RU2758536C1 - Method for reducing inflammatory hyperactivation of neutrophils - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, namely to critical condition medicine, resuscitation, anesthesiology and clinical pharmacology; it can be used, if necessary, to reduce inflammatory hyperactivation of neutrophils. To do this, inhalation is carried out with a gas mixture: xenon of 30 vol.%, oxygen of no more than 40 vol.%, the rest is nitrogen, along a closed circuit lasting 60 min.EFFECT: method reduces the activity of neutrophils, reduces inflammatory processes, prevents or reduces the severity of multiple organ failure, as well as reduces the duration of treatment and increases the survival rate of patients with critical conditions.1 cl, 2 dwg

Description

The invention relates to medicine, namely to anesthesiology, resuscitation and clinical pharmacology, and can be used to treat patients with pathological conditions in which there is a systemic inflammatory response syndrome (SIRS) after extensive operations, surgical interventions using a heart-lung machine, concomitant trauma , severe sepsis and septic shock, tissue ischemia-reperfusion syndrome in stroke and myocardial infarction, diabetes mellitus, tumor metastasis, pulmonary edema and acute respiratory distress syndrome, burn disease, etc. The method allows you to reduce the activity of neutrophils, reduce inflammation, prevent or to reduce the severity of multiple organ failure, shorten the duration of treatment and increase the survival rate of patients in critical conditions.

State of the art

For critically ill patients, relief of the systemic inflammatory response is relevant.

A number of methods are known that involve the use of pharmaceuticals to solve the problem of stopping SIRS. These include in particular:

- a drug aimed at stopping SIRS containing mg: diphosphopyridine nucleotide 0.3-100 and inosine 40.0-1200, as well as an angiotensin-converting enzyme inhibitor, mainly lisinopril 2.5-100, and a cardiac glycoside, mainly digoxin 0.07- 0.3 (RU2280455);

- a method and a pharmaceutical composition for the prevention and / or treatment of a systemic inflammatory response syndrome, comprising a partial inhibitor of factor VIII, such as a monoclonal antibody, useful for the prevention and / or treatment of a disease selected from the group consisting of SIRS, sepsis, septic shock, education blood clots in the microvascular network and disseminated intravascular blood coagulation in the mammalian body, partially suppressing the formation of thrombin (Method and pharmaceutical composition for preventing and / or treating systemic inflammatory response syndrome EP1222929);

- a method for treating cytokine release syndrome, including the introduction of a mast cell stabilizer (Methods of treating cytokine release syndrome WO2020051322);

- a drug for the prevention or treatment of systemic inflammatory response syndrome (SIRS), including a cytokine storm inhibitor having HRG as an active ingredient (Cytokine storm inhibitor WO 2016104436);

- the use of mesenchymal stem cells for the treatment of SIRS (Uses of mesenchymal stem cells AU2015268704);

- a therapeutic or prophylactic agent for the treatment or prevention of SIRS, which contains a polypeptide containing an amino acid sequence the same or substantially the same as the amino acid sequence of the N-terminal domain of pentraxin 3, capable of binding to histone to form a polypeptide aggregate or a pharmacologically acceptable salt thereof ( Agent for treating or preventing systemic inflammatory response syndrome US2015299277).

The use of the above pharmaceutical preparations for solving the problem of stopping SIR does not allow achieving the therapeutic effect of the claimed invention, since the role of neutrophils, cells of innate immunity, which play an important role in the development, regulation and resolution of inflammation, is not defined in them. The immune response to tissue damage or infection begins with the secretion of pro-inflammatory and anti-inflammatory cytokines by neutrophils, which aims to remove the damaging agent and restore homeostasis.

At the same time, methods of xionotherapy are known, including the use of inhalation therapy with xenon or gas mixtures containing xenon.

There is a known method of autoanalgesia with a xenon-oxygen mixture, which consists in the fact that the patient is offered to independently inhale the xenon-oxygen mixture in the ratio of xenon to oxygen in vol. % from 30:70 to 50:50 before pain relief. (RU 2271815). The result of using this method is a pronounced analgesic effect while maintaining consciousness, which, in turn, contributes to the normalization of hemodynamics, the function of the neuroendocrine system, the normalization of metabolic processes, by improving the delivery of oxygen to the tissue under conditions of xenon vasoplegia.

The disadvantages of the known method, which do not allow to achieve the therapeutic effect of the claimed invention, is its use only with an adequate condition of the patient, which prevents the use of the method in the case of an unconscious state of the patient; also the purpose of such use is analgesia, rather than arresting the systemic inflammatory response and not affecting the activity of neutrophils. The use of xenon in accordance with the known method is not regulated in terms of duration and optimal appointment time, which can lead to ambiguous, incomparable and unpredictable results.

There is also known a method of xenon therapy for general somatic diseases, which can be used in the prevention, treatment and rehabilitation of a wide range of human somatic diseases (RU 2305565). For this, a periodic inhalation of a mixture of oxygen with an inert gas xenon is carried out at a ratio of (50:50) - (30:70) vol. %. Each inhalation is carried out 2 times a day - in the morning and in the evening for 7-10 days. In this case, the content of xenon in the mixture is increased from the second to third days, increasing the duration of inhalation from 3-5 minutes in the first two days to 7-15 minutes on the next day. The method allows to restore the regulatory functions of organs and systems as a whole, from the central nervous system to the regional level, by activating the compensatory capabilities of the body by creating a mode of stable postxenonic nonspecific general somatic reaction of the body due to the application of the developed mode of periodic inhalations with a mixture of xenon and oxygen.

The disadvantage of this method, which does not allow to achieve the therapeutic effect of the claimed invention, is its use in planned medicine, and, therefore, the impossibility of using this technique in the event of an SSVO.Moreover, the method is aimed at the regulatory functions of organs and systems and cannot influence in this mode on the activity of neutrophils and the severity of systemic inflammation.

Other examples of the therapeutic use of xenon or xenon-containing gas compositions by inhalation are known. These include:

- the use of xenon to prevent or treat the neurological consequences of a septic shock (WO 2014057179).

- gas mixtures containing xenon and argon at low concentration as organ protectant, in particular neuroprotectant WO 2019008015).

The above-mentioned known methods of xenon therapy are also not related to the solution of the problem posed - to reduce the inflammatory hyperactivation of neutrophils that accompanies SIRS.

No methods of reducing the inflammatory hyperactivation of neutrophils in critically ill patients by means of therapeutic anesthesia with a gas mixture containing xenon have been found in the information database available to the authors.

Disclosure of the essence of the invention

The claimed invention is aimed at reducing the pro-inflammatory activity of neutrophils in critically ill patients, accompanied by the development of a systemic inflammatory response due to the correction of spontaneous apoptosis of neutrophils and a decrease in the ability of neutrophils to pro-inflammatory activation due to a decrease in the expression of CD11b and CD66b on their surface.

This problem is solved by the fact that the correction of neutrophil activation in critically ill patients accompanied by a systemic inflammatory response, as well as a decrease in the expression of CD11b and CD66b on the surface of neutrophils and optimization of neutrophil apoptosis are achieved by inhalation of xenon at a dose of 30 vol. % within 60 minutes.

According to modern concepts, neutrophils are fairly short-lived leukocytes: after leaving the bone marrow, the average lifetime of neutrophils is 4-5 days. During this time, neutrophils are released from the bone marrow, mature and age. Aging of neutrophils is accompanied by an increase in the expression of the CXCR4 chemokine receptor, which leads to migration of neutrophils back to the bone marrow, liver and other organs, where they undergo apoptosis and are phagocytosed by macrophages. Apoptosis is an extremely important and difficultly regulated stage in the life cycle of neutrophils, since too rapid and massive death of neutrophils will lead to neutropenia and the organism's susceptibility to infections, and an unnecessarily long life, on the contrary, can cause prolonged and poorly controlled inflammation.

The inert gas xenon affects the most significant and sensitive markers of the systemic inflammatory response, which are the CD11b and CD66b molecules located in the intracellular granules of neutrophils. CD11b interacts with ICAM-1 receptors on endothelial cells, which ensures the adhesion of neutrophils and their subsequent migration through the endothelial barrier to the inflammation focus [Parkos, A. Interaction of Neutrophils With Epithelial Cells: Lessons From the Intestine / A. Parkos, P. Colgan, JL Madara // J Am Soc Nephrol. - 1994. -Vol.5, No. 2. - P. 138-152].

The essence of the proposed method is that when exposed to xenon, there is a decrease in the expression of CD11b and CD66b on the surface of the membrane of neutrophils, which results in a decrease in excessive activity of neutrophils (hyperinflammation) in patients with critical conditions accompanied by a systemic inflammatory response. Thus, the therapeutic effect is achieved by optimizing the inflammatory activity of neutrophils and restoring their ability to spontaneously apoptosis.

, T.

, K. Brandenburg, A.L. Reiners, J. Zinserling, N. Schnitzler // J. Leukoc. Biol. - 2012. - Vol.91, №5. - P. 791-802. DOI: 10.1189/jlb.0911483 Muller, K.A. Leukocyte activation in sepsis; correlations with disease state and mortality / K.A. Muller, J.E. Tulleken, J.G. Zijlstra, W. Sluiter, J. Hermans et al // Intensive Care Med. - 2000. - Vol.26, №7. - P. 883-892].Neutrophil activation is one of the stages of inflammation development, in which, under the action of external signals, neutrophils undergo morphological and physiological changes (flattening, degranulation, migration, cytokine secretion, oxidative burst, and others). Sensitive markers of activation and degranulation are the CD11b and CD66b molecules, which are located in the intracellular granules of neutrophils [Schmidt, T. CD66b overexpression and homotypic aggregation of human peripheral blood neutrophils after activation by a gram-positive stimulus / T. Schmidt, J.

, T.

, K. Brandenburg, AL Reiners, J. Zinserling, N. Schnitzler // J. Leukoc. Biol. - 2012. - Vol.91, No. 5. - P. 791-802. DOI: 10.1189 / jlb.0911483 Muller, KA Leukocyte activation in sepsis; correlations with disease state and mortality / KA Muller, JE Tulleken, JG Zijlstra, W. Sluiter, J. Hermans et al // Intensive Care Med. - 2000. - Vol.26, №7. - P. 883-892].

The advantage of the proposed method, in comparison with the known, is the therapeutic effect of anesthesia with a gas mixture consisting of: xenon - 30 vol. %, oxygen - no more than 40 vol. %, the rest is nitrogen, in a closed circuit on the "XENA" anesthesia machine, which makes it possible to neutralize the influence of pathogenic factors in the form of excessive activation of neutrophils against the background of a systemic inflammatory response.

For an experimental assessment of the anti-inflammatory properties of xenon, a mode of action tested in practical medicine was used [Burov NE, Potapov VN, Makeev GN. Xenon in anesthesiology: Clinical and experimental research / M .: Pulse, 2000 - 291 p. - ISBN 5-93486-006-2 Molchanov IV, Nikolaev LL Application of xenon in medicine. (Materials of the international scientific-practical conference Chisinau, Republic of Moldova, article 12-13 2019 t)].

The specified therapeutic effect was found by the authors of the proposed method using an example as close as possible to the clinical picture of the occurrence of neutrophil hyperactivation in critically ill patients by incubating a suspension of neutrophils with lipopolysaccharide (LPS) at a dose of 200 ng / ml for 22 hours in a nutrient medium.

The inventive method for reducing inflammatory hyperactivation of neutrophils using inhalation of xenon at a dose of 30 vol. % within 60 minutes is of high practical importance, since clinicians in their work can count on the correction of neutrophil hyperactivation in critically ill patients accompanied by a systemic inflammatory response. Moreover, the proposed method can be successfully used when carrying out emergency measures in an ambulance.

Brief Description of Drawings

FIG. 1 is a diagram showing the change in the expression level of CD1 lb and CD66b by neutrophils as a result of the action of LPS 200 ng / ml and xenon.

FIG. 2 shows a diagram showing the results of a study of the effect of LPS 200 ng / ml before xenon anesthesia and after xenon anesthesia (60 minutes 30 vol.%) On the level of apoptosis and necrosis of human neutrophils.

Description of the experiment procedure

In the experiment, 20 blood serum samples from 10 practically healthy donors were used (10 blood serum samples were taken from donors before xenon inhalation and 10 blood serum samples were taken from the same donors after xenon inhalation). Healthy volunteers underwent a session of inhalation anesthesia in a hospital with a gas mixture containing xenon in a closed circuit on the XENA anesthesia machine (xenon - 30 vol.%, Oxygen - no more than 40 vol.%, The rest - nitrogen). The duration of anesthesia in all patients was 60 minutes. The course of anesthesia in all cases was smooth, the hemodynamics of the patients were stable.

The scheme of the in vitro experiment used by the authors is generally accepted today and provides for the activation of neutrophils by LPS (a component of the cell wall of gram-negative bacteria) at a dose of 200 ng / ml, which is estimated by the level of expression of the CD11b and CD66b molecules. The chosen model fully simulates the processes occurring in the body of a patient with critical conditions, accompanied by a systemic inflammatory response, i.e. is an adequate model providing for the achievement of the claimed technical result due to inhalation of xenon.

Neutrophils from healthy donors after inhalation anesthesia with a gas mixture containing xenon for 60 minutes were isolated according to the following procedure: heparinized venous blood was mixed with 5% T-500 dextran solution (Pharmacosmos, Denmark) in PBS in a ratio of 4: 1 (i.e. to a final dextrin concentration of 1%) and left at room temperature for 30 minutes for passive sedimentation of erythrocytes. The upper plasma layer (enriched with leukocytes and devoid of erythrocytes) was layered on Ficoll (PanEco, Russia) with a density of 1.077 g / ml and centrifuged at room temperature at 400g for 30 minutes in a centrifuge with the inhibition mode turned off. Then the supernatant was removed and all further procedures were carried out on ice and using chilled solutions. Removal of contaminated erythrocytes was performed by resuspending the sediment in 2 ml of deionized sterile water for 45 seconds, and then adding 2 ml of 2x PBS to restore tonicity. It was centrifuged at 200g, + 4 ° C, 10 minutes. The precipitated neutrophils were washed with PBS and resuspended in culture medium (RPMI-1640 + 10% FBS). The purity of the neutrophil population (95%) was assessed by forward and lateral light scattering using flow cytometry.

Neutrophil activation (degranulation) was assessed by the increase on the cell surface of CD lib, CD66b molecules (membrane components of specific, gelatinase and quaternary granules) after their incubation with LPS at a dose of 200 ng / ml for 30 minutes at 37 ° C, then 2, 5 μl of antibodies to CD lib, CD66b, CD63 conjugated with a fluorescent dye FITC (ThermoFisher, USA), and left for 30 minutes on ice. Then, at least 50 thousand cells were analyzed using a Beckman Coulter CytoFLEX flow cytometer.

The level of apoptosis and necrosis of neutrophils was assessed by the staining of the annexation of V-positive and propidium iodide-negative cells according to the following method. Donor neutrophils were incubated with LPS at a dose of 200 ng / ml for 22 hours at 37 ° C in a humidified CCh incubator. Then the cells were centrifuged at 400 g for 5 minutes and the pellet was resuspended in 70 μl of buffer (10 mM HEPES, 120 mM NaCl, 2.5 mM CaCl ₂ , pH = 7.4). To each sample, 2.5 μl of annexin V conjugated with the fluorescent dye FITC (ThermoFisher, USA) was added and left for 25 minutes at + 37 ° C. Then propidium iodide was added to a final concentration of 5 μg / ml, incubated for another 5 minutes, after which at least 50 thousand cells were analyzed using a Beckman Coulter CytoFLEX flow cytometer.

In this experiment, the level of expression of CD11b on the surface of intact neutrophils from healthy donors before inhalation of xenon was taken as 100% relative to the level of fluorescence. Pro-inflammatory stimulation of LPS at a dose of 200 ng / ml, on average, 4.5 times (p <0.001) increased the expression of CD1 lb on the surface of previously intact neutrophils. After anesthesia with xenon 30 vol. % for 60 minutes and LPS stimulation at a dose of 200 ng / ml, the expression of CD11b on the neutrophil surface increased only 1.8 times (p <0.05 - when compared with the group where LPS stimulation was performed, but xenon was not inhaled).

Thus, it has been established that inhalation of xenon at a dose of 30 vol. % within 60 minutes more than 2 times (p <0.05) reduces the ability of neutrophils to pro-inflammatory activation.

The level of expression of CD66b on the surface of intact neutrophils (healthy donors before inhalation with xenon) was also taken by the authors as 100% of the relative level of fluorescence. Pro-inflammatory stimulation of LPS at a dose of 200 ng / ml, on average, 1.9 times (p = 0.02) increased the expression of CD66b on the surface of neutrophils (Fig. 1). Joint action of anesthesia with xenon 30 vol. % for 60 minutes and stimulation of neutrophils with LPS at a dose of 200 ng / ml had a statistically insignificant effect on the expression of CD1 lb on the surface of neutrophils (p = 0.09).

Thus, inhalation of xenon at a dose of 30 vol. % within 60 minutes more than 2 times (p <0.05) reduced the ability of neutrophils to pro-inflammatory activation.

The results of a study of the effect of LPS 200 ng / ml before xenon anesthesia and after xenon anesthesia (60 minutes 30 vol.%) On the level of apoptosis and necrosis of human neutrophils showed that spontaneous apoptosis of neutrophils 22 hours after isolation is 56.7% (Fig. 2 ). The addition of LPS neutrophils to the incubation medium at a dose of 200 ng / ml reduced the number of neutrophils that underwent spontaneous apoptosis to 22.4%. Thus, the apoptosis of neutrophils under the influence of LPS decreased by 60% (p <0.05), which in clinical conditions contributes to the generalization of inflammation, as well as its uncontrolled course.

Inhalation of xenon at a dose of 30 vol. % within 60 minutes almost 2 times - up to 41% increased spontaneous apoptosis of neutrophils (p <0.05). The results of the study clearly showed that the use of LPS dramatically reduces the spontaneous apoptosis of neutrophils and contributes to the formation of conditions for the maintenance of inflammation, while the use of xenon has an anti-inflammatory effect and leads to the resolution of inflammation.

Thus, inhalation of xenon at a dose of 30 vol. % within 60 minutes, preventing excessive activation of neutrophils and correcting their apoptosis, has an anti-inflammatory effect, helping to reduce the likelihood of developing a systemic inflammatory reaction in critically ill patients.

Claims (1)

A method for reducing inflammatory hyperactivation of neutrophils, including inhalation with a gas mixture: xenon 30 vol. %, oxygen no more than 40 vol. %, the rest is nitrogen, in a closed circuit with a duration of 60 minutes.

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