RU2115428C1 - Method of treatment of human with immunosuppression at sepsis - Google Patents

Abstract

FIELD: medicine, infectious diseases. SUBSTANCE: invention proposes the use of human interleukin-2 prepared from yeast Saccharomyces cerevisiae. Strain is deposited in All-Russian Collection of industrial microorganisms at N VKPM-U 791. Interleukin-2 is administrated at doses from 100 000 to 3 000 000 IU/24 h/1 m² patient body surface. In contrast to other interleukins used for treatment of sepsis the proposed interleukin-2 shows stimulating effect on "resting" lymphocytes, induces production of interleukin-8 and tumor necrosis factor. Method can be used for treatment of severe states in human caused by infection and for correction of IMMUNOSUPPRESSION at sepsis in human also. EFFECT: enhanced effectiveness of treatment. 5 tbl

Description

 The invention relates to medicine and is intended for the treatment of severe conditions in humans caused by infection.

 Sepsis is the most common cause of death after cardiovascular disease and ranks 13th among other causes of death. The mortality rate from septic shock exceeds 40% and has not shown a downward trend over the past three decades. In the last decade, the risk of developing sepsis has increased significantly in immunocompromised, chronically and / or seriously ill and elderly people, especially among hospitalized patients.

 Typically, sepsis is called a non-specific disease of a non-cyclic type, characterized by violations of the body's reactivity against the background of constant or periodic entry into the blood of microbes or their toxins from the site of infection.

According to the latest international agreements, the term "sepsis" refers to the syndrome of a systemic inflammatory process caused by an infection. The criterion for a syndrome of systemic inflammatory process is the presence of more than one of the following conditions in a patient:
increased or decreased body temperature: above 38 or below 36 ^o C;
increased heart rate (tachycardia): over 90 beats / min;
rapid breathing (tachypnea): over 20 breaths per minute;
altered leukocyte formula: more than 12000 or less than 4000 leukocytes in 1 mm ³ or more than 10% of immature forms of neutrophils (with constrictions).

 The term "severe sepsis" refers to sepsis associated with organ dysfunction, hypoperfusion, or hypotension.

 The clinical picture of the disease is characterized by hectic fever, weight loss up to the development of cachexia, severe anemia, which leads to a characteristic pallor of the skin, and intoxication, manifested in varying degrees of encephalopathy. In the late stages of sepsis, a decrease in systolic blood pressure and a coma are observed.

 Sepsis usually occurs in a patient who has a predisposition to such a disease in advance, which is caused either by the characteristics of the body, for example, the slow response of the immune system, or the peculiarity of a disease transmitted by the patient, such as influenza, which weakened the immune system.

 When a person is infected, a response develops to this infection, which the immune system must localize. In a patient with a predisposition to sepsis, such localization does not occur, and a systemic response of the body develops, which turns into an inadequate (unbalanced) response, leading to depression of the immune system and sepsis.

 For the treatment of sepsis, antibiotics, agents that improve microcirculation, infusion therapy, as well as symptomatic therapy (to improve the functioning of the cardiovascular, nervous and other body systems) are used.

 The action of antibiotics is aimed at destroying microorganisms in the wound and in the bloodstream, and microcirculation-improving agents provide increased blood flow in the affected organs in order to increase the delivery of antibiotics and plasma components that neutralize toxins. Symptomatic therapy is aimed at improving the functions of vital organs, and infusion therapy is aimed at washing the body to dilute and remove toxins.

 For the treatment of sepsis, immunopreparations, such as immunostimulants, for example T-activin, leacadin, thymoline and thymogen, were also used. Such drugs are designed to activate the phagocytic link of the immune system (white blood cells, macrophages, lymphocytes); however, the desired stimulation is not achieved in this case, since these drugs are not strong enough. In sepsis, the immune system is so badly damaged that such immunostimulants practically do not act on it. Their ability to stimulate the immune system in conditions of septic immunosuppression is practically not manifested.

 In immunological studies of septic patients, a strong suppression of the immune system is detected. The immunogram shows clear signs of an imbalance in the immune response, a decrease in phagocytosis (a slowing down of spontaneous migration of leukocytes) and the number of T-lymphocytes by almost 2 times compared to the norm. The use of h-IL-2 in sepsis is known (see M.V. Grinev - in the book: Actual problems and prospects for the development of modern resuscitation. Materials of the International symposium on the 85th anniversary of Academician Ross. AMS V.A.Negovsky, 16 -March 18, 1994, pp. 103-104).

 The objective of this invention is to provide a means and method of influencing the components of the immune system, which is in a state of almost complete inhibition caused by a systemic inflammatory response syndrome, in order to restore the activity and number of leukocytes, as well as other components of the immune system that are affected by active lymphocytes, and thereby treat immunosuppression in sepsis.

 This problem is solved by the fact that as an agent for the treatment of immunosuppression in sepsis, human interleukin-2 (h-IL-2) is proposed, as well as by the fact that a method for its treatment by parenteral administration of h-IL-2 to a patient is proposed.

 From experiments in vitro it is known that h-IL-2 is a lymphokine, which is mainly produced by T-lymphocytes; he is considered a substance that plays a central regulatory role in the immune response. In vitro experiments established some of its properties, which lead, for example, to the fact that h-IL-2 stimulates the proliferation of T cells, causes their cytotoxicity, enhances the production of B-cell growth factor, enhances the induction of h-IL-2 receptors and production of gamma interferon.

 The use of h-IL-2 is known to enhance the host's anti-infectious competence. In this case, h-IL-2 is administered to a patient whose immune system basically functions normally to enhance the functioning of this system in the fight against infection. All components of such an immune system that are affected by h-IL-2 (in particular lymphocytes) are in large quantities characteristic of a healthy immune system, and their activity is not inhibited. In this case, h-IL-2 acts directly on the receptors of active lymphocytes, which leads to an increase in their activity and proliferation, and the therapeutic effect is achieved by the fact that the activity of the immune system is further stimulated.

 It is also known that h-IL-2 is used to affect the immune system through the competitive suppression of the production of tumor necrosis factor (TNF), which is found in patients suffering from cachexia associated with cancer, infectious diseases and other catabolic disorders. Moreover, in such patients, a significant increase in the level of TNF, leading to depletion of the body, was noted, and the level of IL-2 production was insufficient to competitively suppress the production of this factor. The therapeutic effect here is mainly due to a decrease in the concentration of TNF in the blood as a result of the administration of h-IL-2, which is responsible for the mechanism of maintaining intoxication that accompanies, in particular, infection.

 It is also known the use of h-IL-2 to compensate for its natural deficiency in the body with congenital or acquired immunodeficiency in children, in which pre- or intrathymic cells are defective (such immunodeficiency is classified as "primary immunodeficiency"). With such immunodeficiency, the body of a child who has undergone infection is not able to fight against it, because for some reason one of the main components of the regulation of the immune system is missing - h-IL-2. The introduction of exogenous h-IL-2 makes up for its deficiency in the body, allowing the stimulation of healthy lymphocytes and other functions inherent in h-IL-2 during the normal immune system. However, when the administration of exogenous h-IL-2 is stopped, the content of h-IL-2 in the body returns to the initial pathologically low level.

 Various experiments were carried out on animals (mice and rats) in which either sepsis was artificially induced or they were artificially injured, after which sepsis was highly likely.

 Studied mice, which are highly susceptible to bacterial sepsis. In such animals, the ability of mitogen-stimulated production of IL-2 by spleen cells is impaired. The addition of in vitro exogenous recombinant h-IL-2 (100 units / ml) restores the mitogenic response of spleen T cells. The effect of recombinant h-IL-2 administered in vivo at different doses (from 16,000 units / day to 256,000 units / day) was studied. Low doses improved the in vitro response of splenic T cells; a dose of 60,000 units / day was itself immunosuppressive.

 The results of experiments conducted on mice that were given a lethal dose of Esherichia coli bacteria cells are known. After a dose of fatal septicemia-causing bacteria, mice were given recombinant h-IL-2 after a certain time. In the group of mice that received rh-IL-2, a significantly higher survival rate was observed during the observation period compared to the control group that did not receive recombinant h-IL-2. Such an experience causes a condition only similar to sepsis. Like any model, it does not take into account, however, very important factors that arise during human sepsis. With such an experiment, a quick artificial infection occurs, and. the number of leukocytes in mice, as well as their activity is not reduced, as is the case with immunosuppression caused by sepsis in humans. That is, there is virtually no suppression of the immune system, and its treatment is not carried out. The effect is due to the binding of recombinant h-IL-2 with receptors of lymphocytes that are in an active state and in sufficient quantity. The effect is achieved only with the introduction of recombinant h-IL-2 no later than 1 hour after the introduction of a dose of bacteria, leading to the development of a condition similar to sepsis, which is impossible in case of sepsis in a person who develops gradually.

 It is known that h-IL-2 in a relatively healthy body stimulates the proliferation of T-lymphocytes (not in a state of immunosuppression), other substances, for example, thymus preparations, also perform a similar function.

 However, in sepsis, the number and function of lymphocytes is sharply reduced, which is the main reason why thymus preparations do not show the effect of stimulation of the immune system in sepsis. Almost all lymphocytes during immunosuppression are “resting” lymphocytes. Such “resting” lymphocytes do not have receptors for h-IL-2, and in vitro experiments have shown that h-IL-2 (like other immunomodulators) does not act on “resting” lymphocytes.

 As a result of experiments conducted by the authors of the present invention, it was unexpectedly found that, contrary to the prevailing opinion of specialists in the human body, recombinant h-IL-2 has a stimulating effect on such "resting" lymphocytes. Apparently, this is due to the fact that recombinant h-IL-2 acts on some other cells of the human body, such as monocytes, endothelial cells or stromosepleen cells, which produce stimulation factors that lead to the appearance of receptors in “resting” lymphocytes, which allows h-IL-2 contact these receptors, causing the activation and proliferation of T cells. At the same time, the number of lymphocytes increases, their activity increases and a complex of immune reactions is triggered, leading to the conclusion of an almost completely suppressed immune system to a normal level of functioning and the resumption of all its components.

 As a h-IL-2, you can use any substance with the activity of h-IL-2, in particular various dosage forms of recombinant h-IL-2. Commercially the most affordable are rh-IL-2 preparations, obtained on the basis of recombinant strains of microorganisms (bacteria or yeast).

 Obviously, for medical purposes, it is advisable to use recombinant h-IL-2 produced by non-pathogenic microorganisms whose cells do not contain endotoxins such as lipopolysaccharides. Such are, for example, baker's yeast-saccharomycetes. In particular, the yeast recombinant h-IL-2 obtained from the recombinant strain of the yeast Saccaharomyces cerevisiae, deposited in the All-Russian collection of industrial microorganisms under the number VKMG-791, is of interest.

It is advisable to introduce h-IL-2 in an amount of 100,000 to 3,000,000 IU / day per 1 m ² of the patient’s body surface.

 The introduction of a dose below 100,000 IU does not enhance the proliferation of T-lymphocytes to a clinically significant one, a dose of more than 3,000,000 may already cause side effects in the form of a threatening decrease in blood pressure, severe allergic reactions, allergic shock, diarrhea caused by the action of the drug recombinant p-IL -2, which inevitably contains ballast proteins.

 It is advisable to measure the number of T-lymphocytes before and after the introduction of h-IL-2 and to reduce their number relative to the previous measurement to stop the introduction.

 Moreover, an increase in the number of blood lymphocytes before the next administration of the drug compared with the previous study indicates an effective effect on the immune system and serves as the basis for the next administration of h-IL-2. The absence of an increase in the number of blood lymphocytes reflects the depletion of the reserves of the immune system and is the basis for stopping the administration of h-IL-2.

The criteria for selecting patients for the treatment of immunosuppression caused by sepsis are as follows:
the presence of established foci and sources of infection;
increased - more than 38.5 ^o C or low - less than 36.0 ^o C body temperature;
tachycardia - more than 90 beats per minute;
shortness of breath - more than 20 breaths per minute;
the presence of at least one of the following symptoms: impaired mental status, hypoxemia, oliguria, increased lactate in the blood;
age of patients from 16 to 65 years.

h-IL-2 was not used to treat immunosuppression in sepsis in the following cases:
cirrhosis of the liver with portal hypertension;
respiratory failure associated with obstructive syndrome (in particular with pulmonary tuberculosis);
oncological diseases;
a history of allergic reactions;
the use of immunomodulatory drugs;
failure to comply with the conditions listed in the previous section.

 The severity of the patient's condition was assessed. The examination of the patient included registration of the main symptoms and assessment of the conditions of internal organs, hemodynamics, body temperature, diarrhea, etc.

 An integrated assessment of the severity of the patient's condition was carried out using the SAPS (Simplified Acute Physiology Score) scale widely used in clinical trials. This scale takes into account 13 main clinical and laboratory signs that reflect the functional state of the main systems and organs. The calculated SAPS values allow, on the one hand, to track the change in the condition of each patient before and after each administration of h-IL-2, and, on the other hand, to compare the control and experimental groups of patients according to the severity of the condition.

 Immediately before using the drug for the patient, the contents of the ampoule with the recombinant h-IL-2 yeast preparation with an activity of 1–2 million units are dissolved in 400 ml of sterile saline.

 The introduction is carried out parenterally by drip intravenous infusion through a central or peripheral vein lasting 5-6 hours. When h-IL-2 is introduced into the body after 5-6 minutes, up to 70% of the active substance already disappears from the bloodstream. To achieve the clinical effect of the administration of h-IL-2, it is preferable to maintain its concentration in the bloodstream at a constant level for some time. Therefore, the dose is administered not simultaneously, but within 5-6 hours. A longer administration is inconvenient from a practical point of view.

 The drug is administered once a day. In order to achieve a lasting clinical effect, it is possible to repeat the administration, but no more than 4 times, since for any particular patient a 4-fold administration should already provide immunostimulation and further doses will only lead to the depletion of the immune system (lymphocytes).

 It is preferable to take breaks of 2-3 days between drug injections, since during this time the previous dose of h-IL-2 is realized, that is, the immune system is in a state stimulated by the previous dose.

 The effect of the drug is evaluated clinically by reducing the pulse rate, stabilizing blood pressure, lowering body temperature and shortness of breath.

 Biochemical analysis shows a decrease in the leukocyte intoxication index, an increase in the number of lymphocytes in the peripheral blood, as well as a decrease in the concentration of keratin in the blood the day after administration.

 Immunological analysis shows on the 3rd day after the introduction of the normalization of the rate of spontaneous migration, stimulation of the bactericidal activity of leukocytes in the blood (according to luminometric analysis).

 The integral indicator of patient severity on the SAPS scale decreased on average by 4 points after the first injection, by 1.6 after the second, and 0.9 after the third.

 A group of patients, including 33 people, was tested, a comparison was made with a control group (50 people). The following are tables 1-5 with the test results.

 Example 1. A patient, 41 years old, was hit by a tram, diagnosed with multiple injuries of both lower limbs. Amputation of the type of "primary surgical treatment" was performed.

Starting from the second day, the minimum distance to the profile of the "metabolic imbalance" has already been diagnosed and positive signs of SSVR (systemic inflammatory response syndrome) have been noted. The temperature increased to 38.8 ^o C, tachycardia was 120 beats / min, tachypnea - 24 dd / min, leukocytosis - 27 • 10 ⁹ k / l. Infusion and antibiotic therapy continued, and anticoagulation therapy was started. Signs of SSVR persisted despite corrections in the therapy. The temperature rises reached 40.1 ^o C, leukocytosis 34 • 10 ⁹ k / l, stab shift 30%. Diagnosed with sepsis.

 On the 6th day after the injury, sutures were removed from the stump of the right thigh, where non-clostridial anaerobic cellulomyositis was diagnosed (Bacteriodes fragilis was found in the wound during microbiological examination). The operation "secondary surgical treatment". The operation was completed by a thorough infiltration of the surrounding tissues with an anti-inflammatory mixture.

The patient began the entire complex of therapy aimed at eliminating the systemic inflammatory response syndrome. The antibacterial therapy was changed - claforan was prescribed 2 g 4 times a day, gentamicin 80 mg 3 times a day, metrajil 100 ml 3 times a day. The amount of contracal increased to 90 thousand units per day. Anticoagulant therapy was prescribed in the mode of moderate hypocoagulation - 1 thousand units per hour, against the background of trental 5 ml 3 times a day, repoliglyukin - 400 ml, native plasma - 2 doses. The temperature decreased to 39.2 ^o C, however, tachycardia persisted - 110 beats / min, tachypnea - 23 dd / min, leukopenia developed - 3.9 • 10 ⁹ k / l, the lactate / pyruvate ratio increased to 48.

Due to the progression of signs of generalization of the inflammatory syndrome, characterized by inhibition of lecopoiesis, a sharp decrease in the relative and absolute number of lymphocytes, i.e. signs of increasing secondary immunodeficiency are visible, it was decided to conduct immunomodulating therapy using recombinant h-IL-2 yeast, obtained from a yeast strain VKPM-U791 (known under the name "Roncoleukin" ^TM ), at a dose of 1 million units per 1 m ² of the patient's body surface, intravenously, drip, 2 00,000 units / hour. The patient received two doses of recombinant h-IL-2 yeast on the 8th and 10th day after the injury. Both infusions suffered without any side effects.

The very next day after the first injection, the temperature decreased to 38.1 ^o C, the tachycardia decreased to 95 beats / min, the number of leukocytes increased to 7 • 10 ⁹ k / l with a persistent stab shift of 12% (after 30%), tachypnea remained in limits 22-23 dd / min. During ligation, there was a lack of additional sites of necrosis.

After repeated administration of yeast recombinant h-IL-2 on the 10th day after the injury, the condition of the victim completely stabilized, the maximum temperature on the 11th day did not exceed 37.5 ^o C, the number of stab neutrophils decreased to 6%, the number of lymphocytes increased to 18%, and in absolute terms - 1.98 • 10 ⁹ c / l, the number of segmented neutrophils increased to 73%.

Example 2. Patient, 42 years old, diagnosis: metroendometritis, pelveoperitonitis, sepsis, septic endocarditis. The condition is serious. Severe intoxication: pulse 108 beats / min, body temperature 38.7 ^o C, respiratory rate 36 per minute.

 Assessment of the severity of the condition on a SAPS scale of 9 points.

A single intravenous drip of yeast recombinant h-IL-2 at a dose of 3,000,000 units per m ² of the patient’s body surface was performed for 5 hours. The patient’s condition improved significantly the next day. Pulse 76 beats / min, body temperature 37.1 ^o C, respiratory rate 18 per minute.

 Assessment of the severity of the condition on a scale of SAPS 3 points.

On the third day, the condition is relatively satisfactory. Body temperature 36.7 ^o C, pulse 72 beats / min, respiratory rate of 16 per min.

 Assessment of the severity of the condition on a SAPS scale of 1 point. Sepsis is discontinued.

Example 3. Patient, 25 years old. Diagnosis: angiogenic sepsis, endomyocarditis, bilateral pleurisy, interstitial nephritis, ascites. The condition is serious. Pronounced edema. Pulse 100 beats / min, shortness of breath 24 breaths per minute, body temperature 38.3 ^o C. The number of blood lymphocytes 0.7 • 10 ⁹ k / l.

 Assessment of the severity of the condition on a SAPS scale of 6 points.

With an interval of 4 days, an intravenous drip infusion of recombinant h-IL-2 yeast was performed twice for 300,000 units per m ² of the patient's body surface. The next day after the first injection, normalization of body temperature (36.9 ^o C), a decrease in shortness of breath to 20 breaths per minute, tachycardia to 92 beats / min. Swelling of the extremities has not changed significantly. On the 5th day, edema decreased, the number of blood lymphocytes increased to 1.1 • 10 ⁹ k / l. After the 2nd injection of yeast recombinant h-IL-2, stabilization of the achieved improvement was noted, body temperature returned to normal. On the 3rd day after the 2nd injection of the drug, the patient's condition is moderate. No swelling. Pulse 88 beats / min, respiratory rate 20 per min, body temperature 36.4 ^o C.

 Assessment of the severity of the condition on a SAPS scale of 1 point.

The number of blood lymphocytes decreased to 0.9 • 10 ⁹ k / l. The infusion of h-IL-2 is discontinued. On the 9th day after the start of treatment using recombinant h-IL-2 yeast, the patient was transferred from intensive care to the general cardiology department.

Example 4. Patient, 45 years old. Diagnosis: skin burns of 2-3 degrees 15% of the body surface, suturing of stressful stomach ulcers, suppuration of the wound, sepsis. The condition is serious. Body temperature 40.0 ^o C. Pulse 108 beats / min., Respiratory rate 22 per minute. SAPS scale 9 points. The number of blood lymphocytes of 0.5 • 10 ⁹ k / l.

Two intravenous infusions of recombinant h-IL-2 yeast were performed, each of 500,000 U per m ² of the patient’s body surface, as described above. The interval between administrations was 5 days.

On the 5th day after the first infusion, the patient's condition improved slightly. Body temperature 39.4 ^o C, pulse 108 beats / min, respiratory rate 20 per min, SAPS scale 7 points. The number of lymphocytes increased to 0.9 • 10 ⁹ k / l.

Completed 2nd infusion of h-IL-2. On the 3rd day after the second infusion, the condition still improved. Body temperature 38.3 ^o C, pulse 96 beats / min, respiratory rate 18 per minute. SAPS scale 4 points. The number of blood lymphocytes decreased to 0.76 • 10 ⁹ k / l. Immunostimulation of interleukin-2 is discontinued.

Example 5. Patient, 57 years old. Diagnosis: polytrauma, frostbite of the lower extremities, shock-1, pneumonia, DIC, sepsis. The condition is serious. Pulse 100 bpm Body temperature 39.7 ^o C. Shortness of breath - 32 breaths / min. SAPS scale 10 points. The number of blood lymphocytes 1.75 • 10 ⁹ k / l.

A yeast recombinant h-IL-2 was infused at a dose of 100,000 IU per m ² of the patient’s body surface according to the procedure described above. The patient's condition improved slightly. Body temperature decreased. On the 3rd day, body temperature - 38.1 ^o C, pulse - 74 beats / min, respiratory rate 22 / min, SAPS scale of 8 points, the number of blood lymphocytes increased to 2.05 • 10 ⁹ k / l.

The 2nd infusion of recombinant h-IL-2 yeast was performed in the same dosage. The patient's condition has stabilized. 2 days after the 2nd infusion of h-IL-2: pulse 76 beats / min, body temperature 37.6 ^o C, respiratory rate 20 per min, SAPS scale 5 points, the number of blood lymphocytes 2.15 • 10 ⁹ k / l

A third infusion of yeast recombinant h-IL-2 at a dose of 100,000 units per m ² of the patient’s body surface was carried out. On the 3rd day after infusion, the patient's condition without significant changes. The number of blood lymphocytes decreased to 1.9 • 10 ⁹ k / l. Immunostimulation of h-IL-2 is discontinued.

 The invention can be used in medicine for the treatment of immunosuppression in sepsis, which is one of the most dangerous complications arising in the postoperative and post-shock period.

Claims (1)

A method for the treatment of immunosuppression in sepsis in humans by parenteral administration to a patient of recombinant human interleukin-2 obtained from yeast, characterized in that the latter is used interleukin-2 obtained from a yeast strain of the species Saccharomyces cerevisiae, deposited in the All-Russian collection of industrial microorganisms under the number VKPM -V791, which is administered in a dose of 100,000 to 3,000,000 IU / day per 1 m ² of the patient’s body surface.

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