RU2766340C1 - Peptide tolerogenic compound - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology and discloses an anti-inflammatory preparation based on mammalian spleen peptides, wherein the preparation is presented in the form of a dermal water-alcohol lotion of systemic anti-inflammatory action, containing a fraction of purified peptides of spleen of mammals with molecular weight of not more than 12 kDa in amount of 1÷5 wt. % to the total weight of the composition, 96 % ethanol in amount of 40 wt. %, sodium chloride in amount of 0–0.5 wt. %, zinc chloride in amount of 0–0.05 wt. %, calcium sulphate in amount of 0–0.2 wt. %, water is the rest, at pH 7.4 ± 0.2. Group of inventions also relates to a method of suppressing the inflammatory response and to the use of an anti-inflammatory preparation.

EFFECT: achieving anti-inflammatory therapeutic effect with underlying permanent forced overexpression of the most powerful pro-inflammatory cytokine - tumour necrosis factor (TNF-alpha - TNFα), that is, the preparation according to the invention provides not only an anti-inflammatory effect, but also exhibits tolerogenic action - initiates specific tolerance of T-regulatory cells to body own antigens, which causes its effectiveness in therapy of autoimmune inflammatory diseases; more effective stimulation of immature (naive) monocytes to Treg production, compared to cytokines GM-CSF + M-CSF, which are used in the classical Treg induction technique and can be present in peptide fractions from the spleen with molecular weight of more than 12 kDa, specifically about 30 kDa for GM-CSF and about 60 kDa for M-CSF, but are absent in the disclosed preparation based on the spleen peptide fraction with molecular weight of not more than 12 kDa; achieving systemic anti-inflammatory therapeutic action when using the preparation in the form of a water-alcohol lotion when applied to the skin, which causes a decrease in the concentration of pro-inflammatory cytokines TNFa, IL-1b and IL-2 in the blood; prevention of development of psoriatic and rheumatoid arthritis with efficiency of about 70 % due to systemic tolerogenic anti-inflammatory therapeutic action; achieving a tolerogenic anti-inflammatory therapeutic effect in a form suitable for out-of-hospital use; achieving systemic tolerogenic anti-inflammatory therapeutic action without undesirable side effects - allergic reactions and suppression of adaptive immunity.

14 cl, 2 tbl, 8 ex, 10 dwg

Description

The proposed invention relates to biotechnology, namely to the production of an anti-inflammatory agent based on a biologically active peptide fraction from the spleen of mammals. The drug in the form of a water-alcohol lotion can be used in medicine and veterinary medicine as an anti-inflammatory agent to prevent the development of inflammation in autoimmune and inflammatory diseases.

A method has been developed for obtaining a unique composition of an anti-inflammatory drug with the following characteristics:

Dosage form: water-alcohol solution of purified mammalian spleen peptides in the range of molecular weights up to 12 kDa with a concentration of at least 1% (lotion) for skin application. The medicinal lotion is produced using an original technology that allows unlimited scaling by eliminating critical process and hardware methods, namely, acetone, chromatography, dialysis, lyophilization and ultrafiltration. The absorption efficiency of peptides of intact human skin of about 60% in the presence of 40% ethanol was demonstrated for the first time in vivo using chromatographic analysis of residual material on the skin.

Physiological action: purified peptides of not more than 12 kDa from the spleen of mammals in the presence of ethanol penetrate the keratin layer of the skin 10-15 μm, reach the next basal layer with Langerhans cells located in it (the primary biological target) and direct the differentiation of Langerhans cells according to the tolerogenic method, which then migrate to the lymph nodes and initiate specific tolerance of T-regulatory cells to the body's own antigens, which ensures the manifestation of an anti-inflammatory effect.

The physiological action of spleen peptides was first deciphered using a genetically modified in vivo mouse model with forced expression of tumor necrosis factor (TNF-alpha) and in experiments with mixed cultures of dendritic cells, T-lymphocytes and immature (naïve) monocytes in vitro .

With skin application of the proposed drug, spleen peptides trigger a systemic therapeutic process with the achievement of a tolerogenic anti-inflammatory effect, which was demonstrated in vivo in mammals with rheumatoid arthritis (dogs) and psoriatic arthritis (mice).

Prior Art

Known anti-inflammatory drugs

Anti-inflammatory and immunosuppressive agents such as glucocorticosteroids, NSAIDs, cytostatics and specific anti-inflammatory monoclonal antibodies are symptomatic agents, i.e. they do not cure the disease, but only mitigate its manifestation.

Long-term use of all the drugs described is accompanied by side effects, which over time often become more threatening than the diseases themselves. Recently, anti-inflammatory monoclonal antibodies have shown hope, but they also have serious side effects that increase over time - allergic reactions, flu-like syndrome (fever, malaise, muscle pain), nausea, diarrhea and other dyspeptic disorders, a decrease in the number of blood cells - platelets, leukocytes, erythrocytes, as well as cardiotoxic effects. In addition, the high cost of monoclonal antibody preparations makes them practically inaccessible for mass use.

Non-hormonal anti-inflammatory drugs - NSAIDs, such as aspirin, ibuprofen, paracetamol and others have moderate side effects, mainly affect the gastrointestinal tract (NSAID-gastropathy), but are of little effectiveness. When applied externally, they can cause irritation of the skin in the form of redness, swelling, rashes or itching; with prolonged use - systemic adverse reactions.

Glucocorticosteroid drugs - natural adrenal hormones or synthetic analogues of anti-inflammatory hormones, such as cortisol, hydrocortisone, prednisolone, dexamethasone and others have a strong anti-inflammatory effect, but suppress the immune system and have severe, often irreversible side effects with long-term use. Systemic side effects when taking glucocorticosteroids are manifested from the central nervous system (psychosis, depression, increased nervous excitability, and others), from the cardiovascular system (thromboembolism, increased blood pressure, myocardial dystrophy, and others), from the reproductive system (hirsutism, delayed sexual development and others), from the digestive system (fatty liver, pancreatitis, bleeding from the gastrointestinal tract, steroid ulcers of the intestine and stomach), from the endocrine system (diabetes mellitus, Cushing's syndrome, obesity, atrophy of the adrenal cortex due to inhibition of its function) and other body systems. Also, during therapy with glucocorticosteroid drugs, exacerbations of infectious and inflammatory processes of the chronic form are observed.

Cytostatics, such as methotrexate and others, also have a strong specific effect and severe irreversible side effects with long-term use.

Monoclonal antibodies to anti-inflammatory cytokines and/or their receptors, as noted above, have a strong specific effect, but are extremely expensive and eventually lose their effectiveness due to the appearance of secondary antibodies in the patient's body.

The spleen of mammals was previously identified as the most promising source of the active principle for the treatment of autoimmune and inflammatory diseases. Known injection drug "Splenopid", developed by A. B. Tsypin and co-authors in 1998 [1], obtained in the form of an ultrafiltrate extract of porcine spleen on a membrane with a cut-off of 50 kDa, with which it was possible to cure a number of autoimmune and inflammatory diseases. Also known is an earlier patent by V.A. Chernov and co-authors in 1993 [2], where the protein-peptide compound of bovine spleen in the range of 10-140 kDa was used to treat canine distemper.

However, both drugs have 4 critical drawbacks:

(1) In the range up to 50 kDa and, moreover, up to 140 kDa, almost all known effectors (cytokines, chemokines, interferons) of both pro-inflammatory and anti-inflammatory nature are present. For this reason, such preparations, in principle, cannot be unambiguously characterized and cannot be assigned to the category of medicines, despite the declared positive physiological effect.

(2) Both preparations are extremely dirty in their composition and, in addition to potential effector molecules, contain more than 80% of ballast components of a protein, polynucleotide, polysaccharide and other nature.

(3) Their injectable use is unacceptable not only because of the prevalence of ballast, but also because biopolymers from xenobiotic organs and tissues over 20-30 kDa are immunogenic for humans, which is fraught with the production of autoimmune antibodies (for example, to minimize the appearance of secondary autoimmune antibodies, therapeutic monoclonal antibodies are specifically "humanized"). The authors of both patents did not test the transdermal form, but with the described composition, the transdermal form is also dangerous for the occurrence of autoimmune dermatitis after repeated use.

(4) Both drugs have not been studied in any way at the molecular-cellular level, their biological targets have not yet been known.

Moreover, in the patent and medical literature, the authors did not find a single example where the primary biological target would be determined and the mechanism of action of splenic and other natural low molecular weight peptides with a molecular weight less than 10-12 kDa would be deciphered. This applies to all known commercial peptide preparations from natural sources (Cortexin, Polyerga, Splenopid, Samprost, Timalin, Taktivin, Pinalex, etc.).

Also, no anti-inflammatory drugs based on spleen peptides of systemic anti-inflammatory action in the transdermal (cutaneous) dosage form were found.

Transdermal drug delivery

It would seem that transdermal drug delivery theoretically allows the drug to be administered in an accessible non-invasive way. Due to ease of use and self-administration, transdermal delivery should be characterized by a rate-controlled release of the drug, unique for such a desirable route [3,4].

However, despite the impressive breadth of research in this area [5], the market for systemic therapeutics using passive non-invasive transdermal transfer is still minor.

Substances that can be delivered transdermally, namely nicotine, corticosteroids, scopolamine and others, are small and hydrophobic molecules. Bypassing natural barriers to allow drug penetration is a coveted step towards successful transdermal delivery [6]. Therefore, attempts to advance in this direction continue constantly, namely, transdermal delivery technologies are being developed: using a nanoemulsion [7], using transferosomes [8], [9], using nanoparticles [10], using an ethylcellulose/polyvinylpyrrolidone glipizide matrix, and Eudragita RL-100/Eudragita RS-100 [11]), using iontophoresis [12], using electroporation [13]). There is a method [14], where ethanol is indicated in the composition of skin products at a concentration of 0.01 to 60%, but this application considers only low molecular weight substances up to 0.5-0.6 kDa and there is no mention of peptides. On the whole, despite numerous attempts, there are no methods of passive deep delivery of peptides to the vascularization layer so far in real practice.

The only effective method of transdermal administration of protein hormones and peptides is the technique using patches with soluble microneedles [15, 16, 17, 18, 19]. The technology is aimed at community use, but it goes beyond the topic of non-invasive passive transdermal administration and is a high-tech development of injection methods.

Bioavailability and biological target

It seems not entirely logical that the inventors of transdermal methods for the delivery of active proteins and peptides did not set themselves the task of identifying the primary biological target, i. effector cell. A likely reason is the fact that the term "bioavailability" is generally understood to mean the ability of a transdermal drug to reach the capillaries of the systemic circulation, although in some cases this is not a necessary and sufficient requirement.

Anatomically, the skin consists of three main layers. The outermost part and the first line of defense against the penetration of foreign substances is the stratum corneum. This layer mainly consists of dead cells (keratinocytes), has a thickness of approximately 10–15 µm, and is surrounded by a lipid extracellular matrix [20]. Under the stratum corneum there is a viable epidermis (basal and spiny layer in total), the thickness of which is approximately 50-100 microns. Taken together, these three layers are called the complete epidermis. Beneath the complete epidermis is the first layer of vascularity present in the layer known as the dermis. It is a fibrous layer, approximately 1-2 mm thick, consisting of capillary layers, which are the site of drug entry into the circulation [21] if the drug is able to reach them. However, it has been repeatedly experimentally proven that the skin does not let molecules exceeding 500 Da [22] deeper than the basal layer [22], especially for molecules of a hydrophilic nature [23].

The authors of the proposed invention set the task to find out whether there are effector cells in the upper layers of the skin that can be affected in a therapeutic sense. In the skin directly under the keratin layer, i.e. in the basal and spinous layers, there are dendritic cells (Langerhans cells). The critical point is that Langerhans cells do not exist as a permanent epidermal class of dendritic cells. They migrate to the epidermis and back to the lymph nodes, where they transport "captured" foreign agents, such as antigens, and present them to lymphocytes. This position was demonstrated in a series of fundamental works by GFMurphy et al., performed since 1986 [24], where the transformation of dermal actively phagocytic macrophages into epidermal Langerhans cells through intermediate forms was shown, which combined both the properties of monocyte-macrophages and typical dendritic cells.

It is Langerhans cells that take part in the induction of immune tolerance to the skin's own antigens, which are released during injuries and inflammatory processes, linking two organs into a single functional system - the skin and lymph nodes [25].

Based on the analysis of these data, the authors of the present invention concluded that if the term "bioavailability" means the ability of a drug substance to reach a biological target, i.e. effector cell, then among all peptide candidates for the role of a transdermal anti-inflammatory agent with passive transfer, it is logical to preselect those that directly interact with Langerhans cells.

Methods for assessing the absorption of peptides by intact skin in vivo

Known methods for evaluating the effectiveness of transdermal delivery in vitro (more correctly, evaluating the effectiveness of skin absorption) allow you to control experimental variables using the simplest protocols. In vitro assessments are expected to be important tools for developing and screening formulations that predict in vivo skin absorption [26, 27, 28, 29, 30, 31]. However, the dissemination of the results of this approach, incl. using modifications of the Franz cell [32], the efficiency of peptide transfer through uninjured skin in vivo is controversial.

To better quantify the penetration of substances into the skin in vivo , methods are used that are practically inaccessible for routine analysis, namely: attenuated infrared total reflection spectroscopy with Fourier transform [33, 34] and confocal laser scanning microscopy (CLSM) as a modification of fluorescence microscopy [35 , 36, 37].

A review of methods for assessing drug absorption by the skin in vivo shows that this is the most methodologically difficult problem [38, 39, 40, 41, 42]. At the same time, unreasonably exotic ones are used, incl. microtraumatic procedures such as skin microdialysis, stripping adhesive tape, thermal exposure, etc., described in detail in the review [43]. As a result, it became necessary to develop our own original correct method of evaluation.

Obtaining peptide drugs from natural sources

Known methods for producing peptide drugs, cosmetics and biologically active additives from organs and tissues of mammals, for example: SU 944191 [48], Patent RU 2104702 [49], Patent CN 1557465A [50]. Common features of the known methods for obtaining peptide drugs is the use of a significant amount of acetone and/or ultrafiltration and/or chromatography and/or lyophilization, which is unacceptable for mass production in the required range of therapeutic courses, millions or more doses of the drug.

As the closest analogue, the anti-inflammatory drug "Splenopid" is proposed [1]. The drug is intended for intramuscular administration and is a peptide fraction from the spleen of cattle or pigs with a molecular weight of 0.4-50 kDa in the form of a lyophilisate with gelatinol and gentamicin, the therapeutic effect of which is shown in rodents and humans. "Splenopid" has a pronounced immunocorrective effect in various pathologies associated with dysfunction of the immune system, stimulates the metabolic and functional activity of peripheral blood leukocytes, promotes the elimination of autoimmune complexes from the body in systemic lupus erythematosus, which is associated with an increase in the activity of phagocytic cells and a decrease in the inflammatory response.

As noted above, Splenopid has a number of disadvantages, of which at least two of the most significant stand out:

- due to the presence in the composition of the drug of an indefinite mixture of biopolymers in the range up to 50 kDa, including effectors of both pro-inflammatory and anti-inflammatory nature, such a drug, in principle, cannot be unambiguously characterized, which does not allow it to be classified as a drug;

- due to the presence in the composition of the drug, in addition to potential effector molecules, more than 80% of ballast components of protein, polynucleotide, polysaccharide and other nature from xenobiotic organs and tissues over 20 kDa, repeated injection use is unacceptable, since it is fraught with the production of autoimmune antibodies.

The proposed invention solves the technical problem of the lack of an effective peptide anti-inflammatory agent in the form of a transdermal preparation, which can be obtained in a simple way suitable for mass production.

The technical task is to develop a composition of a therapeutically effective peptide anti-inflammatory agent with minimization of side effects, suitable for community use, as well as a method for its preparation.

Disclosure of the essence of the invention

The authors of the proposed invention set the task of finding a biologically active substance or molecular compound suitable for the stable treatment of autoimmune and inflammatory conditions without side effects characteristic of glucocorticosteroids, NSAIDs, cytostatics and specific anti-inflammatory monoclonal antibodies.

At the same time, the following 4 restrictions were guided in advance:

1. The active substance or molecular compound must be of natural origin. This is the cheapest and unlimited source of raw materials, which is important for mass production. In addition, synthetic compounds, such as peptides, do not take into account the necessary natural requirements, such as glycosylation, phosphorylation, branching, formation of ring forms, synergistic action in the complex (compound). The latter statement is justified by the fact that synthetic substances have not yet been created that stably cure autoimmune and inflammatory diseases.

2. In the case of peptides, their molecular weight should not exceed 20 kDa to avoid autoimmunization.

3. The dosage form must be non-invasive for the possibility of out-of-hospital individual use. The oral form was excluded due to the inevitable degradation of biomolecules in the gastrointestinal tract. Given the need for preclinical studies, the candidate dosage form was reduced exclusively to transdermal (cutaneous), because the rectal and sublingual forms are difficult to test in animals.

4. The method for obtaining a substance or molecular compound must be standardizable, economical, environmentally acceptable and scalable without limit due to the extremely wide demand for the desired drug.

In the process of implementing these tasks, a product with specified target properties was obtained, and an effective technology for the method of its production and application was developed.

The technical result of the proposed invention consists in:

- achieving an anti-inflammatory therapeutic effect against the background of permanent forced overexpression of the most powerful pro-inflammatory cytokine - tumor necrosis factor (TNF-alpha - TNFα), i.e. the drug according to the invention provides not only an anti-inflammatory effect, but also exhibits a tolerogenic effect - it initiates a specific tolerance of T-regulatory cells to the body's own antigens, which determines its effectiveness in the treatment of autoimmune inflammatory diseases;

- more effective stimulation of immature (naïve) monocytes to produce Treg, compared to GM-CSF + M-CSF cytokines, which are used in the classical method of Treg induction and can be present in peptide fractions from the spleen with a molecular weight of more than 12 kDa, namely about 30 kDa for GM-CSF and about 60 kDa for M-CSF, but are absent in the proposed preparation based on a fraction of peptides from the spleen with a molecular weight of not more than 12 kDa;

- achieving a systemic anti-inflammatory therapeutic effect when using the drug in the form of a water-alcohol lotion when applied to the skin, which causes a decrease in the concentration of pro-inflammatory cytokines TNFa, IL-1b and IL-2 in the blood;

- prevention of the development of psoriatic and rheumatoid arthritis with an efficiency of about 70% due to systemic tolerogenic anti-inflammatory therapeutic action;

- the possibility of achieving a tolerogenic anti-inflammatory therapeutic effect in a form suitable for the possibility of out-of-hospital individual use;

- the possibility of achieving a systemic tolerogenic anti-inflammatory therapeutic effect without the manifestation of undesirable side effects - allergic reactions and suppression of adaptive immunity.

The specified technical result is achieved due to the development by the authors of the present invention of a unique composition and therapeutic form of an anti-inflammatory drug.

An anti-inflammatory drug based on mammalian spleen peptides is proposed, presented in the form of a systemic anti-inflammatory skin water-alcohol lotion containing a fraction of purified mammalian spleen peptides with a molecular weight of not more than 12 kDa. In this case, the working range is represented by a fraction of peptides with a molecular weight in the range from approximately 0.4 kDa to 12 kDa.

The fraction of purified mammalian spleen peptides with a molecular weight of not more than 12 kDa is presented in the preparation with a concentration of at least 1%, which provides an effective amount of the active principle, namely, 1 ÷ 5 wt % to the total weight of the composition.

The composition of the proposed drug also includes 96% ethanol in the amount of 40 wt % and water - the rest, at pH 7.4±0.2.

In addition, the composition of the proposed preparation may contain sodium chloride in an amount of 0 - 0.5 wt%, zinc chloride in an amount of 0 - 0.05 wt% and calcium sulfate in an amount of 0 - 0.2 wt%.

In the most preferred embodiment, an anti-inflammatory drug based on mammalian spleen peptides is proposed, presented in the form of a systemic anti-inflammatory skin water-alcohol lotion composition, containing a fraction of purified mammalian spleen peptides with a molecular weight of not more than 12 kDa in an amount of 1 ÷ 5 wt % to the total weight of the composition , 96% ethanol in an amount of 40 wt%, sodium chloride in an amount of up to 0.5 wt%, zinc chloride in an amount of up to 0.05 wt%, calcium sulfate in an amount of up to 0.2 wt%, water - the rest, at pH 7.4 ±0.2.

The proposed drug normalizes the balance of inflammatory and anti-inflammatory reactions, which is determined by the in vivo normalization of the level of TNF-alpha, IL-1-beta and IL-2 cytokines, including during inflammatory LPS provocation.

In addition, the proposed drug prevents the development of autoimmune or inflammatory diseases, including psoriatic and rheumatoid arthritis, which allows it to be used in the prevention, therapy, or to reduce the severity of symptoms of these diseases in both humans and animals.

Accordingly, the proposed aqueous-alcoholic lotion can be presented as a preparation, composition or agent for pharmaceutical or veterinary use.

The specified technical result is achieved due to the development by the authors of the present invention of an effective technology for producing an anti-inflammatory drug.

The proposed method for obtaining an anti-inflammatory drug involves the sequential implementation of the following steps:

mixing the spleen of a mammal crushed to the state of minced meat with a 0.1% solution of zinc chloride in a ratio of 2 mass parts of the solution to 1 mass part of minced meat,

fixing the large molecular fraction by acidifying the resulting homogenate with vigorous stirring to a pH value of 3.0 ± 0.2 with a 5 M hydrochloric acid solution,

extraction of peptides by adjusting the pH of the homogenate to pH 8.0±0.2 with 5 M sodium hydroxide solution,

obtaining a clarified extract using freeze-exudation or flow centrifugation,

precipitation of large-molecular ballast by adding a 5 M solution of sulfuric acid to the extract to a pH value of 2.5 ± 0.2 with a cut-off of the ballast using a suction filter and obtaining an acid filtrate,

sulfate precipitation by neutralizing the filtrate with a 30% aqueous suspension of calcium hydroxide to a pH value of 7.4 ± 0.2 with a cut-off of the precipitate using a suction filter and obtaining a neutral filtrate,

precipitation of the residual ballast and partial desalination by adding a calculated volume of 96% ethanol to the filtrate to a content of 40 wt % with a cut-off of the residual protein-salt precipitate using a suction filter and obtaining a composition of a cutaneous aqueous-alcoholic lotion of systemic anti-inflammatory action containing a fraction of purified peptides spleens of mammals with a molecular weight of not more than 12 kDa in an amount of 1 ÷ 5 wt % of the total mass of the composition, 96% ethanol in an amount of 40 wt %, sodium chloride in an amount of up to 0.5 wt %, zinc chloride in an amount of up to 0.05 wt %, calcium sulfate in an amount up to 0.2 wt %, water - the rest, at pH 7.4 ± 0.2.

In this case, the determination of the cutoff limit of the molecular weight of the peptides is carried out using displacement chromatography and using SDS electrophoresis in a gradient polyacrylamide gel.

To obtain the drug using the spleen of mammals, preferably cattle or pigs.

The specified technical result is achieved due to the development by the authors of the present invention of an effective technology for the use of an anti-inflammatory drug.

The proposed method for suppressing an inflammatory response involves applying an effective amount of an anti-inflammatory peptide preparation to the patient's skin.

An effective amount of the proposed drug is provided, for example, by administering at a dose of at least 2 mg/kg of body weight with daily, weekly, every 2 weeks and monthly. The course of treatment is from one to several months, preferably three, six or nine months. The most preferred dose is 2 mg/kg body weight daily.

At the same time, the peptides in the composition of the proposed drug, without penetrating into the systemic circulation, act on intradermal dendritic cells, directing them to the tolerogenic differentiation pathway with subsequent transmission of the tolerogenic signal to T-regulatory lymphocytes (Treg).

In this case, the inflammatory response may be associated with psoriatic arthritis, including those caused by artificial constitutive synthesis of tumor necrosis factor alpha (TNF-alpha), or activation of pro-inflammatory cytokines, including those caused by bacterial endotoxin (LPS), or rheumatoid arthritis, in including spontaneous rheumatoid arthritis, or a systemic inflammatory response.

The anti-inflammatory drug used in the method for suppressing the inflammatory response can be obtained by the above-described production method.

Also proposed is the use of an anti-inflammatory drug in the treatment of an inflammatory disease selected from a chronic inflammatory disease, a systemic inflammatory response (reaction) syndrome or an autoimmune disease, namely psoriatic arthritis or rheumatoid arthritis.

An anti-inflammatory drug used in the treatment of an inflammatory disease can be obtained by the production method described above.

Brief description of drawings:

Fig.1. Absorption efficiency of porcine spleen peptides in intact human skin (wash analysis).

Column: HW-50 1x25cm

Mobile phase: Monoethanolamine 2%, citric acid 10 mM, pH 10.3

Designations of the tested samples:

0 - Initial peptide sample ≤ 12 kDa, 20 µg

1 - Remaining peptides on the skin after washing with saline

2 - Residue after washing with 15% DMSO

3 - Residue after rinsing with 3% Tween-80

4 - Residue after rinsing with 40% ethanol

5 - Residue after rinsing with 40% ethanol + 5% DMSO (dimethyl sulfoxide)

Fig.2A. The development of psoriatic arthritis in genetically modified ihTNFtg mice with the tumor necrosis factor gene - TNFα (TNFα), the synthesis of which in mice is induced by doxycycline (Dox) when administered orally, where

PsA - psoriatic arthritis

ctr - control mouse (healthy)

Dox is the result of TNFα activation by Doxycycline

Fig.2B. Demonstration of the application of the skin form of the tested peptide samples.

Fig.2C. Restoration of physiological parameters in vivo under the influence of the tested peptide fractions.

Simultaneously with the induction of TNF-alpha (TNF-α), starting from day 0, mice are administered daily:

- ● - saline solution (placebo);

- □ - test peptides intraperitoneally in the form of acetone precipitate dissolved in saline at a dosage of 2.0 mg/kg of body weight per day.

- ∆ - the test peptides skin in the form of a lotion with 40% ethanol at a dosage of 2.0 mg/kg body weight per day, as shown in Fig.2B;

Every 7 days up to 21 days, four standard parameters are observed:

WG - Weight Gain - weight gain index.

PASI - Psoriasis Area and Severity Index - psoriasis severity index.

SFP - Swelling (Fore Paws) - front paw edema index.

GS - Grip Strength - an index of grip strength with the front paws.

Figa. Determination of the PTC peptide molecular weight cutoff and the total peptide concentration of the PTC sample using analytical size exclusion chromatography.

Column: HW-50 1x25cm

Mobile phase: Monoethanolamine 2%, citric acid 10 mM, pH 10.3

Designations of samples and standard proteins:

1 - BSA (bovine serum albumin) 69 kDa (40 µl, 10 mg/ml)

2 - Cytochrome C 12 kDa

3 - Serial PTC sample (40 µl)

4 - Control sample of the peptide extract after ultrafiltration on a 10 kDa membrane

5 - Insulin 5.7 kDa

Fig.3B. Determination of the cutoff limit of the molecular weight and control of the purity of the PTC preparation (PTC) using SDS electrophoresis in a gradient polyacrylamide gel 10–18% according to the Laemmli method [52]. The complete absence of protein components with a molecular weight over 12 kDa was shown.

Fig.4. PTC peptides (PTC) do not directly activate CD4+ T cells.

CD4+ T cells, selectively isolated from the spleen of C57Bl/6 mice, are stimulated in vitro for 5 days with various amounts of PTC peptides.

After incubation, cells are analyzed by flow cytometry for proliferative activity (left panel) or expression of Foxp3 or Tbet nuclear proteins that are characteristic of activated Treg suppressor or immunogenic Th1 cells.

Foxp3 and Tbet expression is only shown for cells stimulated with 5000 μg/ml PTC peptides.

Figure 5 PTC (PTC)-activated DCs induce differentiation of Foxp3+ Treg cells

(A) C57Bl/6 mouse bone marrow cells are differentiated into DCs by stimulation with GM-CSF + IL-6 for 6 days. After removal of cytokines, DCs are activated with PTC peptides (PTC) or the indicated cytokines for an additional 24 hours. The DCs are then co-cultured with C57Bl/6 mouse CD4+ cells for 5 days. T cells are analyzed for proliferation and for a fraction of Foxp3+ and Tbet+ cells.

(B) Representative images illustrating the gating strategy including FMO controls (flourescence minus one) to quantify the parameters shown in (A).

Means ± SEM of five replicate experiments are shown. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison to analyze the significance between curve shapes over time. Significant differences between PTC and GM-CSF+IL-4 curves are shown (*P < 0.05 and **P < 0.01).

Fig.6. The class of dendritic cells (DCs) is the main biological target of PTC peptides.

(A) Cells derived from bone marrow differentiate into DCs with GM-CSF + IL-4 (10 ng/ml both) for 6 days. After removal of cytokines, mature DCs are activated with PTC peptides (PTC) or the indicated cytokines for an additional 24 hours and analyzed by flow cytometry. Means ± SEM of five replicate experiments are shown. Statistical analysis is performed by one-way ANOVA followed by multiple Tukey comparison. Only values related to DCs treated with PTC peptides are shown (*P < 0.05, **P < 0.01 and ***P < 0.001).

(B) Representative images illustrating the gating strategy including FMO controls (flourescence minus one) to quantify the parameters shown in (A).

Fig.7. PTC-mediated Treg induction depends on the interaction of DCs with PD-1 and CTLA4 receptors on CD4+ T cells

(A) C57Bl/6 mouse bone marrow cells are stimulated with GM-CSF + IL-4 (10 ng/ml each) for 6 days to generate mature DCs. After removal of cytokines, DCs are activated with PTC for another 48 hours. Sensitized DCs are washed twice with cytokine-free medium and incubated with syngeneic CD4+ cells of the spleen in a ratio of 1:5 and in the presence of specific monoclonal antibodies (monAbs) (20 μg / ml) within 5 days. Cells are harvested and analyzed for the indicated parameters by flow cytometry.

The mean values of the analyzed parameters in cells incubated with IgG control are assigned a value of 1. Mean ± SEM values from four replicate experiments are shown.

Statistical analysis is performed by one-way ANOVA followed by multiple Tukey comparison. (*P < 0.05, **P < 0.01 and ***P < 0.001).

(B) Representative images highlighting the gating strategy, including FMO controls, to quantify the parameters shown in (A).

Fig.8. The effect of PTK (skin application) at a dosage of 2.0 mg/kg body weight per day on the in vivo concentration of pro-inflammatory cytokines TNFα (TNF-alpha), IL-1b, IL-2 after LPS challenge.

Fig.9. Effects of PTC (skin application) at 2.0 mg/kg body weight per day in an in vivo anti-inflammatory activity test. Saline is used as a placebo. A post-mortem microscopic examination of a transverse section of the skin in areas of psoriatic deformity is shown. Control is a healthy mouse.

Fig.10. Arthritis-prone dog joints.

EXAMPLES OF CARRYING OUT THE INVENTION

Example 1. Selection of the composition of the dermal dosage form

The biological target of anti-inflammatory peptides was determined in several stages, which turned out to be dendritic cells represented in the skin by Langerhans cells (see Example 4 below), which determined the sufficiency of assessing dermal absorption (indelibility of the sample from the skin surface). In order for the peptides to quantitatively reach Langerhans cells, they must be able to overcome at least a thin keratin layer of 10-15 µm. The calculation that peptides that are absorbed into the skin are able to penetrate the keratin layer and reach Langerhans cells was further confirmed by a clear unexpected physiological effect (see further Examples 2, 6, 7, 8).

To demonstrate the effect, a concentrated sample of porcine or cattle spleen peptides is prepared, obtained by the micromethod, which is then used to determine the sources of the active principle (see Example 2 below), namely: the spleen sample is homogenized in 2 volumes of 0.1% zinc chloride , adjust the pH of the homogenate with a 10% ammonia solution to a value of 8.0 ± 0.2, freeze at a temperature of ─20 ° C, thaw on a grid, add 1.5 volumes of acetone to the resulting extract (exudate) (desalting and removing large-molecular ballast above 12 kDa), filtered on a paper filter, 2 volumes of acetone are added to the filtrate, the precipitate is collected on a paper filter, washed with acetone, dried and dissolved in 0.9% sodium chloride solution (saline), filtered through a 0.2 μm syringe nozzle, diluted to the required concentration.

Based on this formulation of the problem, a solution composition was developed that best promotes the passive absorption of macromolecules by uninjured skin. Non-ionic detergents, ethanol, dimethyl sulfoxide and combinations thereof were tested. The absorption efficiency of peptides in vivo was measured by chromatographic determination of the residual amount and composition of peptides on intact human skin after passive absorption of the sample. Unexpectedly, this optimal solvent turned out to be an aqueous solution containing 40 wt % ethanol. The efficiency of skin absorption of peptides less than 12 kDa was about 60% without noticeable discrimination by molecular weights (Figure 1).

In the experiment, a desalted sample of peptides in the range up to 12 kDa with a concentration of 40 mg/ml, obtained from the total extract according to the above method with fractional acetone purification, is used as the initial control sample. For the preparation of test samples, the original sample is diluted in saline with the addition of the components indicated in the diagram.

On clean intact human skin in vivo , 10 μl of samples with a total amount of 20 μg of peptides are applied. After spontaneous absorption and drying of the sample, the rest of the peptides are washed off the skin twice each time with 30 µl of the mobile phase (2% monoethanolamine, 0.01 M citrate, pH 10.3), taken with a microdoser and collected in a microtube. Washouts are analyzed by size exclusion chromatography.

The choice of analytical chromatography is determined by two reasons, namely:

(a) high sensitivity and reproducibility,

(b) the ability to evaluate absorption discrimination depending on the molecular weight of the peptides.

Result: The most effective was 40% ethanol (sample No. 4), incl. with the addition of DMSO up to 5% (sample No. 5). At the same time, discrimination by molecular weight was unexpectedly absent. Thus, the dosage form was chosen as an alcohol-containing composition, specifically, a water-alcohol lotion.

Example 2. Determination of a natural source of a medicinal principle to optimize the technology for its production

Peptides were chosen as the initial object of study. For this, indirect data were known. Subsequently, the choice was justified.

Despite the fact that the experiments of A.B. Tsypin et al. [1] previously showed that spleen ultrafiltrate up to 50 kDa has anti-inflammatory properties; due to the risk of developing side effects, primarily autoimmunization, a cutoff of 50 kDa is completely insufficient, and it was necessary to accurately determine the source of the active principle, and it was detected in a safe range up to 12 kDa.

In the first series of experiments, peptides from blood plasma and various organs and tissues of a pig (4 sources in total) were used, obtained from the total extract according to the method with fractional acetone purification described in Example 1.

Peptide samples were formed from the obtained fractions in two versions:

(a) skin version with a total concentration of 6 mg/ml in 40% ethanol,

(b) injection intraperitoneal version with a concentration of 1 mg/ml in saline.

To test the fractions that have the best therapeutic effect against artificial psoriatic arthritis, genetically modified ihTNFtg mice [51] carrying an additional gene for tumor necrosis factor (TNF-alpha, TNF-α), the main pro-inflammatory cytokine, were used. The gene contains a promoter activated by doxycycline (Dox), which mice get from their drinking water. Upon activation of the TNF-α promoter, mice develop severe psoriatic arthritis (FIG. 2A). These mice are used as a specific sensitive model in the selection of fractions that have a systemic therapeutic effect in transdermal treatment form.

Simultaneously with the induction of TNF-alpha (TNF-α), starting from day 0, mice are administered daily:

- ● - saline solution (placebo);

- □ - test peptides intraperitoneally in the form of acetone precipitate dissolved in saline at a dosage of 2.0 mg/kg of body weight per day (30 g per mouse, injection of 60 μl at a concentration of peptides of 1 mg/ml);

- ∆ - test peptides skin application in the form of a lotion with 40% ethanol at a dosage of 2.0 mg/kg body weight per day, as shown in Fig.2B (30 g per mouse skin application 2x5 μl at a concentration of 6 mg/ml);

Every 7 days up to 21 days, four standard parameters are observed:

WG - Weight Gain - weight gain index.

PASI - Psoriasis Area and Severity Index - psoriasis severity index.

SFP - Swelling Fore Paws - front paw edema index.

GS - Grip Strength - an index of grip strength with the front paws.

Figure 2C shows the result for the most adequate porcine spleen peptide variant, which was further taken as the basis for the development of technology for the production of an anti-inflammatory drug (see Example 3). Similar data were obtained using bovine spleen.

Results:

In this series of experiments, the richest sources (organs and tissues) containing the therapeutic principle were identified. Since the extracts of not a single organ and tissue of mammals (cattle and pig) had therapeutic activity, it was concluded that anti-inflammatory regulatory peptides are ubiquitous in the body, but for the best standardization of the subsequent industrial production of the peptide compound, the spleen was chosen, which is the richest in the peptide compound with anti-inflammatory action.

When administered cutaneously and intraperitoneally, spleen peptides equally effectively restore physiological parameters, overcoming psoriatic arthritis caused by constant exposure to TNF-α (TNF-α) overexpression. It is important to note that intraperitoneal administration is fundamentally different from intravenous or intramuscular, because. the introduced peptides do not enter directly into the systemic circulation, but act on the cells of the intestinal wall.

In addition, a systemic all-organismal effect of peptides was revealed, when the application of peptides to the local area of the ears or intraperitoneally causes the distal areas of the body - joints and skin - to normalize. An unequivocal therapeutic effect of spleen peptides was shown against the background of permanent forced overexpression of the most powerful pro-inflammatory cytokine - tumor necrosis factor (TNF-alpha - TNFα), which indicates that the peptide compound has not only an anti-inflammatory effect, but acts at the level of a deeper normalization of immunity, those. has a tolerogenic effect. This was further confirmed in experiments in vitro (see Fig.4-7). On this basis, he received the code name "Peptide Tolerogenic Compound" - PTC (Peptide Tolerogenic Compound - PTC).

Using a test model of genetically modified mice carrying an additional gene for tumor necrosis factor (TNF-alpha - TNF-α) [51], the optimal technology for obtaining PTK was developed (see Example 3).

Example 3. Description of the technology for obtaining PTC (PTC)

The raw material (cattle or pig spleen) is crushed in a meat grinder, mixed with a 0.1% solution of zinc chloride in the ratio of 2 mass parts of the solution per 1 mass part of minced meat, the resulting homogenate is acidified with vigorous stirring to a value of 3.0 ± 0.2 using a 5 M solution hydrochloric acid to fix the large molecular fraction, extract the peptides by adjusting the pH of the homogenate to a value of 8.0 ± 0.2 using 5 M sodium hydroxide solution, obtain a clarified extract by freezing-exudation or flow centrifugation (using standard techniques for clarification of peptide extracts, known, for example, from [ 1], [2], [50]), add 5 M sulfuric acid solution to pH 2.5±0.2 (second fixation of large-molecular ballast), obtain an acidic filtrate using a suction filter, neutralize the filtrate with a 30% aqueous suspension of calcium hydroxide to a pH value of 7.4±0.2 (sulfate precipitation), a neutral filtrate is obtained using a suction filter, an equal volume is added to the filtrate 96% ethanol (final precipitation of large-molecular ballast and desalting) to obtain a calculated ethanol content of 40.1 wt % (calculated taking into account the density of 96% ethanol 0.806 and the decrease in the resulting volume of the water-alcohol mixture with a coefficient of 0.964 [55]), the filtrate is obtained using a suction filter (prepared PTK in the form of a lotion).

Determination of the PTC peptide molecular weight cutoff and the total sample concentration of the peptides was performed using analytical size exclusion chromatography (FIG. 3A).

The correct determination of the total concentration of peptides in PTC is carried out by comparing the integral of curve "1" (sample solution of BSA with a concentration of 10 mg/ml) and the integral of curve "3" (sample of PTC of equal volume with BSA). In the present case (FIG. 3A), the total concentration of peptides in the obtained PTC sample is ≈ 15 mg/ml ≈ 1.5%.

Determination of the cutoff of the molecular weight and control of the purity of the PTC preparation (PTC) is carried out using SDS-electrophoresis in a gradient polyacrylamide gel 10-18% according to the method of Laemmli [52] (Fig.3B). The complete absence of protein components with a molecular weight over 12 kDa was shown.

The proposed method for obtaining a preparation of low molecular weight peptides has the following advantages:

- The method eliminates critical hardware and process limitations, namely: acetone precipitation, dialysis, chromatography, lyophilization, ultrafiltration. As a result, it provides the most economical and unlimited industrial scaling.

- Using an empirically selected combination of concentrations of acids, zinc chloride, calcium hydroxide and ethanol, the proposed method allows you to clearly limit the molecular weight of the target peptides from above at a level of 10 to 12 kDa, but not higher than 12 kDa, without the use of toxic explosive acetone and expensive low-performance ultrafiltration methods and chromatography, which are used in prior technologies. Zinc in this case plays a dual role as a differential precipitant and an essential cofactor for regulatory peptides.

- Due to the combination of sulfuric acid and calcium hydroxide as sulfate precipitant, and ethanol as a secondary precipitator, the salt content in the final product does not exceed 0.05% zinc chloride, 0.2% calcium sulfate (maximum solubility [53]), 0, 5% sodium chloride at pH 7.4±0.2 without dialysis and/or desalting on columns. This determines the physiologically comfortable composition of the finished lotion for skin application.

Table 1. The composition of the proposed water-alcohol lotion obtained by the proposed method

Lotion components, at pH 7.4±0.2 quantitative content,
% wt. to the total weight of the composition The fraction of purified peptides of the spleen of mammals with a molecular weight of not more than 12 kDa, namely, 0.4-12 kDa at least 1, namely 1 ÷ 5 96% ethyl alcohol (ethanol) 40 sodium chloride 0 - 0.5 zinc chloride 0 - 0.05 calcium sulfate 0 - 0.2 water rest

All subsequent experiments were carried out using PTC standard serial samples (PCT) as a standardized source of peptides to maintain reproducibility of results.

For in vitro experiments , peptides are precipitated from PTC using a two-step acetone procedure. First, 1 volume of acetone is added to the PTC solution to precipitate residual salts and filtered. 2 volumes of acetone are added to the filtrate to obtain a peptide precipitate, the precipitate is washed with acetone, collected on a filter paper and dried under sterile laminar air flow. The dried precipitate is dissolved in saline, the stock solution is brought to a concentration of 10 mg/ml, sterilized using a syringe filter nozzle with a 0.2 μm membrane, packaged and frozen in small portions.

Example 4 Determination of the biological target of PTC in vitro

To determine the effect of PTK peptides (PCT) directly on T cells, CD4+ cells are isolated from the spleen of C57BL/6 mice using MACS (Magnetic Cell Population Separation Technology Based on Surface Antigens) and stimulated in vitro for 5 days with various amounts of peptides. PTK.

Since differentiation and/or activation of lymphocytes is usually associated with extensive proliferation [56], CD4+ cells are prelabeled with CFSE fluorochrome, and the degree of cell proliferation (quantified as the proportion of CFSE ^lo cells), and the number of Foxp3- or Tbet-positive cells are analyzed by flow analysis. cytometry. Foxp3 and Tbet are the main transcription factors that initiate and maintain the development of Treg and Th1 lineages from naïve Th progenitor cells, respectively. They are commonly used as markers for anti-inflammatory Treg and inflammatory Th1 cell sublines.

Result (Figure 4): Stimulation of CD4+ cells with PTK does not affect T cell proliferation or differentiation into Treg or Th1 subtypes.

Full activation of CD4+ T cells usually requires the involvement of the T cell receptor and costimulatory molecules, which are usually mediated by interaction with antigen presenting cells such as dendritic cells (DCs). Under these conditions, T cells begin to proliferate and, depending on the cytokine environment, differentiate into a specific subset of T helpers.

Thus, the next step is to analyze whether PTK peptides can stimulate T cells indirectly through the activation of DCs. For this, bone marrow cells isolated from C57Bl/6/c mice are differentiated into mature DCs by stimulation with GM-CSF+IL-4, as described by Varga et al. [54] and then activated for 2 days with PTK peptides or IL-10, TGFβ and GM-CSF+IL-4, i.e. known tolerogenic and immunogenic triggers for the activation of dendritic cells (DCs). The DCs are then co-cultured for 5 days with naïve CD4+ cells isolated from the spleens of C57Bl/6 mice in the so-called MLR (Mixed Lymphocyte Reaction) assay, and the number of proliferating and Foxp3- and Tbet-positive cells is determined by the flow-through method. cytometry.

When DCs are activated with PTK peptides, they promote significantly higher CD4+ T cell proliferation than any other stimulus used. Moreover, the number of Foxp3+ tolerogenic Treg cells produced by PTK-activated dendritic DCs is as high as that of DCs activated by IL-10 or TGFβ and significantly higher than that of DCs activated by the immunogenic combination GM-CSF + IL. -4. On the contrary, the number of Tbet-positive immunogenic cells remains the lowest when CD4+ cells are co-cultivated with PTK-activated DCs and differs significantly from the number of Tbet+ CD4+ cells induced by DCs dendritic cells activated by the sum of cytokines GM-CSF + IL-4.

Result (Figure 5): PTK peptides stimulate T cells indirectly through the activation of dendritic cells (DCs). PTK peptides directly affect DCs and differentiate them into tolerogenic cells, which in turn promote polarization of immature (naïve) CD4+ T cells into tolerogenic Foxp3+ Treg cells.

Example 5. Deciphering the cascade of tolerogenic activation in vitro

This example examines whether PTK peptides directly stimulate DCs. For this, bone marrow cells are incubated with cytokines GM-CSF + IL-4 for 6 days to obtain mature DCs. After cytokine washout, cells are stimulated with PTK (PCT), tolerogenic (IL-10, TGFβ) or immunogenic (GM-CSF + IL-4) cytokines for another 24 h, and CD11c+/MHCII+ dendritic cells DCs are analyzed by flow cytometry .

Compared to GM-CSF + IL-4, PTC peptides appear to be only weak inducers of CD80, a ligand typically highly expressed by immunogenic DCs, and thus the phenotype of PTC stimulated T cells more closely resembles that obtained after stimulation with tolerogenic IL-10 and growth factor TGFß.

In CD86 induction, PTC peptides are as effective as GM-CSF + IL-4, but slightly less effective than TGFβ. In contrast, the number of cells expressing typical tolerogenic markers, such as surface ligands PD-L1 and CD205, but not intracellular IDO, was significantly increased when DCs were stimulated with PTC peptides compared with GM-CSF + IL-4 stimulation. Of the analyzed tolerogenic markers, the CD205 receptor was most strongly induced by PTC peptides (PTC). Neither IL-10 nor TGFβ can activate this marker as strongly. PTC peptides were intermediate in their ability to stimulate the intracellular IDO protein or the CD86 surface receptor compared to IL-10 and TGFβ.

Result (Figure 6): PTC peptides (PTC) indeed stimulate mature DCs to tolerogenic differentiation and the mechanism of DCs activation by PTC peptides is fundamentally different from that of IL-10 or TGFβ.

The question remains whether blocking PD-1 - PD-L1/2 and/or CTLA4 - CD80/86 interactions between DCs and T cells, which are known to be important for DCs-mediated Treg cell differentiation, would prevent PTK-mediated tolerogenic activity of DCs.

To answer this question, bone marrow-derived GM-CSF + IL-4 matured and PTK-activated DCs are co-cultured with syngeneic immature (naïve) CD4+ T cells in the presence or absence of functionally blocking anti-PD- 1 and/or anti-CTLA4 monoclonal antibodies (monAbs).

While the number of proliferating T cells is slightly increased in the presence of antibodies compared to IgG control, the proportion of Foxp3+ Treg is significantly reduced in the presence of anti-PD-1 monAb and, to a lesser extent, anti-CTLA4 monAb. In addition, the presence of both functionally blocking antibodies provides the strongest effect, suggesting an additive effect.

This effect is observed in both proliferating and non-proliferating cells, but is more pronounced in resting CD4+ cells. Expression of the immunogenic transcription factor Tbet shows the opposite result. The number of Tbet+ cells increases in the presence of functionally blocking anti-PD-1 and anti-CTLA4 antibodies, and the effect is observed only in proliferating CD4+ T cells.

Result (Figure 7): PTC peptides (PTC) primarily target DCs and induce the expression of ligands such as PD-L1 or CD86. Activated DCs, in turn, convert naïve CD4+ cells into tolerogenic Tregs through direct PD-1⇔ PD-L1/2 and/or CTLA4 ⇔ CD80/86 interactions.

Example 6 Effect of PTC (skin application) on concentration in vivo proinflammatory cytokines.

Outbred white mice are used, which are injected with LPS (lipopolysaccharide - pyrogen) in combination with cutaneous administration of PTC at a dosage of 2.0 mg/kg of body weight (as in Fig.2B). Measure the concentration of cytokines in blood plasma.

The effect of PTK peptides on the concentration of cytokines in vivo is shown in Fig.8. The plasma concentration of the following cytokines is measured: TNFα (TNF-alpha), IL-1b, IL-2.

Result: With daily applications of PTC for a month in the form of a water-alcohol lotion at a dosage of 2.0 mg/kg of body weight, the blood concentration of pro-inflammatory cytokines TNFa, IL-1b and IL-2 was significantly reduced compared with placebo. None of the animals had an allergy at the site of application of the drug, as well as the development of a systemic allergic reaction to the drug.

Example 7 Conducting a Post-mortem Examination to Demonstrate the Effect of PTC in an Anti-Inflammatory Activity Test in vivo

Also used for this experiment are genetically modified ihTNFtg mice [51] with forced overproduction of tumor necrosis factor - TNF (TNFα), as a result of which the mice develop severe psoriatic arthritis (see Fig. 2A).

Simultaneously with the induction of TNF-alpha (TNF-α), starting from day 0, mice are daily applied saline (as placebo) or PTK peptides cutaneously on the hairless part of the ears, as shown in Fig.2B, at a dosage of 2.0 mg/kg weight per day. After 10 days postmortally conduct a microscopic examination of the transverse section of the skin in the distal areas of psoriatic deformity (Figure 9).

Result: When exposed to PTK in the form of a skin lotion at a dosage of 2.0 mg/kg of body weight per day, arthritic-psoriatic deformity is suppressed with an efficiency of about 70%. In an additional control experiment without TNF-α induction, none of the 5 animals had an allergy at the site of application of the drug, as well as the development of a systemic allergic reaction to the drug.

Example 8 Treatment of Rheumatoid Arthritis in Large Mammals.

Nine test dogs of various breeds and outbreds, aged 4 to 8 years and weighing 12 to 25 kg, were provided for treatment by their owners. Before the use of PTC, the condition of the animals was documented by veterinarians, where they were regularly treated. In all animals, arthritis was not the result of injury. Purulent arthritis and chlamydial arthritis were also excluded.

Prior to peptide application, all nine dogs were treated for 1.5 to 3 years either unsuccessfully or with a temporary therapeutic effect with physiotherapy and known anti-arthritic drugs (Stop Arthritis, Arthroglycan, Horsepower, Ketofen, Rimadyl, Chondartron, Chondroitin Complex, Chondrolone, Rumalon). Corticosteroid preparations were not previously administered to the test dogs.

For the purity of the experiment, all antiarthritic drugs were canceled before starting the use of PTC.

Due to the specifics of the behavior of animals, the application of PTC lotion was carried out only on the hip region and in the region of the spine in order to avoid intense licking (Figure 10). The application was carried out as follows: PTC lotion was applied to the joints at a concentration of 1% to 5% with a disposable polyethylene pipette under the coat directly on the skin in a volume of 0.5-1.5 ml at each point (at least 3 points) and pressed with a plastic film on 3 minutes. According to experience, this time is sufficient for the development of a therapeutic effect. Due to the presence in the dosage form of 40% ethanol, PTK lotion spreads easily over the skin under the coat. When applying the PTK solution, each time the dosage was not less than 2 mg/kg of weight, based on the concentration of peptides and the weight of the animal. The drug was used daily, weekly, every 2 weeks and monthly.

Dogs Nos. 1,2,3 (the mildest group) underwent a 3-month course followed by follow-up for 6-9 months after completion of treatment (weekly use).

Dogs Nos. 4,5,6,7 received a 6-month course followed by follow-up for 3-6 months after completion of treatment (Nos. 4.5 with application every 2 weeks, Nos. 6.7 - monthly).

Dogs No. 8,9 (initially poorly responding to treatment) underwent a 9-month course followed by observation for 3 months after completion of treatment (first quarter - weekly application, in the middle of the course (second quarter) - daily, at the end (third quarter) ) - once a week).

The results are shown in Table 2.

Table 2. Data on the therapeutic effect of PTC in dogs

1-3 months 4-6 months 7-9 months 10-12 months Dogs PTK State PTK State PTK State PTK State #1 + +++ ─ +++ ─ +++ ─ N/A #2 + +++ ─ +++ ─ +++ ─ +++ No. 3 + +++ ─ +++ ─ +++ ─ N/A #4 + ++ + +++ ─ +++ ─ N/A #5 + + + +++ ─ +++ ─ +++ #6 + + + +++ ─ +++ ─ N/A #7 + ++ + +++ ─ +++ ─ +++ #8 + + + + + +++ ─ +++ #9 + + + ++ + +++ ─ +++

Designations:

"+" - insufficient therapeutic effect, pain in the joints persisted when probing the joints;

"++" - a satisfactory therapeutic effect, when probing the joints, pain reactions were not observed, but when moving, there was some limitation of mobility or impaired coordination;

"+++" - the maximum therapeutic effect, there were no pain reactions when probing the joints and noticeable restrictions on mobility or impaired coordination during movement;

"N/A" - no data.

It is also important that during the entire observed period during treatment and in the subsequent period, side effects were not observed. Animals after treatment showed healthy active behavior, temperature and stool were normal. No fungal, viral, or bacterial infections were noted, suggesting that the peptides did not exhibit immunosuppressive effects. None of the animals had an allergy at the site of application of the drug, as well as the development of a systemic allergic reaction to the drug.

It is noteworthy that the effect of peptides only on the hips and spine leads to a clear therapeutic effect in other diseased joints, although in the distal joints the therapeutic effect was achieved with a 1-1.5 month delay, which confirms the fact of the systemic body-wide action of the cutaneous dosage form. PTK.

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Claims (14)

1. An anti-inflammatory drug based on peptides of the spleen of mammals, characterized in that the drug is presented in the form of a skin water-alcohol lotion of systemic anti-inflammatory action, containing a fraction of purified peptides of the spleen of mammals with a molecular weight of not more than 12 kDa in an amount of 1-5 wt. % to the total weight of the composition, 96% ethanol in the amount of 40 wt. %, sodium chloride in the amount of 0-0.5 wt. %, zinc chloride in the amount of 0-0.05 wt. %, calcium sulfate in the amount of 0-0.2 wt. %, water - the rest, at pH 7.4 ± 0.2. 2. An anti-inflammatory drug according to claim 1, characterized in that it contains a fraction of purified mammalian spleen peptides with a molecular weight of not more than 12 kDa in an amount of 1-5 wt. % to the total weight of the composition, 96% ethanol in the amount of 40 wt. %, water - the rest, at pH 7.4 ± 0.2. 3. An anti-inflammatory drug according to claim 1, characterized in that it contains a fraction of purified mammalian spleen peptides with a molecular weight of not more than 12 kDa in an amount of 1-5 wt. % to the total weight of the composition, 96% ethanol in the amount of 40 wt. %, sodium chloride in an amount up to 0.5 wt. %, zinc chloride in an amount up to 0.05 wt. %, calcium sulfate in an amount up to 0.2 wt. %, water - the rest, at pH 7.4 ± 0.2. 4. An anti-inflammatory drug according to any one of claims 1 to 3, characterized in that the drug normalizes the balance of inflammatory and anti-inflammatory reactions, which is determined by the normalization of the in vivo level of the cytokines TNF-alpha, IL-1-beta and IL-2. 5. An anti-inflammatory drug according to any one of claims 1 to 3, characterized in that the drug prevents the development of psoriatic arthritis or rheumatoid arthritis. 6. A method for suppressing an inflammatory response, comprising applying to the patient's skin an effective amount of an anti-inflammatory peptide drug according to any one of claims 1 to 5. 7. The method according to claim 6, characterized in that the peptides in the composition of the anti-inflammatory drug, without penetrating into the systemic circulation, act on intradermal dendritic cells, directing them to the tolerogenic differentiation pathway with subsequent transmission of the tolerogenic signal to T-regulatory lymphocytes. 8. The method of claim 6, wherein the inflammatory response is associated with psoriatic arthritis or rheumatoid arthritis. 9. The method according to claim 6, characterized in that the specified drug is used at a dose of 2 mg/kg of body weight with daily application. 10. The use of an anti-inflammatory drug according to any one of claims 1 to 5 in the treatment of an inflammatory disease. 11. Use according to claim 10, characterized in that the inflammatory disease is selected from a chronic inflammatory disease or an autoimmune disease. 12. Use according to claim 10, characterized in that the inflammatory disease is psoriatic arthritis or rheumatoid arthritis. 13. Use according to claim 10, characterized in that the drug is used at a dose of 2 mg/kg of body weight when applied daily to the patient's skin. 14. The use according to claim 10, characterized in that the peptides in the composition of the anti-inflammatory drug, without penetrating into the systemic circulation, act on intradermal dendritic cells, directing them to the tolerogenic differentiation pathway with subsequent transmission of the tolerogenic signal to T-regulatory lymphocytes.

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