Zinc Complexes, Their Production and Use

Abstract

The present invention relates to a novel zinc complex of γ-L-glutamylhistamine with a metal / ligand ratio of 1 / 1. In particular, the present invention relates to compounds of formula (I). The complex according to the invention promotes the restoration of the epithelial tissue barrier function and inhibits the abnormal activity of immune system cells. The present invention also relates to the production of the complex and the use of the zinc complex for the treatment of atopic dermatitis and other diseases associated with the disruption of the epithelial tissue barrier function and the occurrence of abnormal inflammatory responses. The present invention also relates to the use of the obtained complex for inhibiting glutaminyl cyclase. The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the invention. #imgabs0#

Description

Technical Field

[0001] The present invention relates to novel zinc complexes of γ-L-glutamylhistamine, which restore the barrier function of epithelial tissues and inhibit the abnormal activity of immune cells. The present invention also relates to the preparation of specific zinc complexes and their use for the treatment of atopic dermatitis and other diseases associated with impaired epithelial tissue barrier function and the occurrence of abnormal inflammatory responses. Background Art

[0002] The epithelial barrier plays a crucial role in human and animal organisms. The epithelial barrier prevents the penetration of various substances, allergens, and bacteria into the internal environment of the body. Impairment of the barrier function of epithelial tissues greatly contributes to the pathogenesis of several diseases (e.g., oral inflammatory diseases (stomatitis, gingivitis, pharyngitis, etc.), gastrointestinal diseases (colitis, enteritis, intestinal autointoxication, irritable bowel syndrome, malabsorption syndrome, recurrent diarrhea, etc.), allergic diseases (allergic rhinitis, bronchial asthma, atopic dermatitis, etc.)) [Recent Pat Antiinfect Drug Discov. 2015; 10(2): 84-97; Current Pediatrics. 2013; 12(2): 12-19; Bulletin of Siberian Medicine. 2017; 16(2) 32-46]. Given the key role of the epithelial tissue barrier function in the development of inflammatory and allergic diseases, strategies aimed at restoring the epithelial tissue barrier function are crucial for the treatment of many pathological conditions in humans. In particular, topical treatment is extremely important in the treatment of atopic dermatitis, which aims to restore the damaged epithelium, improve the skin barrier function, hydrate the skin, and prevent and eliminate secondary infections [Pediatrics. December 2014; 134(6): e1735-44].

[0003] Zinc is one of the key trace elements for maintaining skin barrier function. Therefore, its anti-inflammatory, antioxidant, and antibacterial properties make it one of the most commonly used trace elements in dermatology [FEMS Microbiol Lett. February 2008; 279(1): 71-76]. Zinc is an essential component of matrix metalloproteinases that control epithelial remodeling and is a key participant in epithelial cell growth, wound healing, and maintenance of skin barrier function [Front Biosci (Landmark Ed). March 1, 2017; 22: 1469-1492]. Recent studies have shown that the severity of atopic dermatitis symptoms is negatively correlated with the zinc concentration in the red blood cells of patients [Postepy Dermatol Alergol. October 2016; 33(5): 349-352]. In addition, the use of zinc compounds in preclinical and clinical studies has led to a reduction in the severity of atopic dermatitis symptoms [Biol Trace Elem Res. September 2017; 179(1): 110-116; Clin Cosmet Investig Dermatol. May 6, 2013; 6: 115-21; Acta Derm Venereol. September 2014; 94(5): 558-62]. In animal models of atopic dermatitis, the use of zinc compounds has been shown to restore the thickness of the epithelial tissue and reduce the severity of local skin inflammation [Dermatol Ther. July 17, 2018: e12659].

[0004] It should be noted that in many cases, impairment of the barrier function of epithelial tissue is associated with the occurrence of abnormal inflammatory responses, which in turn induce further cell damage and degradation of the epithelial barrier function. For example, in patients with atopic dermatitis, impairment of the skin barrier function leads to its colonization by opportunistic bacteria, including Staphylococcus aureus (S. aureus) [Br J Dermatol. December 1998; 139 Suppl 53: 13-6]. Bacterial contamination of the skin by opportunistic microorganisms leads to an excessive inflammatory response and the occurrence of abnormal chemotaxis of immune cells that produce pro-inflammatory cytokines (mainly lymphocytes, eosinophils, dendritic cells, and mast cells). Pro-inflammatory cytokines and reactive oxygen species secreted by immune cells induce further degradation of the skin barrier function, which serves as a self-maintaining positive feedback to ensure the progression of the disease to the chronic stage [J Clin Diagn Res. December 2013; 7(12): 2683-2685]. Therefore, inhibiting the abnormal activity of immune cells by suppressing their chemotaxis can be a key point in the treatment of atopic dermatitis and other diseases in humans and animals.

[0005] Chemokines of the CCL family (CCL2, CCL7, CCL8, CCL13) are potent factors for the chemotaxis of monocytes, macrophages, eosinophils, T lymphocytes, and dendritic cells in mammals [Biochem. J. March 1, 2012; 442(2):403-12; Postepy Dermatol. Alergol. May 2014; 31(2):84-91]. Members of the CCL family (CCL2, CCL7, CCL8, CCL13), fractalkine, and many other hormones and secreted proteins contain pyroglutamic acid (pE) residues, which serve to protect them from degradation by aminopeptidases [Chem Immunol. 1999; 72:42-56; Biochemistry. October 5, 1999; 38(40):13013-25]. The pyroglutamination of the N-terminal residue is catalyzed by the enzyme glutaminyl cyclase (QPCT or QC) [J Biol Chem. December 12, 2003; 278(50):49773-9; J Mol Biol. June 20, 2008; 379(5):966-80]. During the course of experimental studies, it has been shown that glutaminyl cyclase inhibition results in the non-pyroglutamylated forms of the chemokines CCL2, CCL7, CCL8, and CCL13 [Biochem. J. (2012) 442, 403-412] and fractalkine (Biosci Rep. August 23, 2017; 37(4)) with a sharp decrease in chemotactic activity. Thus, pyroglutamination of CCL family chemokines is a necessary step in CCL-mediated immune cell chemotaxis, and thus strategies aimed at inhibiting glutaminyl cyclase may represent possible strategies for modulating aberrant inflammatory responses and reducing immune cell activity.

[0006] To date, zinc complexes of glutaminyl cyclase inhibitors, their preparation and therapeutic use have not been described. At the same time, glutaminyl cyclase inhibitors are known, including thioesters [WO 2017 / 046256, published on March 23, 2017, HOCHSCHULE ANHALT, DE], flavonoid derivatives [Bioorg Med Chem. May 15, 2016; 24(10):2280 - 6], pyridine derivatives [US 2015 / 0291632, published on October 15, 2015, DowAgroSciences LLC, US] and some recently described small molecules [J.Med.Chem. March 23, 2017; 60(6):2573 - 2590; WO 2014 / 193974, US 2015 / 0291557]. Additionally, glutaminyl cyclase inhibitors are described in the publication of Probiodrug Aktiengesellschaft [J.Biol.Chem. December 12, 2003; 278(50):49773 - 9]. These articles describe glutaminyl cyclase inhibitors based on imidazole derivatives. However, the publication of Probiodrug Aktiengesellschaft does not include zinc complexes of glutaminyl cyclase inhibitors in the first place, and the structures of the compounds disclosed by Probiodrug Aktiengesellschaft contain imidazoles with aliphatic substituents at one of the nitrogen atoms. The introduction of the aliphatic substituent reduces the metabolic stability of the compound. Additionally, the presence of the aliphatic substituent increases the hydrophobicity of the compound and increases the systemic bioavailability of the compound, which is clearly not necessary for the treatment of atopic dermatitis and other diseases associated with impaired epithelial tissue barrier function.

[0007] To date, no drugs based on zinc complexes of glutaminyl cyclase inhibitors that can be used to treat atopic dermatitis or other diseases associated with impaired epithelial tissue barrier function and / or abnormal activity of immune cells are known, and thus there remains an unmet need for the development and therapeutic use of new and effective drugs based on zinc complexes of glutaminyl cyclase inhibitors.

[0008] The object of the present invention is to develop new and effective compounds that are effective in restoring the barrier function of epithelial tissue and capable of inhibiting glutaminyl cyclase in the treatment of atopic dermatitis or other diseases associated with impaired epithelial barrier function and abnormal activity of immune cells. Summary of the Invention

[0010] The object of the present invention is to develop new drugs that are effective for the treatment of diseases associated with impaired barrier function of epithelial tissues and abnormal activity of immune cells, in particular atopic dermatitis and other diseases.

[0011] The technical result of the present invention is to provide new compounds that are effective for the treatment of atopic dermatitis and other diseases associated with impaired barrier function of epithelial tissues and / or abnormal activity of immune cells.

[0012] The specific technical result is achieved by providing a compound of zinc complex of γ-L-glutamylhistamine (metal: ligand ratio = 1:1).

[0013] Based on the studies conducted and described in detail in this specification, the applicant proposes that the structure of the obtained complex can be described by the following structural formula:

[0014]

[0015] A specific zinc complex (Compound 1) or its hydrate, solvate or pharmaceutically acceptable salt is effective for the treatment of atopic dermatitis and other diseases associated with impaired barrier function of epithelial tissues and abnormal activity of immune cells.

[0016] Another technical result of the present invention is the use of the prepared zinc complex of γ-L-glutamylhistamine (Compound 1) or its hydrate / solvate for the prevention and / or treatment of atopic dermatitis and other diseases associated with impaired barrier function of epithelial tissues and abnormal activity of immune cells.

[0017] Another technical result of the present invention is the use of the new zinc complex of γ-L-glutamylhistamine or its hydrate, solvate or pharmaceutically acceptable salt for the preparation of a pharmaceutical composition for the prevention and / or treatment of conditions associated with abnormal activity of immune cells, in particular abnormal chemotaxis of immune cells.

[0018] In addition, the present invention provides a pharmaceutical composition for the prevention and / or treatment of atopic dermatitis and other diseases associated with impaired barrier function of epithelial tissues and abnormal activity of immune cells, which comprises an effective amount of the compound of the present invention and at least one pharmaceutically acceptable reagent / excipient. In some embodiments, the reagent is a pharmaceutically acceptable carrier and / or excipient.

[0019] The present invention also includes a method for the prevention and / or treatment of atopic dermatitis and other conditions associated with impaired barrier function of epithelial tissues and abnormal activity of immune cells in an organism, the method comprising administering to a subject a pharmaceutical composition according to the present invention. In a specific case of an embodiment of the present invention, the subject is a human or an animal.

[0020] The zinc complex of γ-L-glutamylhistamine has not been described in the prior art. However, the pseudopeptide of γ-L-glutamylhistamine was first isolated in trace amounts from rat and mollusk nerve tissues [J. Neurochem. - 1976. - Vol. 27. - pp. 1461 - 1463; J. Neurochemistry. - 1977. - Vol. 29. - pp. 633 - 638.]. The production of γ-L-glutamylhistamine is described in patent RU2141968 (published on November 27, 1999). The use of a cream based on γ-L-glutamylhistamine is described in patent RU 2233152 (published on July 27, 2004). Brief Description of the Drawings

[0022] Figure 1 . IR spectrum of a sample of γ-L-glutamylhistamine pseudopeptide (ligand).

[0023] Figure 2 . IR spectrum of a sample of the product prepared by mixing a solution of γ-L-glutamylhistamine and zinc chloride according to Example 1.

[0024] Figure 3 . IR spectrum of a sample of the zinc complex of γ-L-glutamylhistamine according to Example 2.

[0025] Figure 4 . IR spectrum of a sample of the zinc complex of γ-L-glutamylhistamine related to Synthesis "A" of Example 3.

[0026] Figure 5 . IR spectrum of a sample of the zinc complex of γ-L-glutamylhistamine related to Synthesis "B" of Example 3.

[0027] Figure 6 . 1H nuclear magnetic resonance spectrum of a sample containing the zinc complex of γ-L-glutamylhistamine according to Example 2.

[0028] Figure 7 . Pharmacological effect of a cream containing Compound 1 (0.01%) applied to skin lesions in a mouse model of atopic dermatitis according to histological analysis data.

[0029] Figure 8 . Tissue section of the affected mouse ear skin (animal model of atopic dermatitis). Detailed Description of the Invention

[0031] As described above, although the zinc complex of γ-L-glutamylhistamine has not been described in the prior art, the pseudopeptide γ-L-glutamylhistamine is known as a substance first isolated in trace amounts from rat and mollusk nerve tissues [J. Neurochem. - 1976. - Vol. 27. - pp. 1461 - 1463; J. Neurochemistry. - 1977. - Vol. 29. - pp. 633 - 638]. The preparation of γ-L-glutamylhistamine is also disclosed in patent RU 2141968. These works describe γ-L-glutamylhistamine as a pseudopeptide having antioxidant, free radical scavenging, lipid-regulating, hypoglycemic, anti-asthmatic, antiviral, antibacterial, anti-tumor, anti-inflammatory, anti-metastatic, adaptogenic effects, which is capable of regulating the metabolism of arachidonic acid and preventing the manifestations of diabetes, obesity, coronary heart disease, stress disorders, hepatitis, cirrhosis, toxic liver damage, alcoholism, radiation damage and geriatric changes.

[0032] At the same time, these works do not provide any data on the zinc complex of γ-L-glutamylhistamine, its preparation method, or research data on its biological activity. At the same time, it should be noted that the zinc complex of γ-L-glutamylhistamine cannot be obtained by simply mixing a zinc salt with γ-L-glutamylhistamine, because under these conditions, a mixture of the zinc salt and free γ-L-glutamylhistamine is formed.

[0033] Devoting to the present invention, the author / applicant has developed a method for producing a stable zinc complex of γ-L-glutamylhistamine with a metal / ligand ratio of 1 / 1.

[0034] (Compound 1)

[0035] During the study focusing on the specific pharmacological activity of the zinc complex of γ-L-glutamylhistamine (Compound 1) prepared by the applicant in a model of atopic dermatitis, it was shown that Compound 1 exhibited significantly higher therapeutic activity compared to the free ligand of γ-L-glutamylhistamine. Applying Compound I (0.01 wt%, cream) to the skin reduced the influx of inflammatory cells (monocytes, dendritic cells, eosinophils and neutrophils) into the epidermis and dermis of the skin to the level of normal non-injured animals, and reduced other microscopic manifestations of atopic dermatitis. Thus, it has been shown that Compound 1 affects the chemotaxis of immune cells. Reducing the influx of immune cells can be used to treat various diseases associated with abnormal activity of immune cells.

[0036] The research study conducted by the applicant showed that the observed therapeutic effect of Compound 1 is related to the ability of this compound to inhibit the activity of glutaminyl cyclase.

[0037] Therefore, Compound 1 (zinc complex of γ-L-glutamylhistamine with a metal / ligand ratio of 1 / 1) is a new compound that can be used to treat atopic dermatitis and other diseases associated with impaired epithelial tissue barrier function and abnormal activity of immune cells.

[0038] Terms and Definitions

[0039] The term "Compound I" refers to the zinc complex of γ-L-glutamylhistamine with a metal:ligand ratio of 1:1.

[0040] In particular, the complex or its pharmaceutically acceptable salts can be represented by the following structural formula:

[0041]

[0042] The term "°C" when used in reference to temperature means the centigrade or Celsius scale.

[0043] The term "IC 50 " represents the concentration of a test compound that results in 50% maximum inhibition of an enzyme.

[0044] The term "solvate" is used to describe a molecular complex that contains a compound of the present invention and one or more molecules of a pharmaceutically acceptable solvent (e.g., ethanol). When the specific solvent is water, the term "hydrate" is used.

[0045] The term "abnormal activity" of immune cells in this document refers to an activity that is significantly different from the baseline level of immune cell activity in a subject without a pathological condition. Abnormal activity can be caused by an excessive influx of immune cells into an organ or tissue, disruption of processes leading to immune cell activation, dysregulation of processes related to immune cell death, and other factors.

[0046] The term "excipient" means any pharmaceutically acceptable substance of inorganic or organic origin that is included in the composition of a pharmaceutical product or used in the manufacturing process, preparation of a pharmaceutical product to impart the necessary physicochemical properties.

[0047] The term "glutaminyl cyclase" refers to an aminotransferase that is involved in the conversion of N-terminal glutamine to pyroglutamine in a variety of peptide substrates. The formation of the N-terminal pyroglutamate moiety protects bioactive peptides, hormones, and chemokines (e.g., thyrotropin-releasing hormone, β-chemokine ligand-2) from degradation by exopeptidases and, in some cases, can increase the affinity of the ligand for its receptor.

[0048] The term "chemotaxis" refers to the directed movement of cells in response to a chemical stimulus. Chemotaxis is based on the ability of cells to respond to a concentration gradient of a chemotactic mediator. Chemotaxis is the process by which immune cells leave the vascular bed and migrate to damaged tissue. Chemotactic substances or chemoattractants play a dominant role in chemotaxis. The CCL2 chemokine is one of the most potent chemoattractants for monocytes and macrophages.

[0049] The term "treatment" encompasses treating a pathological condition in a mammal, preferably a human, and includes: a) improving, b) blocking (terminating) the disease process, c) reducing the severity of the disease, i.e., the onset of disease regression, d) reversing the disease or disorder to which the term is applied or one or more symptoms of the disease or disorder.

[0050] The term "prevention" or "prophylaxis" encompasses the elimination of risk factors, as well as prophylactic treatment in the subclinical stage of a disease in a mammal, preferably in a human, aimed at reducing the likelihood of the clinical stage of the disease occurring. Subjects for prophylactic treatment are selected based on factors known to be associated with: an increased risk of clinical disease compared to the general population. Prophylactic treatment includes a) primary prevention and b) secondary prevention. Primary prevention refers to prophylactic treatment / prophylactic measures for patients who have not yet reached the clinical stage of the disease. Secondary prevention is the prevention of recurrence of the same or similar clinical manifestations of the disease.

[0051] The subject compound I of the present invention shows promise for use in treating diseases associated with impaired barrier function of epithelial tissue and abnormal activity of immune cells, and in particular, for treating diseases associated with abnormal chemotaxis of immune cells, preferably for treating atopic dermatitis. In some specific embodiments, the compounds of the present invention can be used to treat other diseases caused by impaired barrier function of epithelial tissue and abnormal activity of immune cells.

[0052] Method for the therapeutic use of a compound

[0053] The subject matter of the present invention also includes administering a therapeutically effective amount of a compound of the present invention to a subject in need of appropriate treatment. A therapeutically effective amount is the amount of the compound administered or delivered to a patient in such a manner that the patient is most likely to exhibit the desired response to the treatment (prevention). The exact amount required may vary from subject to subject and depends on the age, weight and general condition of the subject, the severity of the disease, the method of drug administration, whether used in combination with other drugs, etc.

[0054] The compounds of the present invention or pharmaceutical compositions comprising said compounds can be administered to a subject in need thereof in any amount effective for treating or preventing a disease and by any route of administration effective for treating or preventing a disease (preferably a topical route of administration).

[0055] After mixing the compound with a specific suitable pharmaceutically acceptable carrier at a desired dose, the composition of the present invention can be topically administered to humans or other animals, etc.

[0056] Administration can be daily, once or several times a week (or by any other time interval), or from time to time as needed. In addition, the compound can be administered to the subject daily for a specified number of days (e.g., 2 to 10 days), followed by an interval period (e.g., 1 to 30 days) in which the drug is not administered.

[0057] In the case where the compounds of the invention are used as part of a combination therapy regimen, dosages of each of the combination therapy components are administered over the desired treatment period. The active ingredients constituting the combination therapy may be administered to the subject in a dose containing all components at once or in separate doses of the components.

[0058] Pharmaceutical composition

[0059] The present invention also relates to pharmaceutical compositions comprising a compound of the present invention (or a prodrug or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, diluents and / or excipients, such as those that can be co-administered to a subject in combination with the compound of the present invention under conditions that do not affect the pharmacological activity of the compound of the present invention and that are non-toxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0060] The present invention also relates to a method for producing a complex of zinc and γ-L-glutamyl histamine, the method comprising stirring a mixture of zinc acetate and γ-L-glutamyl histamine in a polar solvent, preferably in a polar solvent, separating the product precipitate and drying the resulting product, which represents a zinc complex of γ-Lβ-glutamyl histamine with a metal / ligand ratio of 1 / 1. Preferably, the solvent is methanol or water, or a combination thereof. The method preferably uses aqueous zinc acetate, in particular zinc acetate dihydrate. If methanol is used as a solvent, the stirring of the mixture is carried out at a temperature range of 15 to 50 degrees Celsius, preferably 15 to 40 degrees Celsius, or if water is used as a solvent, at a temperature of 15 to 90 degrees Celsius.

[0061] The compounds of the invention can be used in the form of their pharmaceutically acceptable salts. In particular, organic and inorganic acid addition salts known to those skilled in the art, such as hydrochlorides or acetates, can be prepared.

[0062] The pharmaceutical composition according to the present invention comprises the compound of the present invention and a pharmaceutically acceptable carrier, which may include any solvent, diluent, dispersant or suspending agent known to those skilled in the art, surfactants, isotonic agents, thickening agents and emulsifying agents suitable for specific dosage forms, preservatives, binders, lubricants, etc. Suitable substances that can be used as pharmaceutically acceptable carriers include: monosaccharides and oligosaccharides and their derivatives; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, sassafras oil, sesame oil, olive oil, corn oil and soybean oil; diols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic solutions, Ringer's solution; ethanol and phosphate buffer solutions. In addition, the composition may contain other non-toxic compatible lubricants (such as sodium lauryl sulfate and magnesium stearate) as well as coloring agents, film-forming agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants.

[0063] The subject matter of the present invention also includes dosage forms, namely a class of pharmaceutical compositions, the composition of which is optimized for a specific route of administration to an object in need thereof at a therapeutically effective dose, for example, for topical administration to an object.

[0064] The dosage forms of the present invention may include preparations prepared by liposome methods, microencapsulation methods, nano-form preparation methods or other methods known in the art.

[0065] For topical application, forms known to those skilled in the art can be used, such as ointments, creams and suspensions containing pharmacologically compatible agents such as propylene glycol or butylene glycol.

[0066] Some examples of pharmaceutical compositions

[0067] The compounds described in the present invention can be used in the form of the following compositions for preventing and / or treating diseases in humans or animals:

[0068] Ointment, ml

[0069] Active ingredient 40 mg

[0070] Ethanol 300 μl

[0071] Water 300 μl

[0072] 1-Dodecylazacycloheptanone 50 μl

[0073] Propylene glycol, up to 1 ml

[0074] Cream I, wt%

[0075] Active ingredient 0.005 - 0.5

[0076] Oil component 10.0 - 15.0

[0077] Glyceryl stearate for cosmetics 2.0 - 3.0

[0078] Emulsifier 1.0 - 3.0

[0079] Emulsion wax 1.0 - 3.0

[0080] Triethanolamine 0.1 - 0.6

[0081] Vitamin A 0.003 - 0.05

[0082] Distilled glycerol 2.0 - 3.0

[0083] Antioxidant 0.05 - 0.075

[0084] Preservative 0.15 - 0.25

[0085] Tea tree oil 0.3 - 0.5

[0086] Purified drinking water, up to 100%

[0087] Cream II, wt%

[0088] Active ingredient 0.01

[0089] Vegetable oil 15.0

[0090] Glyceryl stearate for cosmetics 0.75

[0091] Emulsifier PG - 3 2.25

[0092] Emulsion wax 2.25

[0093] Triethanolamine 0.1

[0094] Vitamin A 0.028

[0095] Sorbitol 2.0 - 3.0

[0096] Vitamin E 1.5

[0097] 90 - O18 0.75

[0098] Nipazole 0.2

[0099] Nipagin 0.3

[0100] Tea tree oil 0.3 - 0.5

[0101] Purified drinking water, up to 100%

[0102] Cream III, wt%

[0103] Active ingredient 0.1

[0104] Vegetable oil 15.0

[0105] Glyceryl stearate for cosmetics 0.75

[0106] Emulsifier PG - 3 2.25

[0107] Emulsion wax 2.25

[0108] Triethanolamine 0.1

[0109] Vitamin A 0.028

[0110] Sorbitol 2.0 - 3.0

[0111] Vitamin E 1.5

[0112] 90 - O18 0.75

[0113] Nipazole 0.2

[0114] Paraben 0.3

[0115] Tea tree oil 0.3 - 0.5

[0116] Purified drinking water, up to 100%

[0117] Cream IV, wt%

[0118] Active ingredient 0.3

[0119] Vegetable oil 15.0

[0120] Glyceryl stearate for cosmetics 0.75

[0121] Emulsifier PG - 3 2.25

[0122] Emulsion wax 2.25

[0123] Triethanolamine 0.1

[0124] Vitamin A 0.028

[0125] Sorbitol 2.0 - 3.0

[0126] Vitamin E 1.5

[0127] 90 - O18 0.75

[0128] Nipazole 0.2

[0129] Paraben 0.3

[0130] Tea tree oil 0.3 - 0.5

[0131] Purified drinking water, up to 100%.

[0132] In the case of ointments, the preferred oil components are vegetable oils, mineral oils, or mixtures thereof. Vegetable oils can be olive oil, sunflower oil, or a mixture of both. Mineral oil can be liquid petrolatum. Polyester of unsaturated fatty acids in glycerol (emulsifier PG - 3) can be used as an emulsifier. Vitamin A can be present as retinyl palmitate. The preferred antioxidant is tocopheryl acetate, and the preservative is at least one lower alkyl ester of p - oxybenzoic acid, such as methyl ester or propyl ester.

[0133] These preparations can be prepared according to standard pharmaceutical methods.

[0134] Use of Compound I in combination therapy

[0135] Although Compound I of the present invention can be administered as a single active ingredient, it can also be used in combination with one or more other medicaments. In particular, the other medicaments can be represented as antibiotics, NSAIDs (non - steroidal anti - inflammatory agents) or other anti - inflammatory agents, glucocorticoids, calcineurin inhibitors, anti - histamines, membrane stabilizers, immunomodulators, etc. When administered together, the therapeutic agents can be in different dosage forms administered simultaneously or sequentially at different times, or the therapeutic agents can be combined into a single dosage form.

[0136] The phrase "combination therapy" regarding the combination of the compounds of the present invention with other medicaments means administering all the medicaments that provide the beneficial effects of the drug combination simultaneously or sequentially. Co - administration means, in particular, co - delivering the medicaments, for example, in the same ointment, cream, tablet, capsule, injection, or other form with a fixed ratio of active substances, and delivering each compound simultaneously in several independent dosage forms.

[0137] Therefore, the administration of the compounds of the present invention can be combined with additional therapies known to those skilled in the art in the fields of preventive medicine and treatment of related diseases (including the use of antibacterial, cytostatic, and cytotoxic drugs, drugs that inhibit symptoms or side effects of a drug).

[0138] If the preparation is in dosage form, such a combination should contain the compound of the present invention within an acceptable dosage range. When it is not possible to combine these drugs, Compound I of the present invention can also be administered to the patient sequentially with other medicaments. The present invention is not limited to the order of administration; the compounds of the present invention can be co - administered to the subject before or after administering another drug.

[0139] Examples of embodiments of the present invention

[0140] Elemental analysis of samples

[0141] The elemental composition of the samples was studied using a Multi EA 5000 elemental analyzer from Analytik Jena. For this analysis, a weighed portion of approximately 20 mg (accurately weighed) of the sample was placed in an autosampler cell. After filling the equipment with an inert gas (helium), oxygen purified from nitrogen, carbon, and moisture was added, and the sample under study was combusted at a temperature of approximately 1000 °C. To remove the excess oxygen, the combustion products were passed through metallic copper at a temperature of 750 °C. Then, the mixture of gases (CO2, N2, and H2O) was passed through an adsorption trap to collect the moisture and determine the amount of hydrogen. Then, the mixture of nitrogen and carbon monoxide was introduced into a gas chromatographic column and separated into components, which were transferred by a carrier gas to a chemiluminescence detector CLD (to analyze the nitrogen content in the gas mixture) and an NDIR infrared detector (to analyze the carbon monoxide content in the gas mixture). The content of each of the determined elements in the test sample was calculated using the software package multiWin. 3 The IR spectra were recorded on an IFS-113v Bruker IR spectrometer in the range of 4000...400 cm

[0142] with a resolution of 1 cm -1 and used for evaluating the spectra. To prepare the sample, the zinc complex of γ-L-glutamylhistamine was ground with KBr (5 mg of the substance per 100 mg of KBr), pressed into a tablet, and used for evaluating the spectra. -1 Example 1 (not included in the present invention). Zinc chloride was mixed with γ-L-glutamylhistamine

[0143] This example shows that the zinc complex of γ-L-glutamylhistamine cannot be prepared by simply mixing the components (such as a zinc salt and γ-L-glutamylhistamine) because under these conditions, a mixture of the zinc salt and free γ-L-glutamylhistamine is formed. For example, an aqueous solution of zinc chloride was mixed with γ-L-glutamylhistamine in a ratio of 1:2 and ammonia water was added (until the medium was weakly basic), and subsequent precipitation of the product resulted in the formation of a white crystalline substance. The elemental analysis data (see Table 1) showed that the data values were close to the calculated values for the complex compound, but this data could not be regarded as evidence of complex formation. The underestimated contents of carbon and nitrogen in the sample indicated that the possible formation of zinc hydroxide (which partially lost water during analysis) could lead to the underestimation of the values of other elements. After additional washing with cold water, the repeated elemental analysis of the complex resulted in an even greater overestimation of the amount of hydrogen in the sample.

[0144] Table 1 - Elemental analysis data for the sample separated from the reaction of zinc chloride with γ-L-glutamylhistamine

[0145]

[0146] ​

[0147] In addition, according to the IR spectral data, the IR spectra of the original γ-L-glutamylhistamine and the mixed product are consistent (see Figure 1 and 2 ). The absence of displacement of the absorption bands of the C=O, -OH, and -NH groups and the consistency of their relative intensities indicate that in Example 1, zinc chloride was mixed with γ-L-glutamylhistamine, the original γ-L-glutamylhistamine was separated, and zinc was released in the form of an inorganic compound.

[0148] Example 2. Preparation of a zinc complex according to the present invention (Method No. 1)

[0149] In additional studies, the authors developed a method for obtaining a complex of zinc with γ-L-glutamylhistamine, which involves using an aqueous solution of zinc acetate.

[0150] Dissolve 2.20 g (0.01 mol) of Zn(CH3COO)2·2H2O in 20 ml of methanol and add it dropwise to a solution of 2.40 g (0.01 mol) of γ-L-glutamylhistamine and 1.08 (0.02 mol) of freshly prepared sodium methoxide in 50 ml of methanol with vigorous stirring at room temperature. After adding approximately half of the volume of the zinc acetate solution, the formation of a white precipitate was observed. After adding all of the zinc acetate solution, stirring was continued for about 4 hours, the product was filtered out, washed with water (4 × 10 ml), and dried in vacuo at 65 °C until its weight remained constant. Yield, 2.67 g (88%).

[0151] Two test runs of this technique for the implementation of the method were carried out, and samples for elemental analysis were obtained from each run. Based on the analysis of the elemental analysis data of these two samples obtained from the implementation of the method, a complex of zinc with γ-L-glutamylhistamine with a molar ratio of γ-L-glutamylhistamine / zinc of 1 / 1 was formed (see Table 2)

[0152] Table 2 - Elemental analysis data of samples separated from the reaction mixture of zinc acetate and γ-L-glutamylhistamine

[0153] C H N Measured value 1 39.17 4.73 18.02 Measured value 2 37.26 4.80 18.110 Calculated value 39.56 4.65 18.45

[0154] Subsequent complexometric titration of zinc was carried out to determine the complex composition and the obtained metal / ligand ratio. To determine the zinc content, the product was mineralized as follows: A sample of the product weighing approximately 70 mg was heated with five-fold amount of concentrated sulfuric acid until the sample was completely charred. Then, approximately 1.5 ml of 30 volume % hydrogen peroxide was added to the residue and boiled for about 10 minutes. The residue was transferred to a 25 ml conical flask and approximately 10 ml of ammonia buffer mixture (pH = 10) and a small amount of dried Eriochrome Black T indicator were added. The contents of the flask were mixed well until the indicator was completely dissolved and the solution turned raspberry color. The sample was titrated with 0.02 M EDTA solution until the deep red color changed via purple to bright blue. The final result showed that the zinc content in the prepared product was 21.5%, which was consistent with the theoretical calculated value (21.1%) of the zinc complex with a corresponding metal:ligand ratio of 1:1.

[0155] In the IR spectrum of the product (see Figure 3 ), two strong bands of the carbonyl group were observed: 1616 cm -1 and 1646 cm -1 . At the same time, the structure and absorption frequencies were significantly different from those in the spectrum of free γ-L-glutamylhistamine (see Figure 1 ): Due to the coordination with zinc, the band of the carboxyl group shifted to higher frequencies. Additionally, in the IR spectrum of the product, there was a strong absorption band at 3276 cm -1 , which was absent in the spectrum of γ-L-glutamylhistamine. It can be attributed to the vibration of the N-H bond in the amide fragment or the N-H bond in the imidazole system. Moreover, in the IR spectrum of the product, compared with the spectrum of γ-L-glutamylhistamine, there were various changes in the fingerprint region and the vibration of the C-H bond, indicating the absence of free γ-L-glutamylhistamine in the sample.

[0156] The obtained prepared product was insoluble in the solvent used for recording the NMR spectrum.

[0157] As an example, Figure 6 shows the D2O NMR spectrum recorded on a Bruker DRX500,13400 device, where the working frequency was 500.13 MHz.

[0158] It mainly contained the residual signals of the undehydrated solvent and low-intensity signals corresponding to the original γ-L-glutamylhistamine.

[0159] This indicated the absence of impurities in the aqueous suspension and insignificant dissociation of the obtained product.

[0160] Example 3. Obtaining a zinc complex according to the present invention (Method No. 2)

[0161] The aim of modifying this method according to the invention is to improve the filterability of the resulting product and to ensure the most uniform distribution of zinc hydroxide in the reaction mixture.

[0162] For scaling up, the issue of distribution uniformity is extremely important for this technology, because the formation of a complex of zinc and γ-L-glutamylhistamine on the surface of zinc hydroxide prevents the reaction from proceeding further. To solve this problem, it is proposed to obtain zinc hydroxide in situ by simultaneously adding sodium hydroxide and zinc acetate solution.

[0163] Synthesis "A": Using two funnels, simultaneously add dropwise to a suspension heated to 70 °C a solution of 12.0 g (0.05 mol) of γ-L-glutamylhistamine and 11.0 (0.05 mol) of zinc acetate dihydrate in 80 ml of water in 150 ml of water and 50 ml of 2M aqueous sodium hydroxide solution. Stir the reaction mixture at 70 °C for 4 hours, cool to room temperature and leave overnight. Filter off the precipitate, wash it on the filter with 3 × 50 ml of water, and dry it in vacuo at 45 to 50 °C until it reaches constant weight. The yield is 21.5 g (71% of the product).

[0164] The multi-amorphous complex obtained by this method is more easily separated in the form of a filtrate compared to the complex obtained using sodium methoxide in methanol according to Example 2.

[0165] This technology is scaled up and reproduced according to Synthesis "B".

[0166] Synthesis "B": Using two funnels, simultaneously add dropwise to a suspension heated to 70 °C a solution of 24.0 g (0.1 mol) of γ-L-glutamylhistamine and 22.0 (0.1 mol) of zinc acetate dihydrate in 120 ml of water in 300 ml of water and 100 ml of 2M aqueous sodium hydroxide solution. Stir the reaction mixture at 70 °C for 4 hours, cool to room temperature and leave overnight. Filter off the precipitate, wash it on the filter with 4 × 50 ml of water, and dry it in vacuo at 45 to 50 °C until its weight remains constant, to 44.1 g (71% of the product).

[0167] Two test runs of this method were carried out, and samples for elemental analysis were obtained from each run. According to the analysis of the elemental data of these two samples, as a result of the reaction, a complex of zinc and γ-L-glutamylhistamine with a molar ratio of γ-L-glutamylhistamine / zinc of 1 / 1 was formed (see Table 3).

[0168] Table 3 - Results of elemental analysis of the zinc complex of γ-L-glutamylhistamine (Method No. 2)

[0169]

[0170] The IR spectrum of the obtained product (see Figure 4 and 5 ) is similar to that of the zinc complex of γ-L-glutamylhistamine synthesized by Method No. 1, and there are no significant differences in these spectra in the fingerprint region.

[0171] Complexometric titration of zinc was carried out to determine the complex composition and the obtained metal / ligand ratio (see Table 4). As a result, it was shown that the zinc content in the product obtained by Method No. 2 was about 22.0%, which is consistent with the theoretical calculated value (21.1%) of the zinc complex with a corresponding metal:ligand ratio of 1:1.

[0172] Table 4 - Complexometric titration results of the zinc complex of γ-L-glutamylhistamine

[0173]

[0174] Characterization of the biological activity of the complex of Compound 1

[0175] The biological activity of Compound 1 of the present invention has been studied in multiple in vitro and in vivo experiments. In particular, in the experiment focusing on the activity of Compound 1 in an in vivo model of atopic dermatitis, the inhibitory effect of Compound 1 on immunocyte chemotaxis was shown. The study of the in vitro biological activity of Compound 1 has determined that Compound 1 is an inhibitor of the enzyme glutaminyl cyclase. Therefore, the effect of Compound 1 on immunocyte chemotaxis can be mediated by inhibiting the activity of glutaminyl cyclase.

[0176] Example 4. Study on the effect of Compound 1 on the enzymatic activity of human glutaminyl cyclase in vitro.

[0177] In the study on the effect of Compound 1 (which is the subject of the present invention) on the enzymatic activity of glutaminyl cyclase in vitro, a direct inhibitory effect of Compound 1 on recombinant intracellular human glutaminyl cyclase was found.

[0178] The activity of glutaminyl cyclase at various Compound 1 concentrations was studied at 25 °C using the fluorescent substrate L-glutaminyl 2-naphthylamide (Gln-bNA) [Anal Biochem. April 1, 2002; 303(1): 49 - 56]. The reaction mixture with a volume of 100 μl contained 50 μM of the fluorescent substrate; approximately 0.2 unit of human pyroglutamyl aminopeptidase (1 unit is defined as the amount of hydrolyzing 1 micromole of pGlu-bNA per minute) and an aliquot of recombinant intracellular human glutaminyl cyclase (gQC) in 50 micromoles of trisaminomethane HCl and 5% glycerol (pH 8.0). The reaction was started by adding an aliquot of glutaminyl cyclase that had been incubated with Compound 1 for 5 minutes to the reaction mixture.

[0179] The further progress of the reaction was monitored spectrophotometrically (excitation and emission wavelengths were 320 and 410 nm). The enzymatic activity was determined by the amount of 2-naphthylamide (bNA) released, calculated based on the calibration curve. The IC50 value was calculated using non-linear regression of the "inhibitor concentration" - "enzyme activity" curve.

[0180] The experiment showed that compound 1 inhibited the activity of glutaminyl cyclase, with an IC50 = 26 μM.

[0181] Example 5. Study on the activity of compound 1 in a murine atopic dermatitis model

[0182] Experimental techniques. The study on the activity of compound 1 in the atopic dermatitis model was carried out using standard techniques [Evidence-based Complementary and Alternative Medicine. 2012. Article ID 545497, 9 pages]. For the experimental group, we selected animals with no signs of deviation in average appearance, where the body weight of the animals was no more than ±20% from the average within the sex.

[0183] On days 0 and 12 of the experiment, male mice of the balb / c strain were treated with a 100 μl 2% solution of 1-chloro-2,4-dinitrobenzene (DNCB) in ethanol applied to the previously shaved back area to sensitize the body. On day 17, 20 μl of a 2% DNCB ethanol solution was applied to the "tested" right ear of the animals twice at 1-hour intervals. The compound 1 cream with an active substance content of 0.01 wt% was topically applied to the "tested" right ear twice: 1 and 13 hours after the last application of DNCB to the ear. The following substances were used as comparative / reference / control drugs: aceproclomazone (0.1% topical ointment), zinc pyrithione (0.2% topical cream), pimecrolimus (1% topical cream), and a topical cream containing 0.01% γ-L-glutamylhistamine. On day 18, the animals were euthanized and histological analysis of the affected ears was performed. Tissue sections with a thickness of 5 μm were stained with hematoxylin-eosin. The microscopic changes showing dermatitis were evaluated according to the following scale:

[0184] 1) Acanthosis - thickening of the epidermis and epithelium and elongation of the interpapillary projections:

[0185] 0 points - no pathological condition;

[0186] 0.5 points - pathological condition detected, but very weak expression;

[0187] 1 point - moderate to severe pathological condition;

[0188] 2 points - severe pathological condition.

[0189] 2) Hyperkeratosis is a non-inflammatory feature of skin disorders, characterized by a marked thickening of the stratum corneum or a delay in its normal shedding:

[0190] 0 points - No pathological condition;

[0191] 0.5 points - Pathological condition detected, but expression is very weak;

[0192] 1 point - The indicated pathological condition is clearly present.

[0193] 3) Pustule (abscess) - An acute inflammatory element of a cavity with purulent content:

[0194] 0.5 points - A single manifestation of the pathological condition;

[0195] 1 point - Mild pathological condition;

[0196] 2 points - Moderate to severe pathological condition;

[0197] 3 points - Severe pathological condition;

[0198] 4 points - All pathological conditions.

[0199] 4) A cyst is a round or oval structure lined with stratified squamous keratinized epithelium and filled with a stratum corneum mass, which occurs in the presence of epithelial hyperplasia (acanthosis):

[0200] 0 points - No pathological condition;

[0201] 0.5 points - Pathological condition detected, but expression is very weak;

[0202] 1 point - The indicated pathological condition is clearly present.

[0203] 5) Inflammation:

[0204] 0 points - No pathological condition;

[0205] 0.5 points - A single manifestation of the pathological condition;

[0206] 1 point - Mild pathological condition;

[0207] 2 points - Moderate to severe pathological condition;

[0208] 3 points - Severe pathological condition;

[0209] 4 points - All pathological conditions.

[0210] 6) Edema:

[0211] 0 points - No pathological condition;

[0212] 0.5 points - A single manifestation of the pathological condition;

[0213] 1 point - Mild pathological condition;

[0214] 2 points - moderate to severe pathological conditions;

[0215] 3 points - severe pathological conditions;

[0216] 4 points - all pathological conditions.

[0217] The obtained data were verified using the Grubbs test (also known as the maximum normalized residual test, which is a test for detecting outliers in a dataset). Values identified as "outliers" in this test were not used for further analysis. Descriptive statistics were used for all data: the mean (M) and the standard error of the mean (m) were calculated. The normal distribution of the values obtained in the experiment was verified using the Kolmogorov - Smirnov test. In the case of a normal distribution, the Student’s t - test was used to evaluate the differences between groups. In the case of a non - normal distribution, the Kruskal - Wallis test (together with Dunn’s post - hoc analysis) was used to compare several groups. Statistical analysis was performed using GraphPad Prism software 5.0. Differences were determined at the 5% confidence level.

[0218] The experimental results are shown in Figure 7 and 8 . From the data shown in Figure 7 , it can be seen that, according to histological analysis, the pharmacological effects of the cream containing compound 1 (0.01 wt%) on skin lesions in a murine atopic dermatitis model were evaluated as follows: a) acanthosis (score), b) hyperkeratosis (score), c) pustules (score), d) cysts (score), e) inflammation (score), f) edema (score). The experiment was conducted according to Evidence - based Complementary and Alternative Medicine. 2012. Article ID 545497, 9 pages. Note: * - significance of the difference from the complete group (P < 0.05), & - significance of the difference from the control (P < 0.05).

[0219] Figure 8 A histological section of the affected ear skin in a murine atopic dermatitis model stained with hematoxylin - eosin is shown, with a magnification of ×20.

[0220] Group A - intact animals; Group B - pathological control; Group C - Advantan (cortivazol, 0.1% ointment); Group D - pimecrolimus (Elidel 1% cream); Group E - Compound I cream 0.01%. 1 - epidermis; 2 - dermis; 3 - inflammatory infiltration. Experiments were carried out according to Evidence-based Complementary and Alternative Medicine. 2012. Article ID 545497, page 9.

[0221] Application of Compound 1 (0.01 wt% cream) to the skin reduced the influx of inflammatory cells (monocytes, dendritic cells, eosinophils, and neutrophils) into the epidermis and dermis of the skin and other microscopic manifestations of atopic dermatitis to the level of intact animals. Notably, in terms of action, Compound 1 (zinc complex of γ-L-glutamylhistamine) showed a significant improvement compared to the action of the γ-L-glutamylhistamine ligand. Thus, the therapeutic activity of Compound 1 cannot be explained solely by the activity of γ-L-glutamylhistamine. In terms of potency, Compound 1 cream showed superior performance compared to the reference drugs: cortivazol, zinc pyrithione, and pimecrolimus. The results obtained led us to conclude that Compound 1 has shown an improved therapeutic effect in the treatment of atopic dermatitis.

[0222] Conclusion

[0223] Therefore, the studies conducted have shown that Compound 1 is effective in inhibiting glutaminyl cyclase. In the atopic dermatitis model, Compound 1 has shown in vivo inhibition of the influx of inflammatory cells (monocytes, dendritic cells, eosinophils, and neutrophils) and other microscopic manifestations of atopic dermatitis.

[0224] Although the present invention has been described with reference to some of the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the essence of the invention.

Claims

1. A complex of zinc with γ-L-glutamylhistamine or a pharmaceutically acceptable salt thereof, the chemical formula of the complex being 2. A method for preparing the complex of zinc with γ-L-glutamylhistamine according to claim 1, which comprises: Stirring a mixture of aqueous zinc acetate and γ-L-glutamylhistamine in a polar solvent; Precipitating the product; And Drying the obtained product, which is a complex of zinc with γ-L-glutamylhistamine having a metal:ligand ratio of 1:

1.

3. The method according to claim 2, wherein the solvent is methanol or water, or a combination thereof.

4. The method according to claim 2, wherein the aqueous zinc acetate is zinc acetate dihydrate.

5. The method according to claim 3, wherein when methanol is used as the solvent, the mixing of the mixture is carried out at a temperature of 15 to 50 °C, and wherein when water is used as the solvent, the mixing of the mixture is carried out at a temperature of 15 to 90 °C.

6. The method according to claim 5, wherein the mixing of the mixture is carried out at a temperature of 15 to 40 °C and methanol is used as the solvent.

7. Use of the complex according to claim 1 for the preparation of a medicament for the treatment of atopic dermatitis.

Images