammatory process in these animals after surgical intervention. The expression level of the costimulatory molecule CD80 / 86 (a marker of the classic pro-inflammatory polarization of extracerebral phagocytes) was also increased in sham-operated animals. Considering the physiological state of this group of animals and the fact that markers for the population of myeloid-derived suppressor cells (found in the circulating blood of animals exhibiting a systemic inflammatory process) have been described in recent literature and mentioned above [Wang W, et al. Eur J Immunol. 2015;45(2):464-73. doi: 10.1002 / eji.201444799], it can be assumed that the expression of these markers and the increased number of positive cells in the population of circulating phagocytes may be due to an increase in the number of myeloid-derived suppressor phagocytes caused by the activation of repair processes in these animals. Sham-operated animals treated with zinc preparations showed a decrease in the expression of these markers to the levels (and even below) shown by intact animals, which may indicate a homeostatic restoration of the phenotypic profile of blood phagocytes under the action of the test substance. The expression levels of all three analyzed phenotypic markers in the PD animal model were significantly lower than in sham-operated animals, which primarily indicates a functional defect of these cells, given the physiological state of this group of animals. Administration of zinc preparations promoted an increase in the expression levels of all three markers, suggesting an anti-inflammatory functional shift of circulating phagocytes in the PD animal model.

[0461] Functional and phenotypic characteristics of peritoneal phagocytes in the LPS rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0462] Analysis of the phenotypic and functional profiles of peritoneal phagocytes, a subset of MALT (mucosa-associated lymphoid tissue) immune cells, revealed their variation in animals from all experimental groups, indicating the involvement of this immune system compartment in the development of neurodegenerative pathologies and the general nature of the underlying inflammatory process.

[0463] The phagocytic activity of peritoneal macrophages and the relative number of phagocytes in the peritoneal exudate of sham-operated animals were higher than those of intact rats, suggesting that this may be a role for the mucosal immune system in the general inflammatory process. The effects of zinc on the phagocytic activity of peritoneal macrophages in the PD animal model were comparable to those of intact rats and significantly lower than those of sham-operated animals. Given the physiological state of this group of animals, we hypothesize that a systemic inflammatory process with reparative properties is absent. The administration of the test substance contributed to the activation of phagocytic activity of peritoneal exudate cells, which can be attributed to the initiation of a systemic inflammatory repair process.

[0464] The oxidative metabolism of peritoneal macrophages in sham-operated rats was significantly higher than in intact animals, confirming the assumption that mucosal phagocytes are involved in the general reparative inflammatory process (Figure 72). Administration of zinc preparations reduced the production of reactive oxygen species by peritoneal phagocytes, indicating the anti-inflammatory properties of their immunomodulatory effect.

[0465] The oxidative metabolism of peritoneal phagocytes in animal models of PD was significantly lower than that in sham-operated animals. Administration of zinc preparations was accompanied by a decrease in the oxidative metabolism of peritoneal exudate cells, indicating the anti-inflammatory nature of the effects produced by the test substances.

[0466] Analysis of the phenotypic profile of peritoneal phagocytes revealed diverse changes in the expression of the studied markers in the experimental animal groups, which were partially different from those recorded for circulating cells. This may be due to the different origins of peritoneal phagocytes (embryonic origin) and peripheral blood phagocytes (bone marrow origin). The expression levels of all three studied markers in peritoneal macrophages of sham-operated animals were only slightly different from those of intact rats (Figures 73A and 73B). Administration of the zinc preparation caused a sharp increase in the expression of all phenotypic markers.

[0467] A similar situation was observed in a comparative analysis of the phenotypic profile of peritoneal macrophages in animal models of PD. The expression levels of all phenotypic markers studied in Parkinson's disease rats were only slightly different from those in control animals. Administration of zinc preparations caused a significant increase in the expression of all markers. In the context of the clinical scores of animals treated with the test substance compared with the phenotypic parameters of other localized phagocytes, we can argue for metabolic activation of the anti-inflammatory properties of MALT phagocytes involved in the systemic inflammatory response.

[0468] On neuronal nitric oxide synthase expression in astrocytes in the LPS rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0469] The highest activity of this enzyme is found in cerebellar neurons and astrocytes. Lower levels of its activity are observed in the hypothalamus, midbrain, striatum, cortex, hippocampus, and medulla oblongata. In nervous tissue, this enzyme primarily functions as a neurotransmitter, controlling neuronal oscillatory activity, nociception, and other processes. Its role in regulating cerebral vascular tone and trophic mechanisms in nervous tissue is crucial. The results of our study show that sham surgery and the development of LPS-induced PD were accompanied by a slight decrease in the expression level of the nitric oxide synthase enzyme in astrocytes (Figure 74). Administration of zinc preparations caused an increase in its expression, indicating a positive effect of the test substance on neuronal signaling, the impairment of which is characteristic of neurodegenerative processes.

[0470] Knowledge

[0471] In the LPS rat model of parkinsonism, the first and second apomorphine trials There was no significant change in the mean number of rotations between experiments. However, an increase in absolute number of rotations was observed in 66.6% of rats, and a decrease in number of rotations was observed in 33.3% of rats. Immunohistochemical studies showed a threefold decrease in the number of TH-positive neurons in the midbrain of the LPS rat model compared with sham-operated animals.

[0472] Starting on day 9, animals were given LPS to induce parkinsonism. 64 Although Zn-asp administration did not significantly change the mean number of rotations per 30 min between the first and second apomorphine trials, the number of animals that produced a smaller absolute number of rotations between the first and second apomorphine trials increased by 85.7%, suggesting a role for Zn-asp in preventing the intensification of the degenerative process of dopaminergic neurons in the substantia nigra. 64 This demonstrates the significant efficacy of Zn-asp and is confirmed by immunohistochemistry data showing a significant increase in the number of TH-positive neurons in the midbrain.

[0473] The progression of LPS-induced parkinsonism is associated with a significant (up to 30%) loss of body weight in LPS animal models compared with intact and sham-operated animals. 64 Administration of Zn-asp resulted in a statistically significant increase in body weight in the LPS animal model.

[0474] Animals were given LPS to induce parkinsonism over a 10-day period. 64 Administration of Zn-asp did not cause any significant changes in the water content of feces in the LPS rat model.

[0475] Behavioral tests performed as part of the study indicated that the test substances did not significantly affect the levels of locomotor activity, anxiety, or irritability of the experimental animals.

[0476] The progression of LPS-induced Parkinson's disease is accompanied by a functional deficiency of microglial phagocytes caused by a prolonged local inflammatory process. The use of zinc preparations with positive therapeutic effects is associated with an increase in the metabolic activity of microglia, whose significant activation of phagocyte function is involved in the repair process, which may indicate a homeostatic effect of the test substance.

[0477] The progression of LPS-induced parkinsonism is accompanied by significant peripheral inflammation, as evidenced by monocytosis combined with an increased neutrophil-lymphocyte ratio. Therapeutic administration of zinc preparations reduces the systemic inflammatory response.

[0478] The phenotypic and metabolic characteristics of circulating phagocytes indicate a pro-inflammatory shift in their functional activity, as well as their functional deficiency caused by a long-term systemic inflammatory process. Administration of zinc preparations is accompanied by a functional restoration of circulating phagocytes, along with an anti-inflammatory shift in their metabolism.

[0479] The progression of LPS-induced parkinsonism involves the involvement of MALT immune cells in the general inflammatory process, along with their proinflammatory activation. Administration of zinc preparations induces metabolic homeostatic changes in these cells, along with their proinflammatory metabolic shift.

[0480] The progression of LPS-induced parkinsonism is accompanied by a decrease in the level of neuronal nitric oxide synthase expression, and a course of treatment with zinc preparations restores the homeostasis of this neurotransmitter.

[0481] conclusion

[0482] The results of this study demonstrate that PD progression is accompanied by a one-third decrease in the number of viable (tyrosine hydroxylase-expressing) dopamine neurons in the substantia nigra, due to an inflammatory process in microglia. This indicates that the inflammatory process may be caused by the activation of immune cells. This inflammatory process spreads throughout the body, covering the blood and extracerebral compartments of the immune system, such as MALT. Given that the study of immune reactivity parameters was conducted at the end of the treatment course with the test substance, the inventors evaluated them in animals after the prolonged inflammatory process. Therapeutic administration of zinc preparations (after the documented significant progression of PD) resulted in a statistically significant increase in the number of viable dopamine neurons in the substantia nigra, which is the most effective indicator of the therapeutic efficacy of drugs studied using experimental PD models. The significant therapeutic effect of the zinc preparation was accompanied by positive (anti-inflammatory) changes in various localized immune cells, indicating a positive effect of the systemically administered test substance on the local (in the brain) and systemic inflammatory processes that accompany PD progression. Considering the effect of the test substance on mucosal immune cells, the inventors cannot exclude its effect on the intestinal microbiota, either directly (by affecting the metabolism of the microbiota, which is largely dependent on the homeostasis of trace elements) or via a mediation effect (by affecting the ratio of aerobic and anaerobic conditions).The ability of the test substance to affect the intestinal microbiota may be an important mechanism for its efficacy in treating neurodegenerative diseases, which are closely related to gut-brain axis disorders.Furthermore, the inventors cannot exclude the efficacy of this preparation in treating inflammatory bowel disease, and its increasing popularity will make the search for effective multi-target therapeutic agents a reality.

[0483] While the present invention has been described in conjunction with a detailed description thereof, it is to be understood that the foregoing description is illustrative of the invention and not intended to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Thus, while only certain features of the invention have been illustrated and described, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. In one embodiment, for example, the following items are provided: (Item 1) 1. A method of treating, preventing, or slowing the progression of a neurodegenerative disease, comprising administering to a subject a compound of the formula: [ka] (Wherein, R1 to R 14 are independently selected from H, OH, F, Cl, Br, I, C1-C6 alkyl, C1-C6 alkoxy, and NO2; 85 Rb e At least 75% 85 Rb) of 85 Rb e compounds, and salts, and / or 64 Zn e Compound or complex of to a subject in need thereof. (Item 2) The aforementioned 85 Rb e At least 90% 85 Rb, and / or 64 Zn e At least 90% 64 Zn. (Item 3) R1, R2, R4 to R6, R8, R 10 , R 11 , and R 13 The method according to item 1 or 2, wherein all of (Item 4) R3 is selected from H, CH3, OCH3, and NO2; R7 and R9 are each independently selected from H and OCH3; R 12 and R 14 are each independently selected from H, Br, I, and NO2. (Item 5) a) R3 is CH3, and R7, R9, R 12 , and R14 are all H, b) R3, R7, R9, R 12 , and R 14 are all H, c) R3 is CH3 and R 14 is Cl, and R7, R9, and R 12 are all H, d) R3 is CH3 and R 14 is OH, and R7, R9, and R 12 are all H, e)R 14 is OH, and R3, R7, R9, and R 12 are all H, f) R3 is OH, and R7, R9, R 12 , and R 14 are all H, g)R 14 is NO2, and R3, R7, R9, and R 12 are all H, h)R 12 is Br and R 14 is NO2, and R3, R7, and R9 are all H; i) R3 and R9 are both OCH3, and R 12 is Br and R 14 is NO2 and R7 is H, or j) R3 and R9 are both OCH3, and R 14 is NO2, and R7 and R 12 are both H, 4. The method according to any one of items 1 to 3. (Item 6) The method of any of the preceding items, wherein the composition further comprises at least one excipient. (Item 7) R3 is CH3, and R7, R9, R 12 , and R 14 are all H. (Item 8) The method of any of the preceding items, wherein the neurodegenerative disorder is Parkinson's disease. (Item 9) The method of any of the preceding items, wherein the composition is administered to the subject intravenously. (Item 10) Item 11. The method according to any one of items 1 to 8, wherein the composition is administered intraperitoneally to the subject. 9. The method of any of items 1 to 8, wherein the composition is administered orally to the subject. (Item 12) 12. The method of any of items 1 to 11, further comprising administering, prior to, simultaneously with, or after administering the composition, a formulation comprising one or more other therapeutic agents for treating a neurodegenerative disorder. (Item 13) 9. The method of claim 8, further comprising administering a formulation comprising one or more other anti-Parkinson's agents prior to, simultaneously with, or after administering the composition. (Item 14) The aforementioned 64 Zn e The compound or complex of claim 1, wherein the compound or complex is a portion of a zinc finger peptide. A method according to any one of the preceding claims. (Item 15) The method of any preceding item, wherein the subject is a human subject. (Item 16) The aforementioned 85 Rb e The compound was added to 40 mg of 85 Rb e From 2400mg 85 Rb e 10. The method of claim 1, wherein the compound is present in an amount equivalent to between 0.1 and 1. (Item 17) The aforementioned 64 Zn e of compound or complex is administered in an amount of 0.1 to 1.5 mg of pure 64 Zn e is in the range 64 Zn e The method of any of the preceding items, wherein the metal is present in an amount equivalent to a dose in the range. (Item 18) The aforementioned 64 Zn e A compound or complex of (metal) is administered in an amount of 1 to 15 mg of pure metal per kg of human body weight. 64 Zn e 17. The method according to any one of items 1 to 16, wherein the range is

Claims

[Claim 1] The invention described in the specification.

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