Rubidium and / or zinc compounds for treating Parkinson's disease and other neurodegenerative diseases - Patents.com

Abstract

64 Zn concentrated zinc aspartate, etc. 64 Zn-enriched zinc salts, compounds, and complexes, and optionally, a zinc salt, compound, or complex represented by the following general formula (1): 85 Rb-enriched rubidium salt compounds (where R1 through R 14 wherein each of R to N is independently selected from H, OH, F, Cl, Br, I, C1-C6 alkyl, C1-C6 alkoxy, and NO2, for example, a compound of formula 1, wherein R is CH3 and all other R groups are H, for use in treating a neurodegenerative disease (NDD) such as Parkinson's disease (PD). Methods entail administering such a composition to treat an NDD such as PD, optionally in combination with any other treatment for the NDD, such as for PD. TIFF2022551943000021.tif5149

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), in particular Parkinson's disease (PD). [Background technology]

[0002] background Neurodegenerative disorders (NDDs), including Parkinson's disease (PD), are pathological conditions induced by inflammatory processes. No cure is available 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 one another), salts, and complexes, e.g. 64 Zn-enriched zinc aspartate, and 85 Provided is an Rb-enriched rubidium organic salt (structure shown below). The disclosed compositions include one or more than one of the 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 is 85 Rb-rich, R 1 From R 14 Each of the above is independently H, OH, F, Cl, Br, I, C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, and NO 2 is selected from.

[0006] In certain embodiments, R of Formula 1 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R 13 are all H, and

[0007] a)R 3 CH 3 and R 7 , R 9 , R 12 , and R 14 are all H (Compound 1),

[0008] b)R 3 , R 7 , R 9 , R 12 , and R 14 are all H (compound 2),

[0009] c)R 3 CH 3 and R 14 is Cl, and R 7 , R 9 , and R 12 are all H (compound 3),

[0010] d) R 3 CH 3 and R 14 is OH and R 7, R 9 , and R 12 are all H (compound 4),

[0011] e)R 14 is OH and R 3 , R 7 , R 9 , and R 12 are all H (compound 5),

[0012] f)R 3 is OH and R 7 , R 9 , R 12 , and R 14 are all H (compound 6),

[0013] g)R 14 NO 2 and R 3 , R 7 , R 9 , and R 12 are all H (compound 7),

[0014] h)R 12 is Br and R 14 NO 2 and R 3 , R 7 , and R 9 are all H (compound 8),

[0015] i)R 3 and R 9 Both are OCH 3 and R 12 is Br and R 14 NO 2 and R 7 is H (compound 9), or

[0016] j)R 3 and R 9 Both are OCH 3 and R 14 NO 2 and R 7 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 Rb atoms are isotopes. 85 Refers to Rb, which is Rb.

[0018] In certain embodiments, R of Formula 1 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R 13 are all H, and the remaining R groups are as defined above. In another embodiment, R 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R 13 are all H, and R 3 H, CH 3 , O.C.H. 3 , and NO 2 Selected from R 7 and R 9 are independently H and OCH 3 Selected from R 12 and R 14 are independently H, Br, I, and NO 2 In another embodiment, R in formula 1 is selected from 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R13 are all H, and R 3 H, CH 3 , O.H., O.C.H. 3 , and NO 2 Selected from R 7 and R 9 are each independently H and OCH 3 Selected from R 12 H, Br, I, and NO 2 Selected from R 14 are H, OH, Cl, Br, I, and NO 2 is selected from.

[0019] The types of compositions include any known in the art, including but not limited to liquid compositions formulated for intravenous or other parenteral administration, compositions formulated for topical administration, and compositions formulated for oral administration, such as tablets, pills, capsules, lozenges, granules, etc. In certain embodiments, the compositions contain between 0.4 millimoles and 30 millimoles of the disclosed compounds, such as 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 contain at least one of the following suitable solvents, such as water; salts or ions, such as sodium chloride, potassium chloride, potassium ions, sodium ions, chloride ions; sugars, such as glucose and sucrose; buffers; and other excipients, such as DMSO. Topical formulations may include, but are not limited to, ointments, creams, lotions, salves, and may include at least one of the following: a suitable vehicle; a permeation enhancer, such as DMSO and related analogs; and an emulsifier. Tablets may include 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 comprises 64 Zinc 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 to said patient a compound of formula 1, with or without conventional forms of NDD or PD treatment. 85 Rb-enriched rubidium compounds, and / or 64 Zinc e Methods are disclosed that include administering a therapeutically effective amount of a composition comprising a salt, complex, or compound containing: 64 Zinc e A composition comprising a therapeutically effective amount of a compound, salt, or complex containing the formula: 85 A therapeutically effective amount of a composition comprising an Rb-enriched rubidium compound; and 64 Zinc e and a compound, salt, or complex containing 85 In a further embodiment, such a composition comprises at least one excipient. 64 Zinc e Compounds containing 85 Rb-enriched rubidium compounds exist in a specific ratio to each other, e.g., 90% 64 Zinc e Compounds containing 10% of formula 1 85 Rb Enriched rubidium compounds, e.g. 90% 64 Zinc e -asp and 10% 85 Rb e -E2, the percentage being based on the mass of the element. 64 Zinc e A salt, complex, or compound containing64 Zinc 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 steps of: The formula below: [ka] (In the formula, R 1 From R 14 Each of the above is independently H, OH, F, Cl, Br, I, C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, and NO 2 and 85 Rb e At least 75% 85 Rb) of 85 Rb e Compounds and salts, and / or 64 Zinc e Compound or complex of To a subject in need thereof, a therapeutically effective amount of a composition comprising:

[0024] 85 Rb e At least 90% 85 and / or 64 Zinc e At least 90% 64 The method according to

[0023] , wherein the compound is Zn.

[0025] R 1 , R 2 , R 4 ~R 6 , R 8 , R 10 , R 11 , and R 13 The method according to

[0023] or

[0024] , wherein all of are H.

[0026] R 3 But, H, CH 3 , O.C.H. 3 , and NO 2 Selected from R 7 and R 9 are each independently H and OCH 3 Selected from R 12 and R 14 are independently H, Br, I, and NO 2 The method according to any one of

[0023] to

[0025] , wherein the method is selected from the group consisting of:

[0027] a)R 3 CH 3 and R 7 , R 9 , R 12 , and R 14 are all H, b)R 3 , R 7 , R 9 , R 12 , and R 14 are all H, c)R 3 CH 3 and R 14 is Cl, and R 7 , R 9 , and R 12 are all H, d) R 3 CH 3 and R 14 is OH, and R 7 , R 9 , and R 12 are all H, e)R 14 is OH, and R 3 , R 7 , R 9 , and R 12 are all H, f)R 3 is OH, and R 7 , R 9 , R 12 , and R 14are all H, g)R 14 NO 2 and R 3 , R 7 , R 9 , and R 12 are all H, h)R 12 is Br and R 14 NO 2 and R 3 , R 7 , and R 9 are all H, i)R 3 and R 9 Both are OCH 3 and R 12 is Br and R 14 NO 2 and R 7 is H, or j)R 3 and R 9 Both are OCH 3 and R 14 NO 2 and R 7 and R 12 The method according to any one of

[0023] to

[0025] , wherein both of are H.

[0028] The method according to any one of

[0023] to

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

[0029] R 3 CH 3 and R 7 , R 9 , 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] The 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 prior to, simultaneously with, or after administration of the composition.

[0035] The method of

[0030] , further comprising administering a formulation containing one or more other anti-Parkinson's agents prior to, simultaneously with, or after administration of the composition.

[0036] 64 Zinc 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 dissolved in 40 mg of water. 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 between

[0039] 64 Zinc e of compound or complex is administered at a concentration of 0.1 to 1.5 mg of pure 64 Zinc e is in the range 64 Zinc eThe 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 Zinc e A compound or complex of (metal) is administered at a concentration of 1 to 15 mg of pure metal per kg of human body weight. 64 Zinc e The method according to any one of

[0023] to

[0038] , wherein the range is [Brief description of the drawings]

[0041] [Figure 1A] Figure 1A and Figure 1B show the effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on microglial phagocytosis in a rat model of experimental parkinsonism after prophylactic administration. 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 group of animal model of parkinsonism; ^^ - p ≤ 0.01 vs. control group of animal model of parkinsonism. [Figure 1B] Figure 1A and Figure 1B show the effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on microglial phagocytosis in a rat model of experimental parkinsonism after prophylactic administration. 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 group of animal model of parkinsonism; ^^ - p ≤ 0.01 vs. control group of animal model of parkinsonism.

[0042] [Diagram 2]Figure 2 shows the effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on microglial oxidative metabolism in a rat model of experimental parkinsonism after prophylactic administration. 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 group of animal model of parkinsonism; ^^-p≦0.01 vs. control group of animal model of parkinsonism; &-p≦0.05 vs. corresponding index in unstimulated samples; &&-p≦0.01 vs. corresponding index in unstimulated samples.

[0043] [Diagram 3] Figure 3 shows the effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on NO synthesis by microglial cells in a rat model of experimental Parkinsonism after prophylactic administration. 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 / hour.

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

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

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

[0048] [Figure 8A] Figures 8A and 8B show the effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on monocyte phagocytosis in a rat model of experimental parkinsonism after prophylactic administration. Figure 8A - relative number of phagocytic cells (expressed as 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 effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on monocyte phagocytosis in a rat model of experimental parkinsonism after prophylactic administration. Figure 8A - relative number of phagocytic cells (expressed as 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 64Zne-asp and 64Zne-asp containing 10% E285Rbe on oxidative metabolism in circulating monocytes in a rat model of experimental Parkinsonism after prophylactic administration. Legend: *-p≦0.05 vs. corresponding index in unstimulated samples; #-p≦0.05 vs. intact animals; &-p≦0.05 vs. control group of the animal model of Parkinsonism.

[0050] [Figure 10A] Figures 10A and 10B show the effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on neutrophil phagocytosis in a rat model of experimental parkinsonism after prophylactic administration. 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. intact animals. Legend: #-p≦0.05 vs. animal model of parkinsonism. [Figure 10B] Figures 10A and 10B show the effect of 64Zne-asp and 64Zne-asp containing 10% E285Rbe on neutrophil phagocytosis in a rat model of experimental parkinsonism after prophylactic administration. 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. intact animals. Legend: #-p≦0.05 vs. animal model of parkinsonism.

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

[0053] [Figure 13A] Figures 13A and 13B show CD206 expression in a population of peripheral blood phagocytes 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. intact animal group; #-p≦0.05 vs. control group of animal model of parkinsonism. [Figure 13B]Figures 13A and 13B show CD206 expression in a population of peripheral blood phagocytes 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. intact animal group; #-p≦0.05 vs. control group of animal model of parkinsonism.

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

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

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

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

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

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

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

[0067] [Figure 27] Figure 27 shows the effect of E2+85Rbe on 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 collection of brain tissue samples for isolation of 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 collection of brain tissue samples for isolation of 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 collection of brain tissue samples for isolation of microglial cells: Figure 30A: decapitation; Figure 30B: opening of the skull; Figure 30C: removal of brain tissue without bleeding.

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

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

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

[0074] [Diagram 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] [Diagram 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] [Diagram 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 of 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] [Diagram 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] [Diagram 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] [Diagram 42] FIG. 42 shows the effect of 64Zne-asp on arginase activity in non-sensitized rat peripheral blood monocytes.

[0083] [Diagram 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] [Diagram 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] [Diagram 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 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 within 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 within 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 an animal at a given location within 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 an animal at a given location within 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 behavior 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 test.

[0094] [Figure 54-1]Figure 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] Figure 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 of 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 of body weight change during 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 of body weight change during 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 an 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-E show the effect 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-E show the effect 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-E show the effect 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. [Fig. 59D] Figures 59A-E show the effect 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-E show the effect 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-D 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-D 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-D 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-D 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. uninjured 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 the LPS rat model of Parkinsonism, M±SD. Note: #p<0.05 vs. uninjured animals, $p<0.05 vs. sham-operated animals, *p<0.05 vs. Parkinson's disease animal model.

[0105] [Figure 65A]Figure 65A and Figure 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] Figure 65A and Figure 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. uninjured 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] Figure 67A and Figure 67B show the expression of phenotypic markers in the microglial population in the 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] Figure 67A and Figure 67B show the expression of phenotypic markers in the microglial population in the 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] Figure 68A and Figure 68B show the therapeutic effect of 64Zn-asp on 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] Figure 68A and Figure 68B show the therapeutic effect of 64Zn-asp on 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] Figure 70A and Figure 70B show the expression of phenotypic markers by circulating phagocytes in the 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. uninjured animals; $ - p≦0.05 vs. sham-operated animals, * - p≦0.05 vs. control animal model of Parkinson's disease. [Figure 70B] Figure 70A and Figure 70B show the expression of phenotypic markers by circulating phagocytes in the 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. uninjured animals; $ - p≦0.05 vs. sham-operated animals, * - p≦0.05 vs. control animal model of Parkinson's disease.

[0111] [Figure 71A] Figure 71A and Figure 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] Figure 71A and Figure 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 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]Figure 73A and Figure 73B show 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] Figure 73A and Figure 73B show 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 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 + H2O; 3 is sham operated + Zn-asp 1.5mg / kg; 4 is LPS + H2O; 5 is LPS + Zn-asp 1.5mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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] With respect to the terms "for example" and "such as" and their grammatical equivalents, unless expressly stated otherwise, it is understood that they are 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 the 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 Zinc 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 We call it Rb-enriched rubidium organic salt and use it as "E2+ 85 Rb e "or" 85 Rb e -E2". "E2" refers to N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine.

[0119] As used herein, 64 Zinc e At least 50% 64 Zn. Preferably, the zinc compounds, complexes, and salts of the compositions used in the disclosed methods are at least 75% zinc. 64 Zn, e.g. at least 80%, 85%, 90%, 95%, or 99% 64 Zn, 64 Zinc 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 remission of one or more of the signs, symptoms, or causes of a disease or disorder in a patient, or any other desired change in 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 case of discrepancy, the present specification, including definitions, shall prevail.

[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 NDDs is characteristic of the elderly. The pathophysiological basis of this phenomenon has been termed "inflammaging", which is a chronic low-grade systemic inflammatory process accompanied by high expression of inflammatory mediators in tissues, organs, and body fluids, as well as a proinflammatory shift in the metabolic profile of immune system cells, especially effectors of innate immunity, such as mononuclear and polymorphonuclear phagocytes (monocytes, macrophages, and neutrophils). 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), which exert several important biological functions both in normal and in the development of pathological conditions. Myeloid cells of the immune system (monocytes, neutrophils, and macrophages) are the main source of ROS. Monocytes and neutrophils are myeloid cells that constantly circulate in the peripheral blood. Macrophages are resident cells present in all tissues without exception and act as sentinels of the immune system. They are formed through differentiation of monocytes. Under normal conditions, ROS function as signaling molecules that control cellular metabolism. It is ROS-mediated signaling that is involved in tissue repair processes associated with stimulation of angiogenesis and differentiation of stem tissue elements necessary for the reconstruction of tissue lesions. ROS have bactericidal functions and are effector molecules in both intracellular and extracellular killing of pathogenic microorganisms.However, enhanced ROS generation leads to the occurrence of abnormal cell signaling and has a profound effect on the properties of proteins, causing problems in their folding (the formation of their functional tertiary structure), the formation of protein aggregates, and consequently the abnormality / 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 lies in 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 is significantly reduced, which is one of the reasons for the realization of the pathogenic effects of ROS and the onset of inflammation.

[0125] ROS are also pathogenic factors in PD. Targets of the pathogenic action of ROS in parkinsonism are several methionine-containing proteins in the brain, e.g., 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 causes a decrease in its ability to exist in the form of protofibrils, promoting the aggregation of monomers with the formation of pathological forms of this protein. Initially, the inflammatory process accompanying the development of PD was thought to be localized only in 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 with the involvement of phagocytes, localizing it outside the brain in the pathogenesis of the disease. It has been proven that the development of PD in both humans and experimental animals is accompanied by a proinflammatory activation of myeloid cells circulating in the peripheral blood (monocytes and neutrophils). In these cells, there are two types of pathological changes in quantitative and functional changes: monocytosis to neutropenia, accompanied by a proinflammatory shift in monocyte function, and a sharp increase in neutrophil oxidative metabolism. Furthermore, quantitative and functional characteristics of circulating myeloid cells are considered as informative markers to monitor the PD process and evaluate the efficacy of treatments for this pathology. 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 development of PD is also closely associated with phagocytes localized in the gastrointestinal tract, including peritoneal macrophages. Studies carried out over the past few years have convincingly demonstrated the relationship between the development of parkinsonism and dysbiotic disorders of the intestinal microbiota. A simplified scheme involving dysbiosis in the development of PD looks as follows: Dysbiotic changes in the intestine are accompanied by proinflammatory activation of myeloid cells of the intestinal wall and peritoneal phagocytes, which become a source of local ROS generation. The increase in ROS concentration leads to a pathological change in the aerobic / anaerobic ratio in the intestinal microbiota, increasing the relative number of aerobic forms of microorganisms with respect to those without immune tolerance mechanisms. An increase in the number of pathogenic aerobic microorganisms further enhances the proinflammatory activation of myeloid cells. Accelerated ROS synthesis leads to the formation of polymeric forms of α-synuclein in the intestine. Aggregation of α-synuclein 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 most important 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 that stabilize the metabolism of the normal gut microbiota and polyunsaturated plant fatty acids that exert powerful antioxidant functions. 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 a neurodegenerative disease in a subject, such as Parkinson's disease.

[0129] The present disclosure relates to 64 Zn-enriched Zinc-containing compounds (separately or in combination with one another), 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 a neurodegenerative disease in a subject, such as Parkinson's disease.

[0130] In another aspect, the present disclosure provides a method for treating a neurodegenerative disorder (NDD), in particular Parkinson's disease (PD), comprising administering to the patient a therapeutically effective amount of 64 Zn-enriched zinc complexes, salts, and compounds, and / or certain 85Compositions are provided that include Rb-enriched rubidium compounds. The disclosed compositions include one or more than one of the isotopically 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 a compound of formula 1 for treating a neurodegenerative disorder (NDD), particularly Parkinson's disease (PD). [ka]

[0132] The compounds of formula 1 are rubidium salts, and rubidium is 85 Rb-rich, R 1 From R 14 Each of the above is independently H, OH, F, Cl, Br, I, C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, and NO 2 is selected from.

[0133] In certain embodiments, R of Formula 1 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R 13 are all H, and

[0134] a)R 3 CH 3 and R 7 , R 9 , R 12 , and R 14 are all H (Compound 1),

[0135] b)R 3 , R 7, R 9 , R 12 , and R 14 are all H (compound 2),

[0136] c)R 3 CH 3 and R 14 is Cl, and R 7 , R 9 , and R 12 are all H (compound 3),

[0137] d) R 3 CH 3 and R 14 is OH, and R 7 , R 9 , and R 12 are all H (compound 4),

[0138] e)R 14 is OH, and R 3 , R 7 , R 9 , and R 12 are all H (compound 5),

[0139] f)R 3 is OH, and R 7 , R 9 , R 12 , and R 14 are all H (compound 6),

[0140] g)R 14 NO 2 and R 3 , R 7 , R 9 , and R 12 are all H (compound 7),

[0141] h)R 12 is Br and R 14 NO 2 and R 3 , R 7 , and R 9 are all H (compound 8),

[0142] i)R 3 and R 9 Both are OCH 3 and R 12 is Br and R 14 NO 2 and R 7 is H (compound 9), or

[0143] j)R 3 and R 9 Both are OCH 3 and R 14 NO 2 and R 7 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 Rb atoms are isotopes. 85 Refers to Rb, which is Rb.

[0145] In certain embodiments, R of Formula 1 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R 13 are all H, and the remaining R groups are as defined above. In another embodiment, R 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R13 are all H, and R 3 is H, CH 3 , O.C.H. 3 , and NO 2 Selected from R 7 and R 9 are independently H and OCH 3 Selected from R 12 and R 14 are independently H, Br, I, and NO 2 In another embodiment, R in formula 1 is selected from 1 , R 2 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R 13 are all H, and R 3 is H, CH 3 , O.H., O.C.H. 3 , and NO 2 Selected from R 7 and R 9 are independently H and OCH 3 Selected from R 12 H, Br, I, and NO 2 Selected from R 14 are H, OH, Cl, Br, I, and NO 2 is selected from.

[0146] In certain embodiments, the composition comprises 64 Zinc 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 is R 3 CH 3 and the remaining R groups are H. This compound is referred to herein as N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine. 85 Rb-enriched organic salts of rubidium 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 using chemical synthesis methods as disclosed herein and / or 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 of 85 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% 85Rb (e.g., 99.8% 85 Rb). The disclosed compositions may include 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 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 RbEnriched rubidium compounds and / or 64 Zinc e The method includes administering a therapeutically effective amount of a composition comprising a salt, complex, or compound, alone or in combination with a conventional form of NDD or PD treatment. In certain embodiments, the method includes administering 64 Zinc e A composition comprising a therapeutically effective amount of a compound, salt, or complex of formula 1 85 A therapeutically effective amount of a composition comprising an Rb-enriched rubidium compound; or 64 Zinc e A compound, salt, or complex containing 85 In a further embodiment, such a composition comprises at least one excipient. 64 Zinc e Containing compounds and 85 Rb-enriched rubidium compounds exist in a certain ratio to each other, e.g., 90% 64 Zinc e The compound containing and the 10% 85Rb enriched rubidium compound of formula 1, e.g., 90% 64 Zinc e -asp and 10% 85 Rbe-E2, where the percentages are based on the mass of the element. 64 Zinc e The containing salt, complex, or compound is64 Zinc e -aspartate. In some embodiments, the compound is a peptide up to 20 amino acids in length.

[0154] In certain embodiments, the disclosed compositions include 64 Zinc e -aspartate chelate complexes. In some embodiments, the compound is a protein or peptide in the form of a zinc finger, 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 Zinc e and 85 Rb e In a composition comprising both compounds, in certain embodiments, 64 Zinc e and 85 Rb e The ratio of the components is 64 Zinc e is 9 parts (by weight) 85 Rb e is 1 part; in other embodiments, the ratio is 64 Zinc e 7 parts (by weight) and 85 Rb e In some embodiments, the ratio is: 64 Zinc e and 85 Rb e In a composition comprising both compounds: 64 Zinc 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 Zinc e or 85 Rb e Also provided are two compositions each comprising any of the compounds of any of the following: 64 Zince and 85 Rb e The ratio of the components is 64 Zinc e is 9 parts (by weight) 85 Rb e is 1 part; in other embodiments, the ratio is 64 Zinc e 7 parts (by weight) and 85 Rb e In some embodiments, the ratio is: 64 Zinc e and 85 Rb e In a composition comprising both compounds: 64 Zinc e is 1 to 7 parts (by weight) 85 Rb e is 3 to 9 parts (by weight).

[0157] In certain embodiments, any of compounds 1-10 (e.g., N-benzoyl-N'-(4-toluenesulfonyl)-o-phenylenediamine) may be used. 85 Rb enriched rubidium salt), and optionally also containing one or more other anti-NDD agents, e.g., one or more anti-PD agents.

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

[0159] Methods are disclosed 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, the enriched rubidium being 99.8% 85 Rb.

[0161] Combination therapy

[0162] In various embodiments, the disclosed compositions are administered prior to, simultaneously with, or after administration of another NDD or PD treatment or procedure. In some embodiments, when not administered simultaneously, the interval between administrations is about 48 hours or less, such as 48 hours or less, 36 hours or less, or 24 hours or less.

[0163] In other embodiments, the disclosed compositions include 64 Zinc e Compounds, salts, or complexes containing, e.g. 64 Zinc e Aspartate, 64 Zinc e of glutamate, or another amino acid 64 Zinc e Salts, such as any of the 18 other most common naturally occurring amino acids, do not contain a 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 of 85 Rb eThe salts and prior art anti-NDD drugs, e.g., prior art anti-PD drugs, are present, for example, at dosages 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 compounds).

[0165] The anti-PD drug may be any anti-PD drug, such as an anti-PD agent, such as L-dopa (optionally in combination with carbidopa), melevodopa, or etilevodopa; dopamine agonists, such as ropinirole, pramipexole, rotigotine, bromocriptine, pergolide, dihydroergocryptine mesylate, cabergoline, piribedil, and apomorphine; monoamine oxidase B inhibitors, such as rasagiline, selegiline, opicapone, and safinamide; The anti-PD drug composition may be one or more drugs selected from the group consisting of tetracycline-O-methyltransferase inhibitors, such as entacapone, tolcapone; anticholinergics, such as trihexyphenidyl, benztropine biperiden, methixene, procyclidine, profenamine, dexetimide, phenglutarimide, mazaticol, bornaprine, tropatepine, ethanautine, orphenadrine (chloride), ethybenzatropine, budipine; and amantadine. The anti-PD drug composition may optionally contain one or more adjuvants.

[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 include at least one pharma- ceutically acceptable vehicle or excipient, which may include, for example, diluents, fillers, disintegrants, solubilizers, dispersants, preservatives, wetting agents, preservatives, stabilizers, buffers (e.g., phosphates, citrates, acetates, tartrates), suspending agents, emulsifiers, and penetration enhancers, such as DMSO, as needed.

[0169] In certain embodiments, the composition may further contain suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers, or release aids, and other pharma- ceutically 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 dosing 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 pharma- ceutically 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 the 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 the following: a suitable solvent, such as water; a salt or ion, such as sodium chloride, potassium chloride, potassium ions, sodium ions, chloride ions, etc.; a sugar, such as glucose and sucrose; a buffer; and other excipients, 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 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 glycols, sodium lauryl sulfate); and coatings.

[0177] Large macromolecules that are slowly metabolized, 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 models for animal testing. 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 appropriate dose and route of administration in humans. To estimate the human equivalent dose, it is recommended to use the dosage conversion table given 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 a variety of 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 per kg of body weight 85 Rb e / kg body weight. 64 Zinc e The prophylactic dose (of the metal) is 0.1 to 1.5 mg of pure metal per kg of human body weight. 64 Zinc e The range is. 64 Zinc e The therapeutic dose (of the metal) is 1 to 15 mg of pure metal per kg of human body weight. 64 Zinc e The range is.

[0179] In certain embodiments, 85 Rb e The prophylactic dose (of the metal) is 0.1 to 1.25 mg of pure metal per kg of human body weight. 85 Rb eIn certain embodiments, the therapeutic dose of 85Rbe (metallic) ranges from 1.25 to 12.5 mg of pure 85Rbe 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 administration routes may 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 are prepared using methods within common practice applied in the pharmaceutical industry, such as, for example, those exemplified in the latest edition of Remington's Pharmaceutical Science Handbook, Mack Pub. NY, USA.

[0183] Light isotopes may be purchased: 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 schemed below. Step 1. Aryl sulfonation: [ka] Step 2. Acylation: [ka]

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

[0186] Compounds of formula 1 can be prepared using intermediate (v) above, or a salt thereof, as a starting material or as an intermediate in the synthesis of compounds 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] Stage 2.NR 3 Acylation of -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 99% of 85 Rb) is used in the final step shown below. [ka]

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

[0192] Working Example

[0193] In order that the 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 Zinc e -asp and 64 Zinc 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 7 groups of 8 animals each: Group 1 - intact animals; Group 2 - sham-operated animals; Group 3 - 64 Zinc e Group 4 - Sham-operated animals administered -asp; Group 5 - Parkinson's disease animal model induced by stereotactic administration of 6-hydroxydopamine (6-OHDA);64 Zinc e -Animal models of parkinsonism receiving prophylactic injections of asp; Group 6 - 64 Zinc e -asp+10%E2 85 Rb e Group 7 - sham-operated animals, which received injections of 64 Zinc e -asp+10%E2 85 Rb eAnimal models of Parkinsonism received prophylactic injections of . Before the prophylactic administration of the experimental drugs, the animals were subjected to the open field test to evaluate their general locomotor activity level and anxiety. The prophylactic course consisted of 10 daily intraperitoneal injections of the drugs (in case of combined drugs, the components were administered at 2-h intervals). After the prophylactic course was over, Parkinson's disease was modeled in the animals. The degree of destruction of neurons in the substantia nigra was studied kinetically during apomorphine tests 7 and 14 days after the onset of the disease. 10 days after the last (second) apomorphine test, the behavioral response of the animals was evaluated in the open field test, after which they were euthanized and the functional (metabolic and phenotypic) profile of the phagocytes of different localizations was analyzed. Thus, the study of the functional profile of phagocytes in a rat model of Parkinson's disease was carried out 28 days after the end of the course of prophylactic treatment with the experimental drugs. Resident peritoneal macrophages were isolated from sterile cell suspensions of peritoneal exudates; enrichment of the suspension was performed by adhesion for 2 h. Non-adherent 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 anticoagulant. Mononuclear and polymorphonuclear phagocytes were analyzed with a flow cytofluorimeter using a gating method. Microglial phagocytes were isolated using a Percoll density gradient. To evaluate the phenotypic profile of phagocytes, expression indices of the pan-phagocytic marker CD14 (co-receptor for bacterial lipopolysaccharide) and a marker of alternative polarization of phagocytes CD206 (mannose receptor 1) were used. To characterize the metabolic profile, pathways of oxidative metabolism and phagocytic activity of microglia as well as arginine metabolism were studied using flow cytometry and spectrometry, respectively. FITC-labeled Staphylococcus aureus wood 46 was used as a phagocytic target. 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 course of drugs), a reduction in the degree of destruction of neurons 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 Zinc e -asp and 10% E2 85 Rb e Contains 64 Zinc e In the -asp group, only 35% (more than 50% less than in the control group) and 30% (60% less than in the control group) of animals, respectively, showed a complete loss of neurons in the substantia nigra. Furthermore, the destruction of 86% of dopaminergic neurons was observed in 64 Zinc 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 velocity during the apomorphine test in animals with 100% neuronal destruction of the substantia nigra compared with controls was 64 Zinc e It should also be noted that the increase in IL-1 expression 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 phagocytosis is an indicator of their activation, both in repair and in inflammation-initiating processes. The final stage of inflammation is accompanied by a complete loss of the ability of microglial cells to phagocytosis, associated with their acquisition of the properties of antigen-presenting cells, and the activation of adaptive proinflammatory immune responses. See, for example, 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 onset of parkinsonism was accompanied by a decrease in the number of phagocytic cells and a slight decrease in their phagocytic activity compared to sham-operated animals, possibly due to the toxic effects of 6-OHDA. The number of phagocytic cells in microglia of animals that received prophylactic treatment with the drug increased significantly while their endocytic activity decreased. This change in metabolic processes was more pronounced when the combined drug was used (Figure 1A & B).

[0202] for phagocytic activity of microglial cells in intact animals 64 Zinc e The modulatory effect of -asp in vitro was the opposite of that observed in vivo. It can be hypothesized 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.

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

[0204] at the same time, 64 Zinc e In the group receiving -asp, this index was at the level of control intact animals, and at 10% E2 85 Rb e Contains 64 Zinc e In the group that received -asp, the index of reactive oxygen species (ROS) generation was two times higher than in intact animals (Figure 1.1.2). Given the nature of the clinical preventive effect, it can be hypothesized 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 oxidative metabolism of microglial cells 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 proinflammatory (M1) activation of microglia accompanied by destructive inflammatory processes. A decrease in the synthesis of this apoptotic mediator indicates an anti-inflammatory metabolic shift in phagocytes, including in microglia.

[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 groups of animal models of Parkinson's disease given the drug, the levels of NO synthesis did not differ from those in intact animals. This is evidence of the anti-inflammatory immunomodulatory effect of the drug on microglial cells. Such an 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. Considering the fact that an increased synthesis of ROS was recorded in groups of animal models of Parkinsonism given the drug, it is logical to assume that this situation leads to a decrease in the synthesis of apoptosis-inducing reactive nitrogen species by microglial cells and is associated with a reduced destruction of neurons 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, supporting the above hypothesis.

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

[0212] The drugs did not exert any statistically significant effect on arginase activity in non-sensitized microglial cells of intact animals, indicating that the effects of the drugs on arginine metabolism are mediated and dependent on alterations in the oxidative metabolism of phagocytes.

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

[0214] a. 64 Zinc e Prophylactic administration of -asp was accompanied by a decrease in the relative number of CD14+ cells with an increase in their expression level (Figure 5B), which probably indicates the ability of the drug to reduce the occurrence of blood-retinal barrier breakdown and / or phagocyte migration, while stimulating their functional maturation, which can be both pro- and anti-inflammatory. Based on our own experience, we consider that the increase in the number of CD206+ cells in microglia is evidence of the activation of their population as a whole. Such an increase in the level of expression is evidence of an alternative (anti-inflammatory) metabolic polarity of brain phagocytes. According to the results of our study, the onset of parkinsonism was accompanied by an increase in the fraction of CD206+ cells (Figure 6A), without a significant increase in the average level of its expression (Figure 6B), which is evidence of pro-inflammatory microglial activation, taking into account the pathogenesis of the disease. 64 Zinc e Prophylactic administration of -asp caused a significant increase in the relative number of CD206+ cells in microglia, together with an increase in its expression level, indicating an anti-inflammatory shift in the functional profile of these cells. It should be noted that there was significant individual variability in both indices in this group of animals. b. Microglial phenotypic profile in animal models of Parkinsonism 64 Zinc e -asp+10%E2 85 Rb eThe effect of had slightly different characteristics. At the end of the experiment, the relative number of CD14+ cells in the microglia of animals that underwent the preventive course was not significantly different from that of the control group of animal models of Parkinson's disease (Fig. 5B). However, the expression level of this marker was significantly increased (Fig. 6B). Both facts indicate that the drug probably does not exert 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 the microglia after administration of the drug was not significantly different from that of the control animal models of Parkinsonism (Fig. 6A). At the same time, its expression level was significantly increased (Fig. 6B), convincingly demonstrating the ability of the drug to cause an anti-inflammatory activation of brain phagocytes.

[0215] circulating phagocytes

[0216] Parkinson's disease is a multisystem neurodegenerative disorder based on irreversible destruction of dopaminergic neurons occurring against a background of an inflammatory-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 of a systemic nature and involves an inflammatory-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 increased development of monocytic precursors in the bone marrow. 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 decreased 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 an 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 a 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. The disruption of monocyte function is associated with a genetically determined defect in the expression of the LRRK2 kinase (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 the main leukocyte populations (lymphocytes, monocytes, granulocytes (neutrophils)) showed that the cause of leukocytosis in Parkinsonism was probably monocytosis, since 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, taking into account that the study was carried out long after the sham operation, it was considered to be caused by an increase in the fraction of lymphocytes, as a natural sign of the resolution of the inflammatory process. In animal models of Parkinson's disease given drugs containing trace elements, the absolute circulating leukocyte counts did not differ significantly from the same values ​​in the control group of rat models 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 reduction 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 given zinc as a drug, monocyte phagocytosis did not differ from that in intact animals. In rats given the combination drug, monocyte phagocytosis was significantly reduced. The number of phagocytic cells in animals given prophylactic treatment with trace elements was also reduced (Figure 8B), which correlates with the data on the reduction of monocyte counts after a prophylactic course with drugs (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 that received a prophylactic course of trace elements, oxidative metabolism in circulating monocytes was within the normal range.

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

[0222] The modulating effect of drugs containing trace elements on the function of granulocytes (neutrophils) in the peripheral blood of rat models of Parkinsonism was also recorded. The relative number of circulating neutrophils in all animals of the experimental group was lower than in intact animals (Fig. 7B). At the same time, the phagocytic function of these cells in rat models of Parkinson's disease was the same as in intact animals (Fig. 1.1.10). In the group of animal models of Parkinsonism that underwent a preventive course 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 modulating effect of drugs containing trace elements on the phagocytic function of non-sensitized neutrophils in the peripheral blood of intact rats. Fig. 10A and Fig. 10B.

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

[0224] 64 Zinc e -asp and 10% E2 85 Rb e Contains 64 Zinc eProphylactic administration of -asp was associated with the absence of any disturbance of 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 properties. The fraction of CD14+ peripheral blood phagocytes in control animal models of Parkinsonism is statistically significantly lower than 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 is also lower than in intact animals, which is confirmed by the presence of circulating phagocytes with immature forms. Figures 12A and 12B.

[0226] The relative numbers of circulating phagocytes expressing CD206, and the expression levels of this phenotypic marker in the rat model of parkinsonism, were higher than in intact animals, which should be taken as an indication 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 the drug containing trace elements was significantly lower than in the control group of Parkinson's disease animal models and in the group of intact animals. This is additional evidence of the drug's anti-inflammatory effect on phagocytes carried by the circulating blood. In general, analysis of the metabolic and phenotypic characteristics of circulating phagocytes demonstrates the drug's ability to reduce the appearance of a systemic inflammatory response associated with monocytosis, a characteristic sign of Parkinsonism.

[0228] Peritoneal macrophages

[0229] 64 Zinc e -asp and 10% E2 85 Rb e Contains 64 Zinc eThe analysis of the functional and phenotypic profile of peritoneal macrophages in an animal model of Parkinson's disease that underwent a preventive course with -asp arose 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 for the interaction between the brain and the peripheral immune system, and that brain antigens are transported via lymphatic vessels of the central nervous system to regional lymph nodes, nose, and 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 activation scheme of the immune response in MALT, the presence of an antigenic stimulus in any of its compartments may be accompanied by the activation of MALT as a whole. Third, a proven fact is the correlation between neurodegenerative diseases and the disturbance of the normal gut microbiota, i.e. dysbiosis, the development of which is accompanied by the 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 peritoneal macrophages in rats. Thus, peritoneal phagocytes in animal models of Parkinsonism were characterized by a statistically significant increased endocytic activity compared to intact animals. Figures 14A and 14B.

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

[0232] The oxidative metabolism of peritoneal macrophages in the rat model of Parkinson's disease was also higher than that in intact animals.

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

[0234] The greatly enhanced production of reactive nitrogen species also indicates proinflammatory activation of peritoneal macrophages in parkinsonism.

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

[0236] Further evidence of the ability of drugs containing trace elements to prevent the proinflammatory shift in the arginine metabolism of peritoneal macrophages in Parkinson's disease is also 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. An increase in arginase activity is a sign of an anti-inflammatory shift in the metabolism of these cells, while an increase in the production of reactive nitrogen species is a criterion for their proinflammatory activation. According to our results, the arginase activity of peritoneal macrophages in a rat model of Parkinsonism was not different from that of intact animals, which, together with the increase in NO production, indicates a proinflammatory shift in the metabolism of these cells. Figure 17.

[0237] 10% E2 85 Rbe Contains 64 Zinc e -asp significantly increased arginase activity in peritoneal phagocytes, which, together with the 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 that were analyzed in microglial phagocytes and circulating phagocytes. According to the results of our study, the expression of CD14 by peritoneal phagocytes did not change statistically significantly either under the conditions of pathogenesis or under the influence of drugs containing trace elements, which is typical of resident tissue phagocytes, since CD14 expression on the membrane is physiologically at its maximum (Figure 18A and Figure 18B).

[0239] 64 Zinc e -asp and 10% E2 85 Rb e Contains 64 Zinc e CD14 expression in peripheral macrophage populations in a rat model of experimental parkinsonism treated prophylactically with -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 animal models of Parkinson's disease compared to intact animals.

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

[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 Zinc e -asp and 10% E2 85 Rb e Contains 64 Zinc e Prophylactic systemic administration of -asp modulated the metabolic and phenotypic profiles of distinctly localized phagocytes in an animal model of Parkinsonism, reducing the cellular inflammatory metabolic shift against the background of reduced levels of dopaminergic neuronal destruction in the animals.

[0245] 3. The nature of the modulating effect of drugs containing trace elements on phagocytes differed depending on their localization and maturity. Microglial cells in animals that received the drug were characterized by the activation of anti-inflammatory (repair) pathways. Prophylactic administration of the drug 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 leading to a reduction in the destruction of neurons in the substantia nigra. Administration of the drug prevented proinflammatory activation of phagocytes in the peritoneal cavity. The mechanism of this phenomenon may be complex and may involve the effects of the drug on cells of the immune system, as well as on the microbiota and intestinal epithelial cells, leading to the prevention of the formation of a pathological gut-brain axis. Analysis of such complex effects requires the study of the effects of the drug on the 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 Zinc e -asp and 10% E2 85 Rb e Contains 64 Zinc e -asp can be considered a sensitive criterion for monitoring its effectiveness. In these calculations, mass properties of the pure elements are used ( 64 Zinc e and 85 Rb e ). For example, in a mixture 64 Zinc e 9 and 85 Rb e Part 1 means that the calculation is based on the mass of the pure element ( 64 Zinc 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 Zinc e -asp and E2+ 85 Rb eInfluence of preparation

[0248] Materials and Methods: Peritoneal macrophages (PM) were isolated from the peritoneal cavity of male mice of the c57 / B6 strain with an average body weight 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 esterases and the level of phagocyte 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. A 90-minute exposure to drugs according to Kim DK, Pfeifer J. Surg Forum. 1977;28:85-7 was used to analyze their effects on oxidative metabolism and phagocyte activity. An 18-hour exposure to Lastra MD, et al., J Trace Elem Med Biol. 2001;15(1):5-10 was used to analyze the effect 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 JJ. Mol Immunol. 2009;47(2-3):205-14. doi: 10.1016 / j.molimm.2009.09.034.

[0249] result:

[0250] 90 minutes 64 Zinc 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 more exogenous stimulation of 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 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 the lowest drug concentrations were used.

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

[0256] 64 Zinc 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 h 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 towards an inverse dose-dependence of the inhibitory effect of zinc-based drugs on phagocyte-induced production of reactive nitrogen species was recorded.

[0258] E2+ on the metabolic activity of peritoneal phagocytes 85 Rb e The modulatory effect of the compound is 64 Zinc e This was even more pronounced than in the case of -asp. A 90-min exposure of non-sensitized PMs to the drugs in the concentration range of 62-500 μg / ml caused a rapid inhibition of their spontaneous ROS generation as a result of esterase activation (Figure 25). No dose-response relationship was observed. 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 (by bacterial LPS stimulation) intracellular ROS generation by peritoneal phagocytes. No dose-response relationship was observed. FIG. 25.

[0260] E2+ for the activation of NADH and NADPH oxidases in PM 85 Rb eThe nature of the effect of (Fig. 26) was different from that on the reactive metabolic creation of oxygen by esterases (Fig. 26). When used at a concentration of 500 μg / ml, the drug caused a slight increase in the oxidase activity of the phagocytes. All concentrations of the drug lower than 500 μg / ml did not exert a statistically significant effect on this index of metabolic activity of PMs. The nature of the effect of the drug on the induced (by LPS stimulation) 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 towards the ability to reduce the formation of reactive oxygen species by oxidases. However, in view of the non-significant level of its effect and the disproportionate variation in its direction depending on the concentration, it should be considered as non-specific and due to optical density variations. Fig. 26.

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

[0262] E2+ at concentrations of 500 and 250 μg / ml with 18-hour 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 not dependent on additional treatment of the cells with bacterial LPS and was more expressive 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 Zinc e As in the case of -asp, PM was shown to result in the generation of reactive nitrogen species after 18 hours of exposure (Figure 29).

[0264] When used at all of the above concentrations except 8 μg / ml, the drug inhibited spontaneous production of reactive nitrogen species by PM. No dose-response relationship was observed. The drug also showed a tendency to attenuate 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 modulating effect on the metabolic profile of non-sensitized and bacterial LPS-stimulated peritoneal phagocytes. This conclusion is justified by the change in the effect of the drugs on the arginine metabolic pathway in phagocytes towards 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. The reduced activity of iNOS characterizes an alternative phenotype (M2) of phagocytes. There is no clear dose-response relationship.

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

[0268] 3. The high level of variability of 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 effect of zinc-based preparations may also be caused by insufficient exposure of cells to the drug. In this context, since in vitro studies are limited by methodological conditions, it is advisable to carry out additional in vivo studies of the effect of drugs on the oxidative metabolism of phagocytes using intact animals, which would allow a longer effect of the factor on this cellular function.

[0270] Example 3 on the metabolic profiles of non-sensitized microglial cells, circulating monocytes, and neutrophils in healthy rats 64 Zinc 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 (Figure 30A, Figure 30B, and Figure 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 collection of brain tissue, decapitation was performed to avoid bleeding into the brain during sampling (Figure 32). Decapitation was performed immediately after anesthesia to minimize the death of brain tissue, including microglial cells. All operations for isolation of microglial cells were performed using cooling agents. 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) (Figure 33A and Figure 33B). Circulating monocytes were isolated from venous blood collected in tubes containing heparin on 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 levels of NO synthesis measured using a photocolorimetric method), the level of formation of intracellular reactive oxygen species (ROS) by esterases, and the index of phagocyte activity determined by flow cytofluorimetry, as well as according to the activation levels of NADH and NADPH oxidases determined in the NBT test. The duration of treatment of all the abovementioned populations of phagocytes varied according to the methodological characteristics of the metabolic response. A 90-minute exposure to drugs according to Kim et al., 1977 (cited above) was used to analyze their effects on oxidative metabolism and phagocyte activity.Drug effects on the arginine metabolic pathway were analyzed using an 18-hour exposure according to Lastra et al., 2001 (cited above). 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 Zinc e Treatment of non-sensitized microglial cells with -asp had a multidirectional effect 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 at concentrations ≦5 μg / ml, there was 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 did not have any effect on the esterase-dependent oxidative metabolism induced in the cells under study.

[0275] At all concentrations studied, the drug moderately increased the phagocytic activity of microglial cells without affecting the number of phagocytes within 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 (FIG. 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 the synthesis of nitrite.

[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 lack of effect on arginase activity in combination with the 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 Zinc e Treatment of peripheral blood monocytes with -asp resulted in a decrease in spontaneous esterase-dependent intracellular ROS generation 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 ≦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 studied indicators, which was due to the low phagocytic activity of monocytes in isotonic saline. 64 Zinc eThis may be caused by 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 receptors on the membrane of these cells are recommended.

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

[0287] On monocyte arginase activity 64 Zinc e There was no statistically significant effect of -asp, both after treatment of resting cells and after addition of the drug against the background of bacterial LPS cell treatment (Figure 42). The results of the study on monocyte arginase and NO synthase activity show that there is no significant effect of the drug on the arginine metabolic pathway in these cells. According to maturity and functional plasticity, microglial phagocytes differ from circulating monocytes. 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 method used in the study).

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

[0289] peripheral blood neutrophils

[0290] 64 Zinc e Treatment of circulating non-sensitized neutrophils with -asp resulted in a sharp decrease in intracellular ROS production by these cells (Figure 43). The modulating effect had an inverse dose-dependence. Addition of the drug to the cells against the background of their treatment with bacterial LPS also caused a sharp decrease in esterase-dependent oxidative metabolism in neutrophils. Neutrophils 64 Zinc e It should be noted that these were the cells most sensitive to the antioxidant action of -asp. Neutrophils differ in their functional properties from all previous phagocytes. They are functionally mature cells with a highly reactive metabolism, which causes the short duration of their life span. They are exclusively circulating cells and are one of the first of the main effectors of the inflammatory response (associated both with infectious processes and with 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 indicators should also be noted.

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

[0294] It should be noted that the indices of phagocytic activity of neutrophils were 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 features of the intensity of endocytosis by neutrophils in these animals.

[0295] 64Zne-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 practically unchanged under the action of the drugs (FIG. 47).

[0297] FIG. 47 shows the effect of arginase activity on peripheral blood neutrophils from non-sensitized rats. 64 Zinc 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 Zinc 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 the induced oxidative metabolism in all cells of the phagocyte populations studied and inhibited the induced production of reactive nitrogen species in virtually all cells, indicating 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 spontaneous and phytohemagglutinin-induced proliferation of human peripheral blood mononuclear cells 64 Zinc e -asp and E2+ 85 Rb e Effects of preparations.

[0305] Introduction: It is known from 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.). Zinc-transferrin complexes stimulate DNA synthesis in activated T lymphocytes and have 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). According to published data, rubidium salts (chloride) stimulate the process of differentiation of lymphoid and myeloid lineage 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). There are no data in the literature on the influence of rubidium salts on the proliferative activity of differentiated T-lymphocytes.

[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 (adding drugs as single components) and costimulatory (adding drugs 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 evaluated 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+G 2 M) / (G 0 G 1 +G 2 M))×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 Zinc 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 of T lymphocytes, the preparation increased the proliferative activity of the cells compared to samples treated with the mitogen alone and to untreated samples. No dose-response relationship was followed. The data obtained are in agreement with published data on the costimulatory effect of zinc chloride on T cell proliferation.

[0311] E2+ 85 Rb edid not produce any in vitro effect on the spontaneous proliferative 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 measure of T cell activation. In this context, the level of activation-induced apoptosis of T lymphocytes treated with test drugs separately and in combination with PHA was measured. As can be seen in Figure 49, 64 Zinc e -asp had no effect on activation-induced T cell apoptosis. However, when this drug was used in combination with 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 antineoplastic properties, at least with respect to malignant T-lymphocytes. Furthermore, a positive effect of the drug on inflammatory pathological processes accompanied by activation of T-cell proliferation (autoimmune diseases, allergic pathologies) is possible.

[0314] Findings from Example 4:

[0315] 1. 64 Zinc e-asp has stimulatory and costimulatory effects on T lymphocyte proliferation and concomitantly inhibits 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 profile of differently localized phagocytes 64 Zinc e -asp and E2+ 85 Rb e The results of in vitro studies on the effects of demonstrated that microglial cells, circulating phagocytes (monocytes and neutrophils), as well as 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, which is a sign of its activation. 64 Zinc e -asp enhanced ROS generation by resting (unstimulated) microglial cells, indicating the drug's ability to activate the repair activity of brain phagocytes. At the same time, ROS generation by activated microglial cells (a situation simulating an inflammatory process) was reduced under the action of the drug. The arginine metabolic pathway in microglial cells shifted towards increased arginase activity under the action of the drug, again confirming the anti-inflammatory nature of its immunomodulatory action (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 Zinc eThe nature of the changes in the metabolism of circulating phagocytes under the action of -asp differed from that in microglia: the drug caused a decrease in ROS generation by both resting and stimulated monocytes and neutrophils and had a weak effect on phagocytosis and arginine metabolism in these cells. 64 Zinc 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 mature phagocytes of ontogeny origin, as opposed to circulating phagocytes derived from the bone marrow. 64 Zinc e Under the action of -asp, ROS production was enhanced and at the same time the synthesis of reactive nitrogen species was sharply reduced, indicating an anti-inflammatory (repair) shift in their metabolism.

[0319] Differential localization of phagocytes and 64 Zinc 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 decreased by such treatment, and the synthesis of ROS by both resting and stimulated cells was also decreased along with a decrease in the production of reactive nitrogen species, all together indicating an anti-inflammatory modulation of the function of these cells by the combined preparations containing trace elements. 64 Zinc e -asp and E2+ 85 Rb e The nature of the effect of combined treatment of circulating phagocytes with zinc-based drugs was essentially not different from 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 e Treatment of peritoneal macrophages with 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 would be advantageous prior to the onset of PD in in vivo studies. In our opinion, such advanced preparations of phagocytes in the brain, peripheral blood and peritoneal cavity would be able to inhibit their inflammatory activation that accompanies the onset of the disease and attenuate / reduce the extent of destruction of dopaminergic neurons in the substantia nigra in animals.

[0322] This assumption was confirmed by the results on the prophylactic administration of the drug before the induction of PD in rats. Both zinc-based drugs, used as a sole treatment and in combination with a preparation containing rubidium, caused a reduction in the degree of destruction of dopaminergic neurons in animals during the development of PD. Such a reduction in the degree of destruction of dopaminergic neurons was accompanied by a positive change in the different localizations of phagocytes in animals that received the drug under study as a prophylactic medication, in contrast to control animal models of PD, characterized by a proinflammatory activation of phagocytes of different localizations, which was largely abolished by the prophylactic administration of a preparation containing the trace element. Microglia in control animal models of PD differed from those in intact animals by a reduced ROS production, and at the same time, an increased synthesis of reactive nitrogen species, which have a major destructive effect on neurons in the substantia nigra. Prophylactic administration of the drug was accompanied by an increased ROS production by microglial cells, concomitant with a reduced synthesis of reactive nitrogen species. As mentioned above, increased ROS synthesis is a necessary condition for the activation of repair processes. Thus, the enhancement of oxidative metabolism in microglial cells combined with its anti-inflammatory polarization (increased scavenger receptor expression and reduced synthesis of reactive nitrogen forms) indicates the ability of the trace element-based preparation to activate repair processes in the brain of an animal model of PD subjected to its systemic intraperitoneal administration. Circulating phagocytes in a rat model of Parkinsonism are characterized by a pronounced proinflammatory polarization, which confirms literature data on the involvement of these cells in the systemic inflammation that accompanies the development of PD. Prophylactic administration of the trace element-based preparation prevented the inflammatory activation of these cells. The same is true for the alterations in the metabolism of peritoneal macrophages in a rat model of PD after prophylactic administration of the trace element-based preparation.

[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 Compound concentration

[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 - 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] 24 hours after the last addition, cells were seeded into wells of a 96-well plate at a concentration of 1 x 104 cells / well in complete DMEM nutrient medium containing 10% FBS and 40 μg / ml gentamicin. The cells were incubated at 37 °C for 24 h in 5% CO 2 The cells were cultured for 24 hours in a humidified atmosphere at 37°C and 5% CO. After 24 hours, various doses of experimental substances were added to each well. The cells were incubated at 37°C and 5% CO. 2 and incubated for an additional 72 hours.

[0340] After completion of the incubation with the preparations, the proliferation and metabolic activity of the experimental cells was evaluated by a 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 CO2 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 multi-well spectrophotometer at an excitation wavelength of 540 nm. The percentage of live cells was calculated by the formula:

[0343] IR=(A540(experimental) / A540(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 to prepare other compounds of formula 1 by using R 9 OCH 3 The cases where:

[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 washing thoroughly with cold water to remove the residues of unreacted o-phenylenediamine. 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 charcoal and, after cooling, the N-benzenesulfonyl-o-phenylenediamine hydrochloride, which takes the form of fine needle-like crystals, was filtered off (weight 7.11 g per dry substance. Yield 25%). [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 material obtained was recrystallized from isopropyl alcohol and purified with activated charcoal. 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 with 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 99% of 85 Rb) was added and dissolved quickly. The reaction mixture was stirred at room temperature for 30 min and filtered again. The filtrate was made up to 100 ml with deionized water and used for further studies. [ka]

[0352] Among other things, the following methods can be used to identify newly synthesized compounds: DMSO-d 6 Measurement of PMR spectra for TMS in water on a Varian VXR 200 spectrometer; 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); determination of mass spectra 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] R 9 OCH 3 If it is, the following alternative synthesis may be used: 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 proinflammatory activator of cells of the innate immune system, including the brain's resident mononuclear phagocytes, microglia. 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 immunity, as well as by adipocytes, epithelial cells, and many other cells. The interaction of TLR4 with LPS activates the NFκB-dependent signaling pathway, which results, first and foremost, in the activation of the synthesis of proinflammatory mediators, including cytokines, chemokines, eicosanoids, etc. 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. An increased number of circulating LPS molecules is one of the most important factors in the activation of neurodegenerative processes and is considered an essential component of the pathogenesis of Parkinson's disease (PD), Alzheimer's disease (AD), etc. 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, since it allows the assessment of the inflammatory component in the pathogenesis of the disease and, with it, the most appropriate evaluation of the therapeutic efficacy of anti-inflammatory drugs has been presented 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 injection of endotoxin described in the literature, the coordinates ensuring maximal neuroinflammation and subsequent high damage to 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 from Sigma) dissolved in 2 μl of sterile 0.9% NaCl (Infuzia CJSC, Ukraine). The solution was prepared on the day of the surgical procedure. The flask containing the resulting solution was sealed with a drop of silicone oil (STEP-ELECTRONICA LLC, Ukraine) and placed in the refrigerator.

[0365] The animals were anesthetized using a mixture of ketamine (75 mg / kg, diluted in 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, and placed in a stereotaxic apparatus (SEZH-4) modified for rats. The animals were then scalped and a trephine opening was made directly in the substantia nigra with an injection needle (AP=-5.3; ML=±2.0; DV=-7.2). The 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 the animals' scalp soft tissue was sutured with several stitches. 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 movements towards the opposite side of the hemisphere where 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 movements over a period of 30 min indicated the degree of degradation of the nigrostriatal dopamine system (SA Talanov et al., Neurophysiology 38 (2), 128-133). The number of animals whose number of rotations increased, decreased or remained unchanged within 30 min between apomorphine tests I and II 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, 251-261]. Immunohistochemical staining of 5 μm paraffin-embedded midbrain sections was performed using primary TH antibody at 1:200 dilution (Millipore, AB152). Endogenous peroxidase activity was quenched using endogenous peroxidase blocking solution (Dako, EnVision Flex, DM821). Nonspecific binding of the antibody was blocked with a 4% solution of dried milk in Tris-buffered saline (TBS) containing 0.2% Triton X-100.

[0371] Primary antibodies were diluted in TBS containing 0.2% Triton X-100, applied to the sections and incubated overnight (+4°C). Secondary antibodies (biotinylated anti-rabbit, 1:200) were incubated for 60 min. The immunoreaction was developed by application of diaminobenzidine (Dako, EnVision) for 5 min. 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 evaluated 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] Measuring fecal moisture content

[0373] Feces were collected on days 8, 21, and 28 of the study. 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 of the difference between the wet and dry weights of the feces.

[0374] Behavioral testing

[0375] Open field test

[0376] Methods. Open field test is a technique commonly used to measure rodent locomotor activity and their exploration activity in unfamiliar environment, and to evaluate 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 open field test include the distance traveled in outer and inner perimeters, the duration spent in outer and inner perimeters, the total distance traveled, and the percentage of the number of inner squares crossed relative to the total number of squares.

[0377] The open field was a square chamber 100x100cm in size with walls 60cm high. The chamber was illuminated by two LED bulbs of 60 watts each, suspended at a height of 2m. An IP camera was used to record the movement of the rodents. Parameters were analyzed using a 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 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 causes a conflict between fear and the desire to explore this environment. These two behavioral tendencies are characterized by different temporal dispersions and different spatial advantages. In the analysis, the activity area is divided into two zones: inner and outer. The total locomotor activity and the time spent in the inner and outer zones are the most significant parameters to evaluate the rat's level of anxiety. If during the 5-minute test the animal spends most of its time near the walls, on the periphery, and rarely enters inside the inner "open" zone, it is considered normal. An increase in the entry into the inner zone is interpreted as a decrease in the animal's anxiety. The emotional and exploratory behavior of animals is affected by various disorders in brain areas such as the hippocampus and the amygdala. Furthermore, various substances with anxiolytic, stimulating, and muscle relaxant effects can affect the outcome of the experiment. Figure 50A and Figure 50B show an example of the analysis of the rat's trajectory in the experimental activity area.

[0379] elevated cross maze

[0380] Methods. The elevated plus maze is a behavioral assay widely used in rodents that allows to evaluate the sedative or anxiogenic effects of drugs, to define the mechanisms underlying anxiety-related behavior, and to evaluate the level of locomotor activity of rats and their exploratory activity, which can be correlated with the 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 evaluated 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 observed for 5 minutes for the above parameters. Throughout the experiment, animals had free access to food and water. Before the start of the measurements, 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 structure, including two open arms (50×10 cm) crossing each other and perpendicular to two closed arms (50×10×30 cm). The entire apparatus was 50 cm above the floor. In the center of the joint was a central area of ​​10 square cm, where the animal began to face the open arms. The apparatus was illuminated by two LED bulbs, each 60 watts, suspended at a height of 2 m. The rat's location was recorded using an IP camera, and the scoring was completed later using a 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.

[0382] Data analysis and interpretation. The test is based on the rat's natural aversion to the open area and its preference for the closed area of ​​the apparatus. The animal's anxiety level is evaluated as the total time spent in the open and closed arms. Animals that spend more time in the open arms have a lower level 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 causes a conflict between fear and the desire to explore this environment. The data collected from these two tests can therefore be correlated with the distance traveled and the time spent in the outer and inner perimeters. If during a 5-minute test the animal spends most of its time in the closed arms but also makes several entries into the open arms for exploration, this is considered normal. An increase in entries into the open arms is therefore interpreted as a decrease in the animal's anxiety. Figure 51A and Figure 51B show an example of the analysis of the rat's trajectory in the experimental activity area.

[0383] Pickup Test

[0384] The pick-up test is a test for increased irritability. The animal is picked up by grabbing the body. Responses are graded as follows: 1, very easy; 2, easy with vocalization; 3, somewhat difficult, the rat stands on its hind legs and faces the hand; 4, the rat does not move (with or without vocalization); 5, difficult, the rat moves away and avoids the hand; and 6, very difficult, the rat acts 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 house 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 administered 0.1 ml of ultra-light water daily for 10 days after surgery, rats were sham-operated; 64Rats were administered Zn-asp (1.5mg / kg, iv, 10 days) solution daily, rats were sham operated; IV (n=15) - rats were administered 0.1ml of ultra-light water (iv, 10 days) daily after surgery, then LPS-induced Parkinson's disease model was generated; V (n=15) - rats were administered 0.1ml of ultra-light water (iv, 10 days) daily after surgery, then LPS-induced Parkinson's disease model was generated; 64 Rats were administered Zn-asp (1.5mg / kg, iv, 10 days) solution daily, after which 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, for 10 days) solution daily, 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 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 substances for 10 days according to their group allocation. On day 8 of the experiment (before the start of treatment - apomorphine test I) and on day 21 of the experiment (after the administration of the test substances had ended - apomorphine test II), animals were subjected to apomorphine tests to analyze the development of hemiparkinsonism, which correlates with the number of destroyed dopaminergic neurons. Furthermore, a follow-up analysis of fecal water content was performed, i.e. before the start of treatment on day 8 after surgery, as well as on days 21 (after the treatment had ended) and 28 (before necropsy). On day 24 of the experiment, behavioral tests were performed in all groups. All animals were weighed before necropsy. During necropsy, 2 / 3 of the animals from each group were sacrificed by cervical dislocation and brain and blood samples were collected for further biochemical and immunological tests. 1 / 3 of the animals were used for further immunohistochemical studies of the brain; for this purpose, under anesthesia, a procedure of rapid, controlled and uniform fixation of the whole body of the animals was performed using 4% paraformaldehyde perfused through the rat heart to obtain the best preservation of brain structures for immunohistochemistry. Gage GJ, et al., J Vis Exp. 2012 Jul 30;(65).

[0388] Hematological evaluation

[0389] Blood cell counts were analyzed at the end of the study (day 28). Absolute white blood cell numbers, 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 the test substances, microglia and peritoneal macrophages were isolated. The phenotypic and functional characteristics of peripheral blood phagocytes were evaluated without fractionating them (using whole blood). To isolate microglia, brain tissue homogenates were obtained by manual homogenization, after which non-dissociated clumps were removed using a cell strainer (No 70). Microglial cells were isolated from the resulting homogenates by centrifugation on 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 different 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, National Taras Shevchenko University.

[0394] Oxidative metabolism of phagocytes at various localizations

[0395] Oxidative metabolism in various phagocytes is controlled by intracellular esterases, which are non-fluorescent, membrane-impermeable carboxy-H 2 Cell-permeable 2'7'-dichlorodihydrofluorescein diacetate (DHP) (carboxy-H 2 The cells were analyzed by flow cytometry using a DCFDA (Invitrogen, USA).

[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 the data collected from the 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 GraphPad Prism 7 statistical software package. Non-parametric tests were used for statistical data processing. Median values ​​of each group are indicated by horizontal lines. Kruskel-Wallis one-factor test and post-hoc Dunn's test were used to determine significant differences between groups. 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 a significantly lower pronounced rotational behavior compared to the 6OHDA-induced PD model, and this fact was confirmed in this study on a larger number of animals (groups IV, V, VII). In this example, 38 rats (86%) out of 44 LPS models of Parkinson's disease (one rat did not wake up from anesthesia) rotated less than 100 times per 30 minutes.

[0404] The physiological decrease in the number of rotations between the first and second apomorphine trials was indicative of LPS-induced parkinsonism and H. 2 An increase in the number of rotations was observed in 33.3% (5 of 15 animals) of the O-injected control group (group IV) rats, whereas an increase in the number of rotations was observed in 66.6% (10 of 15 animals) of the 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 administration of Zn-asp was started 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 number of 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 the rats injected with ultra-light water, 64 85.7% of rats treated with Zn-asp (12 out of 14 animals) produced fewer rotations between the first and second apomorphine test, and 14.3% of rats (2 out of 14 animals) increased the number of rotations (Figure 53B), which may be due to its role in preventing the intensity 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, the inventors carried out 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 classical marker of dopamine neurons. The study results showed that the color of the substantia nigra of animals from the intact group (group I) and the sham-operated group (group II) was prominent and fell into the score of 3. At the same time, administration of LPS to generate the PD model led to a significant reduction in the number of TH-positive neurons in the midbrain (score 1) compared with the intact group and the sham-operated group (Figure 54A-Figure 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 increase in the number of TH-positive neurons in the midbrain compared to the LPS animal model administered with water (group V) (Figures 54A-54O, 55). These data correlate with the apomorphine test data (Figures 53A and 53B), and indicate the role of Zn-asp in preventing the intensity of 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 an animal is a classic clinical index that allows the assessment of its physiological state. Abundant studies have reported that weight loss may precede the diagnosis of Parkinson's disease by years, and is also associated with the severity and duration of the disease

[26] . In addition, PD-related motor and non-motor symptoms may 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]. In Alzheimer'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 may be considered as 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% body 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 (relative to the difference in values ​​in this group on days 1 and 28 of the experiment (Figure 56A) and 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 a rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0417] Despite the main clinical signs of Parkinson's disease, such as tremor, motor hypofunction, muscle rigidity, and postural instability, it is the early non-motor symptoms of Parkinson's disease that clearly affect the patient's 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. Among the non-motor symptoms, the main discomfort is caused by gastrointestinal (GI) dysfunction, including 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 the feces depends on, and accurately reflects, the digestive function, not on food intake. Intestinal peristalsis and water absorption are related, which in turn relates to water absorption and 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, an increase in fecal water content was recorded in rats of the sham-operated (SO) group compared to intact animals. These impairments were stable over time and were significant even 28 days after surgery (Figure 57). In the LPS rat model of Parkinsonism, a 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 of Parkinson's disease.

[0419] In this study, animals were injected with LPS to induce parkinsonism and then for 10 days 64 Administration of Zn-asp (Figure 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 effect of Zn-asp, the commonly accepted open field test was used. Observation of the behavioral response of rats in this test allows to determine the degree of influence of the test substance on the locomotor and exploratory activity, orientation response, and emotional reactivity of the animals. The test results are shown in Table 3 and Figures 59A to 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 the intact control (group I).

[0424] Significant groups, i.e., group II (SO) and group III (SO+ 64Kruskal-Wallis test used to make comparisons between 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 on the outer periphery and decreased time spent on the inner periphery (P = 0.0020) in groups II (SO) and III (SO+ 64 Zn-asp) (Dunn's multiple comparison test P = 0.0193); - Reduction in total rearing frequency by group (P=0.0160), compared with groups II (SO) and III (SO+ 64 There is a significant difference between the two (Zn-asp) (Dunn's multiple comparison test P = 0.0053).

[0425] Furthermore, the effect of the open field test on behavioral parameters 64 To analyze the effect of Zn-asp, the significant groups, i.e., group IV (LPS) and group V (LPS + 64 Comparisons were made between the groups (Zn-asp) and showed that: treatment with test substance affected the total distance traveled (Kruskal-Wallis test - P value = 0.7031), the time spent in the outer (left chart) and inner (right chart) perimeter (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 an 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 allows 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, i.e., group II (SO) and group III (SO+ 64 Kruskal-Wallis and ANOVA tests used to compare between the IV (Zn-asp) and IV (LPS) groups 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 the IV group (LPS) were reduced by 1.3-, 1.4-, and 1.6-fold, respectively, when compared to group II (SO).

[0432] For behavioral parameters of rats in the elevated plus maze 64 To further analyze the effect of Zn-asp, the significant groups, i.e., group IV (LPS) and group V (LPS + 64 A comparison was made between the Zn-asp and the control group, revealing that administration of the test substances 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 fold, respectively, when compared to group IV (LPS).

[0433] on the 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 grabbing 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 the intact control (group I).

[0436] Significant groups, i.e., group II (SO) and group III (SO+ 64 Kruskal-Wallis and ANOVA tests 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, comparisons were made between the significant groups, i.e., group IV (LPS) and group V (LPS+ 64 Zn-asp) and compared with group IV (LPS), group V (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 a rat model of parkinsonism 64 Therapeutic effect of Zn-asp

[0439] Parkinson's disease is now considered a disease with systemic meta-inflammation. Causes of systemic inflammation include disruption of the blood-brain barrier with excretion of proinflammatory brain mediators to the periphery, transport of inflammatory mediators through the glymphatic system, and peripheral disorders with neurodegeneration, including gastrointestinal dysfunction (digestive ulcers, impaired gut motility, etc.) 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 performed after the completion 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 that there were 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, one of the early markers 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 normalization of the neutrophil to lymphocyte ratio, mainly due to an increase in lymphocyte levels, a criterion for the resolution of inflammation involving regulatory cells.

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

[0444] Sham surgery caused an increase in the relative number of monocytes, which confirms the presence of systemic inflammation in response to surgery. The use of test substances eliminated monocytosis. In addition to the increase in the ratio of neutrophils to lymphocytes, the development of parkinsonism was also accompanied by monocytosis, a fact that is demonstrated by the results of the analysis of the relative numbers of the main populations of leukocytes. Therapeutic administration of test substances reduced peripheral inflammation.

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

[0446] The phagocytic activity of microglia is an indicator of its activation state, and any change in it should be seen 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 should be considered as a sign of activation of the reparative inflammatory process after surgical intervention, given the satisfactory physiological state of this group of animals (Figure 65A and Figure 65B). Administration of zinc preparations to sham-operated animals was accompanied by a decrease in the relative number of phagocytic microglia and a decrease in their endocytic activity, indicating an anti-inflammatory effect. The progression of PD was accompanied by a decrease in the phagocytic activity of microglia, which, given the emergence of disease symptoms, can be considered as a sign of suppression of the scavenger or homeostatic patrol function of tissue-resident macrophages in the brain. Therapeutic administration of zinc preparations had different effects on this functional indicator of microglia in the LPS animal model of PD. We observed a sharp increase in microglial phagocytic activity in animals given zinc, which, taking into account the physiological state of this group of animals, could 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 from 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 a sign of the presence of recruited circulating mononuclear phagocytes in the analyzed population.

[0449] Administration of the zinc preparation to sham-operated animals was accompanied by a decrease in microglial oxidative metabolism, confirming our conclusions regarding its anti-inflammatory action. Oxidative metabolism in the animal model of PD was sharply reduced compared to that of 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 as a sign of a functional defect of microglia. Administration of the test substance slightly but statistically significantly increased microglial oxidative metabolism, confirming our earlier suggestion that the 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 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 proinflammatory activation of extracerebral phagocytes and are also overexpressed by myeloid-derived inhibitory cells, negative regulators of proinflammatory responses of innate and adaptive immunity). According to the results of this study, the expression of all analyzed markers was increased in sham-operated animals, which is indicative of an activated state of microglia. Zinc preparation Administration of the product was accompanied by a decrease in the expression of all phenotypic markers under study. However, the number of CD206+ and CD80+ cells in this group of animals increased sharply. Apparently, this is also a marker of an enhanced repair process. Microglia originate from the yolk sac and the microglial pool settles in the brain early during embryonic development. This unique population of resident phagocytes maintains their numbers by self-healing (proliferation and differentiation of stem tissue elements) and does not need to mobilize immature bone marrow precursors from the circulation. The increased expression of any phenotypic marker is a sign of a cell differentiation process, allowing the conclusion that the increase in 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 (compared to sham-operated animals) expression levels of all the markers studied, which confirms our assumption of a functional defect of these cells caused by a prolonged inflammatory process. However, the fraction of CD206+ cells was very large, which could indicate any healing, reparative inflammation (it should be noted that the analysis of all phagocyte immunoreactivity indicators was performed at the end of the experiment, i.e., 28 days after the induction of the disease). Administration of the zinc preparation was accompanied by an increased expression of CD206, a marker of alternative (anti-inflammatory, reparative) activation of phagocytosis by microglia, 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 phenotypically differentiate the myeloid-derived suppressor cells.

[0453] Thus, 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 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 repair homeostatic effect of the test substances.

[0454] To characterize the function and phenotype of circulating phagocytes in an 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 a systemic inflammation that increases and maintains the perpetuation of the neuroinflammatory process. This situation makes the effector cells of the systemic inflammatory process a less attractive target for anti-inflammatory treatment in PD than the resident intracerebral inflammatory effectors. This was one of the reasons for analyzing the functional and phenotypic properties of circulating phagocytes. Another reason for the analysis was the fact that the zinc preparation was administered intravenously, which makes circulating phagocytes the first line of responding cells. As mentioned above, the results of blood cell counting showed the presence of a systemic inflammatory process in the LPS animal model of PD, with a significant neutrophilia and an increased neutrophil-lymphocyte ratio, a valid indicator of a systemic inflammatory response. The analysis of the functional and phenotypic properties 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, probably indicating an activation of hematopoiesis associated with the inflammatory repair process. Zinc did not have any effect on this parameter.

[0456] The phagocytosis 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 substances did not have any effect on this parameter. The oxidative metabolism of circulating phagocytes of 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 an anti-inflammatory character (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 animal models of PD were lower than in sham-operated rats. Considering the surgical procedure that requires activation of repair, the results obtained from these animals may indicate a deficiency / inhibition of hematopoiesis (myelopoiesis) caused by the progression of the disease. Zinc treatment was accompanied by a slight decrease in the relative number of circulating monocytes and did not result in any effect on the aforementioned granulocyte indices.

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

[0460] Analysis of the expression of phenotypic markers by circulating phagocytes in all groups of animals revealed the following (Figures 70A and 70B): expression of CD206 (a marker of 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 phenomenon revealed supports the abovementioned 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 classical 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 of markers of the population of myeloid-derived suppressor cells (found in the circulating blood of animals exhibiting a systemic inflammatory process) 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 hypothesized that the expression of these markers and the increase in the 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 shown by intact animals (and even below this level), 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 animal model of PD were significantly lower than in sham-operated animals, which indicates primarily 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 animal model of PD.

[0461] To characterize the functional and phenotypic properties 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, which are part of the 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 phagocytosis of peritoneal macrophages and the relative number of phagocytes in the peritoneal exudate of sham-operated animals were higher than in intact rats, which should be considered as the involvement of the mucosal immune system in the general inflammatory process (Figures 71A and 71B). The administration of the zinc preparation reduced the aforementioned values ​​to the level of intact animals, which indicates the homeostatic nature of its immunomodulatory effect. The values ​​of phagocytosis of peritoneal macrophages in the animal model of PD were at the level of intact rats and significantly lower than in sham-operated animals. Considering the physiological state of this group of animals, we can assume that there is no systemic inflammatory process of repair properties. The administration of the test substance contributed to the activation of the phagocytosis 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 the animal model of PD was significantly lower than in sham-operated animals. Administration of the zinc preparation 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 groups of experimental animals, which were partly different from those recorded for circulating cells. This could possibly be due to the different origin 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 the 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 substances compared with the phenotypic parameters of other localizations of 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 mainly exerts the function of a neurotransmitter that controls neuronal oscillatory activity, nociception, etc. Its role in controlling cerebral vascular tone and trophic mechanisms in nervous tissue is of great importance. The results of our study show that performing 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). The 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] Findings

[0471] In the LPS rat model of parkinsonism, there was no significant change in the mean number of rotations between the first and second apomorphine trials. However, an increase in the absolute number of rotations was observed in 66.6% of rats, and a decrease in the number of rotations was observed in 33.3% of rats. Immunohistochemical studies showed a 3-fold decrease in the number of TH-positive neurons in the midbrain of the LPS rat model compared to sham-operated animals.

[0472] Starting on day 9, animals were challenged with LPS to induce parkinsonism. 64 Although administration of Zn-asp did not cause any significant change in 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 intensity of the degenerative process of dopaminergic neurons in the substantia nigra. 64 This shows a 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 to 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 for 10 days. 64 Administration of Zn-asp did not cause any significant change in fecal water content 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 repair of the test substances.

[0477] The progression of LPS-induced parkinsonism is accompanied by prominent peripheral inflammation, as evidenced by monocytosis in combination 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 show a proinflammatory shift in their functional activity as well as their functional deficiencies caused by a long-lasting 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 with their proinflammatory activation. Administration of zinc preparations induces metabolic homeostatic changes in these cells with their proinflammatory metabolic shifts.

[0480] The progression of LPS-induced parkinsonism is accompanied by a reduction in the levels 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 show that PD progression can be caused by the activation of an inflammatory process in microglia, accompanied by a one-third decrease in the number of viable (tyrosine hydroxylase-expressing) dopamine neurons in the substantia nigra. This inflammatory process spreads throughout the body, covering the blood and extracerebral compartments of the immune system, such as MALT. Given the fact that the study of immune reactivity parameters was carried out at the end of the treatment course with the test substances, the inventors evaluated them in animals after a prolonged inflammatory process. The therapeutic administration of the zinc preparation (very large, after the fact of the recording of PD progression) led to 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 remarkable therapeutic effect of the zinc preparation was accompanied by a positive (anti-inflammatory) change in immune cells of various localizations, which indicates 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 gut microbiota as well, either direct effect (by affecting metabolism in the microbiota, which is highly dependent on the homeostasis of trace elements) or mediated effect (by affecting the ratio of aerobic and anaerobic conditions).The ability of the test substance to affect gut 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 prevalence makes the search for effective multi-targeted therapeutic agents a reality.

[0483] Although 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 is 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 patient a compound of the formula: [ka] (In the formula, R 1 From R 14 Each of the above is independently H, OH, F, Cl, Br, I, C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, and NO 2 and 85 Rb e At least 75% 85 Rb) of 85 Rb e Compounds and salts, and / or 64 Zinc e Compound or complex of Administering to a subject in need thereof a therapeutically effective amount of a composition comprising: (Item 2) The above 85 Rb e At least 90% 85 Rb, and / or 64 Zinc e At least 90% 64 Zn. (Item 3) R 1 、R 2 、R 4 ~R 6 、R 8 、R 10 、R 11 , and R 13 The method according to item 1 or 2, wherein all of (Item 4) R 3 But, H, CH 3 , O.C.H. 3 , and NO 2 Selected from R 7 and R 9 are independently H and OCH 3 Selected from R 12 and R 14 are independently H, Br, I, and NO 2 4. The method according to any one of items 1 to 3, wherein the method is selected from the group consisting of (Item 5) a)R 3 CH 3 and R 7 、R 9 、R 12 , and R 14 are all H, b)R 3 、R 7 、R 9 、R 12 , and R14 are all H, c)R 3 CH 3 and R 14 is Cl, and R 7 、R 9 , and R 12 are all H, d) R 3 CH 3 and R 14 is OH, and R 7 、R 9 , and R 12 are all H, e)R 14 is OH, and R 3 、R 7 、R 9 , and R 12 are all H, f)R 3 is OH, and R 7 、R 9 、R 12 , and R 14 are all H, g)R 14 No 2 and R 3 、R 7 、R 9 , and R 12 are all H, h)R 12 is Br and R 14 No 2 and R 3、R 7 , and R 9 are all H, i)R 3 and R 9 Both are OCH 3 and R 12 is Br and R 14 No 2 and R 7 is H, or j)R 3 and R 9 Both are OCH 3 and R 14 NO 2 and R 7 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) R 3 CH 3 and R 7 、R 9 、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) 9. The method of any of items 1 to 8, wherein the composition is administered to the subject intraperitoneally. (Item 11) 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 administration of 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 above 64 Zinc e Item 11. The method of any of the preceding items, wherein the compound or complex of is a portion of a zinc finger peptide. (Item 15) The method of any of the preceding items, wherein the subject is a human subject. (Item 16) The above 85 Rb e The compound was dissolved in 40 mg of water. 85 Rb e From 2400mg 85 Rb e The method of any of the preceding claims, wherein the compound is present in an amount equivalent to between (Item 17) The above 64 Zinc e of compound or complex is administered at a concentration of 0.1 to 1.5 mg of pure 64 Zinc e is in the range 64 Zinc e The method of any of the preceding claims, wherein the metal is present in an amount equivalent to a dose in the range. (Item 18) The above 64 Zinc e A compound or complex of (metal) is administered at a concentration of 1 to 15 mg of pure metal per kg of human body weight. 64 Zinc e 17. The method according to any one of items 1 to 16, wherein the range is

Claims

1. A composition for treating, preventing, or slowing the progression of α-synucleinopathy in a subject in need thereof, comprising a compound represented by the following formula: 【Chemistry 15】 (In the formula, R １ From R １４ Each of the above independently represents H, OH, F, Cl, Br, I, C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, and NO ２ and ８５ Rb ｅ At least 75% ８５ Rb) of ８５ Rb ｅ Compounds and their salts A composition comprising:

2. The above ８５ Rb ｅ At least 90% ８５ The composition of claim 1 , wherein R is Rb.

3. R １ , R ２ , R ４ ~R ６ , R ８ , R １０ , R １１ , and R １３ The composition of claim 1 or claim 2, wherein:

4. R ３ But, H, CH ３ , O.C.H. ３ , and NO ２ is selected from R ７ and R ９ are each independently H and OCH ３ is selected from R １２ and R １４ are each independently H, Br, I, and NO ２ The composition according to any one of claims 1 to 3, wherein the composition is selected from the group consisting of

5. a) R ３ CH ３ and R ７ , R ９ , R １２ , and R １４ are all H, b) R ３ , R ７ , R ９ , R １２ , and R １４ are all H, c) R ３ CH ３ and R １４ is Cl, and R ７ , R ９ , and R １２ are all H, d) R ３ CH ３ and R １４ is OH and R ７ , R ９ , and R １２ are all H, e) R １４ is OH and R ３ , R ７ , R ９ , and R １２ are all H, f) R ３ is OH and R ７ , R ９ , R １２ , and R １４ are all H, g) R １４ No ２ and R ３ , R ７ , R ９ , and R １２ are all H, h) R １２ is Br, and R １４ No ２ and R ３ , R ７ , and R ９ are all H, i) R ３ and R ９ Both are OCH ３ and R １２ is Br, and R １４ No ２ and R ７ is H, or j) R ３ and R ９ Both are OCH ３ and R １４ No ２ and R ７ and R １２ are both H, A composition according to any one of claims 1 to 3.

6. The composition of claim 1 , further comprising at least one excipient.

7. R ３ CH ３ and R ７ , R ９ , R １２ , and R １４ The composition of claim 1 , wherein all are H.

8. The composition of any one of claims 1 to 7, wherein the α-synucleinopathy is Parkinson's disease.

9. The composition of any one of claims 1 to 8, wherein the composition is administered intravenously to the subject.

10. The composition of any one of claims 1 to 8, wherein the composition is administered intraperitoneally to the subject.

11. The composition of any one of claims 1 to 8, wherein the composition is administered orally to the subject.

12. 12. The composition of any one of claims 1 to 11, characterized in that the composition is administered before, simultaneously with, or after a formulation comprising one or more other therapeutic agents for treating a neurodegenerative disorder.

13. 9. The composition of claim 8, wherein the composition is administered before, simultaneously with, or after administration of a formulation containing one or more other anti-Parkinson's agents.

14. The composition of any of claims 1 to 13, wherein the subject is a human subject.

15. The above ８５ Rb ｅ The compound was 40 mg ８５ Rb ｅ From 2400 mg ８５ Rb ｅ 15. The composition of claim 1, wherein the composition is present in an amount equivalent to between

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