RU2205002C1 - Hypoxen as inhibitor of c1q subcomponent and c1 component of human complement system - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: it is suggested to apply preparation to suppress C1q subcomponent and C1 component of human complement system as hypoxen (olifen) of the following structural formula:

Description

 The invention relates to medicine, namely to medical immunology, and can be used in a clinic for the treatment of diseases accompanied by pathological activation of the classical pathway of the human complement system.

 The complement system (SC) consists of more than 30 serum and membrane proteins. SC plays an important role in maintaining the normal cellular status of the body. Being a part of the immune system, complement is involved in maintaining the body’s immune status, in providing an immune response to external aggression, in inflammation reactions, in the clearance of immune complexes, etc. These SK functions are performed in various ways.

 1. SC marks pathogens and accelerates the absorption of foreign cells by phagocytes or causes lysis of certain pathogens as a result of the formation of a membrane-attacking complex.

 2. SC produces a local inflammatory response against pathogens by generating anaphylatoxic peptides.

 3. Prevents immune precipitation, promoting solubilization and clearance of immune complexes from circulation.

 4. Regulates the level of the immune response.

 5. Promotes the elimination of autoreactive B cells from the body.

 SC is activated in three different ways: the classical pathway is activated by antigen-antibody complexes, the alternative and lectin pathways are activated by microbial surfaces. Activation of SC leads to the generation of anaphylatoxins (C3a, C4a, C5a) and to the formation of a membrane-attacking complex (A. Sahu and J. D. Lambris, Immunopharmacology, 2000. V.49. P. 133-148). In this case, activation of the SC is considered only along the classical path.

Activation of SC along the classical pathway is carried out with specific binding of C1, the first component of complement, on immune complexes. The C1 complex includes the C1q subcomponent, which recognizes a specific region on the immunoglobulin molecule and has a unique ultrastructure, as well as two molecules of trypsin-type proteinase zymogen Clr ₂ and Cls ₂ , which transform into enzymatically active forms after activation on immune complexes. The activation of Cl significantly increases the lability of the complex (the association constant of activated Cl is an order of magnitude lower than the association constant of the zymogen form). The C1 inhibitor (Cl-inh) binds quickly and irreversibly to the activated forms of Clr and Cls. When C1 dissociates under the action of Cl-inh, Clq remains bound to the antigen-antibody complex and exhibits various receptor-mediated biological functions, such as enhancing phagocytosis, production of superoxides by neutrophils, synthesis of immunoglobulins, chemotaxis, cell adhesion and regulation of platelet function. All blood cells bind Clq, the receptors of this molecule are also found on endothelial and epithelial cells, fibroblasts and smooth muscle cells (Cooper NR Adv. Immunol., 1985. V. 37. P. 151-216).

 Activation of SC can also cause undesirable phenomena, such as inflammation, damage to normal tissue, autoimmune diseases. Autoimmune conditions are associated with immune complexes formed against their own tissue, which are associated with biologically active complement fragments generated as a result of activation of SC along the classical pathway. Unregulated activation of complement is the essence of a large number of pathologies that affect the immune, renal, cardiovascular, nervous and other systems of the body. Pathological conditions caused by acute unregulated activation of SC include myocardial infarction, hyper-acute transplant rejection, sepsis, asthma, multiple organ dysfunction, trauma, hemorrhagic shock, etc. In chronic activation, the following diseases are observed: paroxysmal nocturnal hemoglobinuria, glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, Alzheimer's disease, transplant rejection, multiple sclerosis (Volonakis JE, Frank MM The Human Complement System in Health and Disease. Marcel Dekker, New York, 1998 P. 455-490).

 From this point of view, the interest in substances that affect the complement system, especially the classical activation pathway, is understandable. An urgent problem is the search and obtaining in an individual state inhibitors of the complement system, which can be useful in the treatment of pathological conditions described above. Inhibitors can become either the basis of drugs for pharmacological effects on the body, or a model for the chemical synthesis of new inhibitors.

 The closest technical solution is the use of factor J as an inhibitor of component C1 (US Patent 5268363, CL C07K 3/28; A61K 37/02, 04/27/1992). Factor J is a protein with a molecular weight of 18,400-22,000 Da. Its effect on Clq is similar to that of a Clq inhibitor. However, factor J acts only on the C1 component and has an insufficiently active effect.

 The objective of the present invention is to expand the range of human complement inhibitors, as well as the search for the most effective inhibitors with a wider spectrum of activity in relation to components of human complement.

 The problem is achieved through the use of Clox and C1 inhibitor components of the complement of the human drug hypoxene.

 Hypoxene is the trade name for the sodium salt of [(2,5-dihydroxyphenylene)] - 4-thiosulfonic acid. The synthetic hypoxene preparation is approved for use in medicine (RF Patent 2105000 C1, 10.30.96. Registration number 99/398/2. VFS 42-3507-99, VFS 42-3508-99, VFS 42-3541-99).

где n=0-4.

where n = 0-4.

 It is known that the drug improves the utilization of oxygen circulating in the blood by the body, increases the body's resistance to oxygen deficiency, blocks free radical reactions, and prevents the formation of toxic lipid peroxidation products (V. S. Smirnov, M. K. Kuzmich. “Hypoxen”, C.- St. Petersburg, Moscow: PHARMindeks, 2001, p. 18-91).

The technical result of the claimed invention is the discovery of a highly specific inhibitor that blocks only the classical pathway of activation of the complement system, inhibits the functioning of component C1, and by contacting Clq inhibits the assembly of the Cl complex [Clq (Clr ₂ Cls ₂ )] with K _i = 7 ng / ml (8.9 • 10 ^-9 M).

In addition, the use of this substance as a complement inhibitor has several advantages characteristic of it: it has unique activity as an antioxidant, the active substance is stable when stored in a dark place at room temperature for 3 years, the drug is harmless (LD ₅₀ = 1700 mg / kg of weight when administered intravenously to white mice), is not carcinogenic and does not have embryotoxicity, any suitable method of application, for example, oral, paren, can be used to introduce an effective dose of the drug into the body eralny, rectal or external, long-term drug was tested in the clinics with posthypoxic conditions and proved to be highly effective therapeutic and prophylactic agent.

 All the above advantages greatly simplify its use as a complement inhibitor.

 We first conducted studies of the effect of hypoxene on the human complement system, which was not known before our studies.

 The studies were carried out as follows.

 Example 1. The effect of hypoxene on the classic pathway of activation of the complement system.

 Hemolytic tests were used to study the functional activity of hypoxene in inhibiting complement activation in vitro.

For the classical route, mutton erythrocytes (10 ⁹ cells / ml) in a veronal saline buffer containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ (VBS ²⁺ ) were sensitized with rabbit anti-Forsman antibodies for 30 min at 37 ^o C. Cells (EA) were then washed with VBS ²⁺ buffer by centrifugation and resuspended to 1.5 x 10 cells / ml.

The effect on SC activity was determined by reducing hemolysis along the classical pathway of diluted human blood serum after its preincubation with 0.1-0.8 μg of hypoxene for 0 and 15 minutes at 37 ^° C in a volume of 0.3 ml and 200 μl of EA suspension (1.5 • 10 ⁸ cells / ml) followed by 30 min incubation. After incubation, 2.5 ml of a cold 0.15 M NaCl solution was added to each sample, centrifuged, and the degree of hemolysis was determined by the value of A ₄₁₂ supernatant. The control sample did not contain hypoxene. Reduced hemolysis in experimental samples compared with the control testified to the presence of effects on SC.

Studies have shown that hypoxene dose-dependently inhibits the classical pathway of activation of the complement system, and pre-incubation of serum with a hypoxene preparation for 15 min at 37 ^o With enhances the effect (see table. 1).

 Thus, the hypoxene preparation has an inhibitory effect on the complement system. An increase in the effect of serum preincubation with the drug may indicate either complement activation or inactivation of one of the components of the classical pathway.

 Example 2. The effect of hypoxene on the formation of the complex EAC1.

The experiments were carried out as follows. 0.1-0.4 μg of the drug was incubated for 15 min at 37 ^° C with a selected concentration of R4 (serum depleted in the C4 component, source of the C1 complex) and an EA suspension (200 μl) in a total volume of 500 μl adjusted with VBS ²⁺ . The reaction was stopped by adding 500 μl of a cold solution of 0.15 M NaCl, centrifuged for 5 min at 1500 g, decanted and 0.5 ml of diluted R1 reagent (human serum depleted in the C1 component) was added to EAC1 precipitate. Thoroughly mixed by shaking and incubated for 30 min at 37 ^o C. After incubation, the degree of hemolysis at A _{412 was} determined, as described above.

The measured value (degree of lysis of y) was used to calculate the value of Z, which is determined by the formula
Z = ln [(HR) / HX)], where H, R and X are the absorbances at A _{412 of the} hemolytic system with complete lysis, in the control (without titratable component) and in the experimental sample, respectively.

 The results of the study convincingly indicate that hypoxene has a dose-dependent inhibitory effect on the formation of the complex EAC1 (see table. 2).

The inhibition constant K _{i was} calculated using the linear equation 1 / Z _i = [I] / (Z _o • K _i ) + 1 / Z _o , plotting 1 / Z _i versus [I]. In this case, the point of intersection of the graph with the abscissa axis corresponds to the value K _i = 1.1 ± 0.1 μg / ml (1.5 • 10 ^-5 M).

 Example 3. The binding of hypoxene with the complex EAC1.

Studies of the effects of hypoxene after the formation of the EAC1 complex were carried out as follows. The EAC1 complex prepared by incubating EA with R4 reagent (serum depleted in the C4 component, the source of the Cl complex) was washed by centrifugation, resuspended in VBS ²⁺ buffer, and the concentration of the EAC1 complex was adjusted to 1.5 • 10 ⁸ cells / ml. 200 μl of EAC1 were incubated with different concentrations of hypoxene (0.1-0.4 μg) for 15 min at 37 ^° C in a total volume of 0.5 ml adjusted with VBS ²⁺ buffer. After incubation, the complex was washed by centrifugation, decanted, and the precipitate was resuspended in a hemolytic system containing reagent R1. Hypoxene, depending on the concentration, inhibited the functioning of Cl in the EAC1 complex (see Table 3).

It was determined To _i = 6.75 • 10 ^-7 M (as described above).

 Thus, the anti-complementary effect of hypoxene is due to interaction with the C1 complex, and after the formation of the EAC1 complex, the inhibition effect is higher than at the stage of complex formation.

 Example 4. Determination of the reversibility of the binding of hypoxene to EAC1.

An EAC1 complex with a limited number of effective C1 molecules was prepared as described in Example 3. 200 μl of a suspension of the EAC1 complex were incubated with 0.1-0.4 μg of hypoxene in a volume of 500 μl for 15 min at 37 ^° C. After incubation with an effector, 500 μl of VBS ²⁺ buffer, the complex was separated by centrifugation and the residual activity of the C1 complex on EA in the hemolytic system with reagent R1 was determined after 30 min of incubation. The degree of cell lysis was determined as described above. The control did not contain hypoxene. The experimental results showed a dose-dependent inhibition of C1 activity, suggesting the irreversible binding of hypoxene to the C1 complex associated with EA (see table 4).

The inhibition constant K _{i was} calculated using the linear equation 1 / Z _i = [I] / (Z _o • K _i ) + 1 / Z _o , plotting 1 / Z _i versus [I]. In this case, the point of intersection of the graph with the abscissa axis corresponds to the value of K _i = 7 ng / ml (8.9 • 10 ^-9 M).

 Thus, the studies of the reversibility of the binding of hypoxene to the EAC1 complex indicate a high affinity and practically irreversible nature of the interaction.

 Inhibition of EAC1 activity can be caused either by direct inactivation or by dissociation of C1 as a result of the interaction of hypoxene with subcomponents of the complex.

 Example 5. The effect of hypoxene on the activity of the subcomponent Clq.

The experiments were carried out as follows. 0.1-0.4 μg of hypoxene was preincubated for 15 min at 37 ^° C with a selected concentration of the purified functionally active subcomponent Clq of the first component of human complement C1 and the hemolytic system was collected after incubation by adding 200 μl of EA suspension (1.5 × 10 ⁸ cells / ml) and 20 μl of Rlq reagent (serum deficient in the Clq subcomponent). The total volume of the hemolytic system was adjusted to 0.5 ml with VBS ²⁺ buffer. After incubation for 30 minutes at 37 ^{° C., the} reaction was stopped by adding 2.5 ml of a cold 0.15 M NaCl solution, centrifuged for 5 minutes at 1500 g, and the degree of hemolysis was determined as described above.

As a result of the studies, data were obtained that indicate the binding of the Clq subcomponent with hypoxene with K _i = 1.66 • 10 ⁸ M (see table 5).

 Thus, studies of the effect of hypoxene on the Clq subcomponent indicate direct inactivation of the recognizing subunit of the C1 complex.

Example 6. Determination of the binding of hypoxene to the complex EAClq. To determine the interaction of hypoxene with Clq associated with EA, the EAClq complex was pre-incubated with various amounts of hypoxene in the dose range between 0.05 and 0.4 μg / ml for 20 min at 37 ^o C. After incubation, the complex was washed by centrifugation, resuspended in the hemolytic system with Rlq reagent. These experiments showed a dose-dependent inhibition of Clq activity, suggesting the binding of hypoxene to Clq associated with EA (see table 6).

The hypoxene preparation interacts with the immobilized subcomponent Clq on EA and thereby inhibits the formation of the C1 complex with K _i = 7.65 • 10 ^-8 M.

 Thus, from the above examples it follows that the inhibitory effect of hypoxene on the C1 component of the complement system is due to binding to the Clq subcomponent in solution, which leads to the dissociation of the C1 complex, and the binding of hypoxene to immobilized Clq prevents the formation of the C1 complex.

Claims (1)

The use of hypoxene having the general formula:

where n = 0 - 4,
as a specific inhibitor of Clq subcomponent and Cl component of the classical pathway of the human complement system.

Images