The way the regulatory impact on the primary mechanisms of basic protective reactions of an organism in norm and pathology: immune responses, inflammatory reactions, free radical process, using substances gamma - glutamylcysteine

 

(57) Abstract:

The invention relates to medicine, in particular to the field of pharmacology, medicinal substances used for the prevention and treatment of diseases, the mechanisms of pathogenesis are the primary biochemical links are release from the depot of histamine and/or hyperactivation of free radical process. To do this, use the gamma glutamylcysteine in doses of 0.01 to 250 mg/kg of body weight per day. The inventive method compared to known allows you to combine immunocorrigirutee, anti-allergic, wound-healing, anti-inflammatory and antioxidant effects on the body. The proposed tool is non-toxic, with minimal doses has significant breadth of therapeutic action. 4 C.p. f-crystals, 15 ill., 7 table.

The invention relates to the field of pharmacology. The invention is a method of adjusting the activity of the primary biochemical protective systems of an organism in norm and pathology using substances gamma-glutamylcysteine. In particular, the regulation of the intensity of the immune response, inflammation, free radical process using gamma glutamylcysteine, without the latter's impact on this receptor mechanisms of regulation of the immune response lead to the development of allergic diseases, which suffers 25% of the population; there is a tendency to increase in their severity and increase in the frequency of occurrence, especially food allergies [1, 2, 3]. The inflammatory process is a major driver in the pathogenesis of many diseases and the primary link in the pathogenesis of post-traumatic and post-surgical conditions. The output from the control of the body's free radical process is a key element in the mechanisms of pathogenesis of radiation and chemical lesions of endo - and exogenous intoxications in the pathogenesis of early skin aging; free radical process also participates in the pathogenesis of major human diseases [4, 5].

Clinical analogues of the proposed method are methods for drug prevention and treatment of these diseases. In these methods, the use of chemical substances belonging to different classes of chemical compounds. The commonality between these methods is:

in their impact on the secondary, delayed stages in the mechanisms of development of pathological conditions;

in the unidirectional nature of their impact on the defensive, which leads, for example, to suppress both destructive and constructive phases vos is deradicalise reaction, with high enough, high toxicity, pronounced side effects, including initiating the ulceration in the gastrointestinal tract, with a wide range of contraindications, exhibiting pharmacological effects, usually in relatively high doses and with low therapeutic range.

The difference of the proposed method to existing methods is that in the present method:

the effect on the key initial steps in the mechanisms start pathological reactions,

the effect is of a regulatory nature, that is enhanced by constructive phase and attenuated destructive phase protective reactions,

used nexinnovations substance prevents hyperactively free radical process, exhibits pharmacological activity at low doses and does not show toxic effects in a physically feasible maximum doses, has no identified contraindications, protects from chemical ulcer formation in the gastrointestinal tract.

In allergic diseases apply competitive inhibitors of H1-histamine receptors. Pharmacological effect occurs when the conditions the drugs: the substance of chloropyramine hydrochloride (preparation "Suprastin"), its side effects - sedation, contra - activities requiring attention.

For the treatment of inflammatory diseases apply steroid and non-steroid drugs. Steroid drugs cause severe hormonal regulation and in this comparison are not used. Examples of non-steroidal drugs: substance ibuprofen (drugs Ibuprofen, Lamedon"), the substance is diclofenac sodium medication Diclofenac, Diclofenac sodium, Ortofen", "Voltaren"). Side effects: allergic reactions, increased blood pressure, dizziness, impaired functioning of the gastrointestinal tract. Contraindications - ulcer of stomach and duodenal ulcers and other

For the treatment of wounds apply two main groups of drugs. On the one hand, it is drugs that stimulate the actual healing of wounds, as a rule, difficult drugs of biological origin, such as "Solcoseryl". And on the other hand, preparations and tools for cleaning wounds from debris and serous effusion, and antimicrobial drugs.

For the prevention and treatment of diseases and lesions in the mechanisms of pathogenesis vadania skin and other - use the so-called "traps", "cleaners", "cleaners" (scavenger) already formed free radicals: antioxidants with polyunsaturated bonds, such as vitamin E, or chelate compounds of ions of metals of variable valency [4, 5].

Substance gamma glutamylcysteine was previously identified in mammals and invertebrates[6, 7, 8, 9].

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The biological role of substance gamma glutamylcysteine and the possibility of its pharmacological use is unknown.

The author of the present invention postulated and experimentally confirmed the regulatory effects of a substance gamma glutamylcysteine on the primary links of the biochemical mechanisms of basic defensive reactions of the organism immune responses, inflammatory reactions, free radical process.

Substance gamma glutamylcysteine and its dichloride salt, by order of the author, synthesized by a group of member-correspondent of the Academy of Sciences of the USSR, Evstigneeva R. P. using chemical peptide synthesis [10, 11, 12] with some modifications. By order of and under the supervision of the author performed a study of identity and purity of the party substances gamma-glutamylcysteine transferred to farmacologicas the m Fig. 1 and 2) and by thin layer chromatography with the results recording on computer densitogram CDS-200 firm Beckman /USA/. By the method of atomic emission spectrometry device GI-70 firms Glben-Ivon /France/ established that the content of heavy metals and toxic elements in weight percent (%) is a calcium - 0.1, lead, boron, copper, silver, zinc, arsenic, aluminum is not more than 0.01, manganese, strontium, chromium, cadmium, cobalt, magnesium, iron, Nickel is not more than 0.001.

EXAMPLES OF PHARMACOLOGICAL REGULATION OF ACTIVITY OF KEY PROTECTIVE SYSTEMS USING SUBSTANCES GAMMA-GLUTAMYLCYSTEINE

1. Regulatory impact substances gamma-glutamylcysteine on the primary links of the biochemical mechanisms of initiation/containment protective reactions.

1.1. Suppression of accumulation in the depot and release from the depot of histamine - the primary biochemical mediator of immune and inflammatory reactions.

Studies were performed on Wistar rats weighing 150-200 grams and Guinea pigs male weighing 250-300 grams. Used chicken ovalbumin production Olinekova NGO Biochimiche", once recrystallized us, and ampoule the drug "Suprastin" production company "Reanal" /Hungary/.

The influence of gamma-glutamylcysteine in vivo on the content of histamine in basophils blood was studied in Guinea pigs sensitized to ovalbumin by the method Shaternikova C. A. et al. [15]. On the 14th day sensitization solution of gamma-glutamylcysteine physiological solution was injected intraperitoneally at a dose of 50 mg/kg 1 hour before blood sampling. 1 ml of blood from the heart was taken in heparin, centrifuged. To the precipitate cells was added 9 ml of distilled water and 20 seconds to 1 ml 10x Hanks solution. The suspension was filtered through cotton wool, washed three times in medium Needle and then in Tris-buffer.

The content of histamine was measured spectroflame spontaneous secretion, which was 2-9%.

Statistical processing of results was performed using student's criterion.

The results are shown in Fig. 3. It is established that gamma-glutamylcysteine statistically reduces allergen-induced secretion of histamine from mast cells in the 3.65 times (0,318 mg of histamine per 1,000,000 cells against 1,161 mg in the control, P<0.01) and the reference product "Suprastin" 1,74 times (0,666 mg of histamine per 1,000,000 cells against 1,161 mg in the control, P<0,05). It is established that gamma-glutamylcysteine statistically significantly reduces the amount of histamine in 1.36 times in the fat cells (1,79 mg of histamine per 100,000 cells against 2,435 mcg in control, P<0.05) and 1.5 times in basophils (0,695 mg of histamine per 1 ml cell suspension against the 1.04 µg/ml in controls, P<0,01), "Suprastin" reduces the amount of histamine in 1.48 times in the fat cells (1,645 mg of histamine per 100,000 cells against 2,435 mcg in control, P<0,05).

The influence of gamma-glutamylcysteine and Suprastin" in vivo on the state of the cytochrome P450 studied on Guinea pigs weighing 250-300 g Gamma glutamylcysteine and Suprastin" was administered intraperitoneally at doses of 50 and the sleep. Geksenal was administered intraperitoneally in saline solution at a dose of 30 mg/kg Activity of cytochromes was measured by the method of Omuro N., R. Sato [17] in the microsomal fraction, isolated by the method of differential centrifugation. Fractional content of water-soluble and lipitorhistory cytochromes were determined by the method Izotova M. C. et al. [18] . The content of microsomal protein was determined by the modified method of Lowry [19]. Statistical analysis was performed using nonparametric criteria Mana Whitney and angular transformations Fisher [20] .

The results are shown in Fig. 4. It is established that gamma-glutamylcysteine statistically increases the specific activity of cytochrome P450 in 1.53 times (P<0.01), and the specific activity of cytochrome b5 - 1.64-fold (P<0.01), and the specific activity of water-soluble fractions of cytochrome P450 1.67-fold (P<0.01), and the ratio of water-soluble and lepidorostrus fractions of cytochrome P450 1.7 times (P<0,01). It is established that gamma-glutamylcysteine statistically significantly reduces the duration geksenalovy sleep 1.75 times (P<0,01).

Experiment to perform is ewww paw culture of Staphylococcus aureus (strain "375") in the amount of 5000000000000 bacterial bodies in a volume of 0.5 ml. In muscle the other paws injected with a solution of muriate of gamma glutamylcysteine physiological solution. On the ninth day, animals were scored, got the serum. In the serum was determined by the content of malonic dialdehyde in the reaction with thiobarbituric acid. To 0.2 ml of serum was added 1.5 ml of 8.1% sodium dodecylsulfate, 3.5 ml of 30% solution of acetic acid (pH 3.0), 1.5 ml of 0.8% solution thiobarbiturate acid, prepared in 30% acetic acid solution. The mixture is incubated for 60 minutes at 96 degrees Celsius, cooled at room temperature, centrifuged for 5 minutes at 1000 revolutions per minute. Spectrophotometry of the supernatant was performed at 535 nm and 560 nanometers. A sample containing biological material, photometrically against samples that do not contain biological material. The content of malondialdehyde was expressed in arbitrary units (ENm-ENm) x 100.

The results are shown in Fig. 15. It is established that the infection leads to two-fold statistically significant increase in the number of malondialdehyde. Hydrochloric acid salt of gamma-glutamylcysteine in the range of doses 12,60-22,07 µg/kg leads to the reduction of the dialdehyde 1.42 radical protection - cytochromes P450 under the influence of gamma-glutamylcysteine.

3. Regulatory impact of substance gamma glutamylcysteine on the activity of the immune system in health and disease.

3.1. Activating substance gamma glutamylcysteine primary immune response intact organism on heterogeneous antigens.

3.1.1. The activated b-cell-mediated humoral response immediate type.

Studies performed on adult mice-females of BALB weighing 18-20 g, were grown in the nursery "Pillar" of the USSR AMS. Mice were immunized intraperitoneally suboptimal dose (50000000) sheep red blood cells (EB). Then oral injected control animals received saline (n= 10) and experimental animals - solution of gamma-glutamylcysteine physiological solution (n=10). On the fifth day of the experiment, animals were scored by cervical displacement of the vertebra. Blood was obtained serum Spleen homogenized in a chilled environment "Hanks". In our experiments we used the individual cell suspensions of splenocytes with the viability of not less than 85-90% on the test with 0.1% solution of the dye tripney blue.

The influence of gamma-glutamylcysteine on the number antigenome volume of a suspension of DL (50000000 DL). The mixture is incubated for 18 hours at 4 degrees Celsius. Then counted the number of rosette cells with five or more red blood cells in preparations crushed drops with a microscope "Gravel" with increasing 25 x 0,50. In each study sample was counted the number of nucleated cells, to avoid non-specific assessment of the status of the cells.

The influence of gamma-glutamylcysteine on the amount of antibody productive cells was evaluated by test local DL hemolysis in gel agarose. Suspension of splenocytes (1,000,000 cells /ml) was mixed with a suspension of DL (0,5%), distributed in a thin layer on a Petri dish with a diameter of 100 mm and incubated 90 minutes at 37 degrees Celsius, was added to complement and incubated at 37 degrees Celsius for at least an hour. Counting the number of zones of hemolysis was performed using magnifier "MBS-9".

The influence of gamma-glutamylcysteine on the amount of synthetic antibodies-hemagglutinin was estimated by the method of titration. The serum was dcomplimentary, serially twofold diluted with microtitration "mostly saddle". Cultivation was mixed in the wells with 0.9% suspension of DL. Agglutination was considered visually in 24 hours. The titer of the test serum in the A.

Variation-statistical analysis was performed using the criterion of validation by the Student.

The results are shown in Fig. 5. It is established that gamma-glutamylcysteine in doses of 25 µg/kg body weight and 250 µg/kg increases the number of antibody productive cells in 2.7-fold (P<0.02) and 2.2 times (P<0,05), respectively, and increases the titer of hemagglutinin 1.3 times (P<0.01) and 1.2 times (P<0,05), respectively, without affecting the number antigenspecific cells and nucleated cells.

3.1.2. Activating substance gamma glutamylcysteine T-cell-mediated cellular response, delayed-type in the intact organism.

The investigations were carried out on outbred mice and mice of BALB weighing 18-20, Mice were immunized intraperitoneally suboptimal dose of a specific antigen - 5000000 DL. Then orally or intraperitoneally injected with a solution of gamma-glutamylcysteine physiological solution or saline. On the fifth day of the experiment in the pads of the hind paw was injected resolving dose (100000000) DL in 50 µl of saline. After 24 hours, the intensity of delayed-type hypersensitivity was assessed by the degree of increase of the pads with the help of Mick is the use of student test.

The results are presented in Fig. 6. As you can see, intact, healthy animals gamma glutamylcysteine using both methods of introducing a statistically reliable increase of delayed-type hypersensitivity to heterogeneous antigens, in particular oral dose of 25 mg/kg - 2.2 times (P<0.01), and a dose of 250 micrograms/kg (2.5 times (P<0.01), and intraperitoneal injection of a dose of 250 micrograms/kg to 2.1-fold (P<0,02). In mice of BALB during oral dose of 250 mcg/kg (2.5 times (P<0,02), intraperitoneal injection of a dose of 250 micrograms/kg to 2.1-fold (P<0,01).

3.2.1. Suppression substance gamma glutamylcysteine allergic reactions of immediate type.

Studies were performed on Wistar rats weighing 150-200 g and Guinea pigs weighing 250-300 g Sensitization in rats was carried out by chicken ovalbumin [13]. On the first and fifth days of the experiment in the neck region subcutaneously injected 20 μg of ovalbumin in 0.5 ml of physiological solution containing 200000 also microbial cells of pertussis vaccine. On the fifteenth day caused active anaphylactic shock by an intravenous injection of 20 mg of ovalbumin in 0.5 ml visitareslovenia cow's milk. Ovalbumin was administered orally within the first three days of the experiment in doses of 50 mg/kg, on the fifteenth day was administered intravenously resolving dose of ovalbumin (7 mg in 0.5 ml saline). Milk is administered orally for three days of 3.6 ml To obtain a resolving dose of milk was centrifuged and used a fraction, free from lipids and cellular elements, a dose of 0.5 ml (7 mg protein) was administered intravenously on the fifteenth day. The weight of active anaphylactic shock was estimated by the mortality rate, the number of seizures (or the state of prostration in rats) and largest anaphylactic index [21].

In Guinea pigs sensitized with ovalbumin and treated with gamma glutamylcysteine within three days prior to the introduction of the resolving dose of allergen, just before permitting the introduction of the allergen received blood. From the last received serum. Serum enzyme-linked immunosorbent method determined the content of Ig G to ovalbumin. To adsorbed in the wells of the blade to ovalbumin was added to the test serum, washed, and made a conjugated to horseradish peroxidase rabbit Ig G antibodies against Guinea pig, incubated, and washed. And iraty buffer with a pH of 5.9), after 20 minutes the reaction was stopped petermannia sulfuric acid and measured the optical density at 492 nm. A standard curve was obtained using dilutions hyperimmune antisera Guinea pigs immunized with ovalbumin.

The number of animals in groups, routes of administration and doses of medications given in the figure captions. Statistical analysis was performed using nonparametric criteria Mana Whitney and angular transformations Fisher [20].

The results of the study are presented in Fig. 7.1-7.5. It is established that gamma-glutamylcysteine starts to show antianaphylactic activity at a dose of 5 µg/kg For both species, when both allergens in various ways, including preventive and curative regimen of administration, doses of 50 mg/kg lead, statistically, to suppression mortality 0.6-4.0 times, reducing the frequency of seizures 1.75-2.4 times, to reduce anaphylactic index 1.6-2.3 times. When this drug comparison Suprastin (H1-blocker) shows significantly less protective activity (see Fig. 7.1-7.5).

In addition, gamma-glutamylcysteine (50 µg/kg 3 times, intraperitoneally) is Pete against 3,190,14 control /n=28/). Suprastin (1 mg/kg 3 times, through the mouth) decreases lg [S] from 3,210,10 control /n=43 to 2,920,06 experience /n=30/, (P<0,05). Under other experimental conditions, reducing the amount of Ig G not observed gamma-glutamylcysteine (5 mcg/kg 3 times, intraperitoneally) - 3,160,09 experience /n= 30/ and 3,190,14 control /n=28/, suprastin (1 mg/kg 3 times, intraperitoneally) - 3,120,18 experience /n=30/ and 3,190,14 control /n=28/, gamma glutamylcysteine (50 µg/kg 3 times, through the mouth) - 3,150,11 experience /n=33/ and 3,210,10 control/n=43/.

3.2.2. The impact of substance gamma glutamylcysteine chronic inflammatory reaction caused by hypersensitivity of the delayed type.

Studies performed on inbred white rats-males weighing 140-160 g To play adjuvant arthritis full beta-blockers (5 mg BCG/ml) at doses of 0.1 ml was injected intradermally at the base of the tail for 22 days. The joints were subjected to ecometry. The severity of arthritis was expressed through the arthritic index index - growth of the affected joints in relation to the original percentage. The function of the joints tested by the ability of rats to remain on the inclined metal lattice. Gamma glutamylcysteine was administered in doses of 50 mg/kg intramuscularly daily. Protivovospalitelno - saline - injected control animals daily. The group contained 10 animals.

It is established that at the peak of the development of adjuvant disease (22-day) arthritic index is (Mm): control - 166,23,9%, in the experiment in animals treated with gamma glutamylcysteine - 139,86,4%, in the experience of animals treated with Ibuprofen - 126,65,8%. That is, the degree of reduction was 26.4% and 39.6%, respectively. Thus, gamma-glutamylcysteine shows antiarthritic activity, but somewhat smaller than the comparison drug Ibuprofen.

4. Regulatory impact of substance gamma glutamylcysteine acute inflammatory response.

Studies, mainly performed according to the "Methodological recommendations" of the Pharmacological Committee of Ministry of health USSR, 1986 [22].

4.1. The decreased activity of acute exudative process of chemical Genesis models edema of the subcutaneous tissue and aseptic peritonitis.

Acute exudative inflammation of the paws of rats caused by subplantar introduction white mongrel intact or adrenal-acetominoven rats (180-200 g) 0.1 ml of 1% solution carragenin, or 2% formalin solution or a 0.1% solution of histamine. Adrenalectomy carried agicheskii solution was administered 4 hours prior to the introduction of phlogogenic agent to identify preventive effect or daily for five days in order to identify therapeutic effect. The volume of paw was measured ecometrics on pletismography "Ugo Basil" /Italy/. The pharmacological activity of the drug was expressed as percent reduction of edema compared with control taken as 100%. Each group had at least 10 animals.

The model of aseptic peritonitis played on outbred white mice (20-22 g) and rats (200-240 g), which was administered intraperitoneally in 0.1 ml of 0.2% solution of silver nitrate and 6 hours were measured amount of ascitic fluid. The solution of gamma-glutamylcysteine physiological solution or saline was administered 40 minutes prior to inoculation of silver nitrate.

It is established that gamma-glutamylcysteine reduces the amount of ascitic fluid 4 times in mice and 3 times in rats (see Fig. 8.1 and 8.2). Gamma glutamylcysteine in doses of 15 to 50 µg/kg and for different routes of administration reduces swelling of the paws of rats by 50-80% (see Fig. 9). Antiexudative activity of gamma-glutamylcysteine manifests itself in adrenalectomized animals. Therefore, this activity develops through mechanisms not involving a well-known mechanisms for the suppression of the inflammatory response of steroid hormones. The effectiveness of gamma-glutamylcysteine in doses up to 50 mcg/kg is comparable to her chemical Genesis model cysteamine stomach ulcers and duodenal ulcers.

Animals were kept in individual cages with wire floors. The day before the experiment, animals were deprived of food, while maintaining access to water. Model of acute chemical ulcer of stomach and duodenum reproduced in outbred female rats weighing 170-245 g by a factor of three (in 10, 14 and 18 hours of the day) intragastric administration of a solution group probably facilitates dose of 300 µg/kg 1 hour before each injection group probably facilitates the animals of the experimental group was administered intragastrically solution of gamma-glutamylcysteine (20 mg/kg 3 times) in physiological solution, and animals of the control group with saline. The slaughter of animals was carried out 48 hours after the first injection group probably facilitates. Disclosed on the greater curvature drugs stomachs with adjacent segments of the duodenum were fixed for 10 minutes in 2% formalin solution. The length of ulcerative lesions was measured using a binocular magnifier with 8-fold increase.

Acute reserpine stomach ulcers in rats reproduced on inbred white rats-females weighing 115 to 170, Reserpine was administered intraperitoneally at a dose of 8 mg/kg Gamma-glutamylcysteine (20 mg/kg 2 times) in saline was administered intragastrically twice in the head after injection of reserpine.

Chronic acetate plague rats reproduced in outbred white rats weighing 135-195, 50 μl of glacial acetic acid through a polypropylene tube with a diameter of 6 mm applicable within 30 seconds preneuring part of the glandular region of the stomach. From the second day of the experiment daily for ten days twice a day (9 and 16 hours) intragastrically injected control animals received saline (5 ml/kg) and experimental animals - gamma glutamylcysteine (20 mg/kg) in adequate amounts of saline. On the twelfth day of the experiment, animals were scored.

Secretory activity was studied in inbred white rats-females with body mass 195-255, the Pylorus of the stomach was ligated under light ether anesthesia. Immediately after ligation vnutriaortalina was injected control animals received saline (5 ml/kg) and experimental animals - gamma glutamylcysteine (100 µg/kg) in the same volume of saline. After 4 hours was carried out the slaughter of animals. Stomach contents were centrifuged (4000 rpm, 20 min). The volume of stomach contents was measured in a graduated centrifuge tubes. The acidity content was measured by titration with 1 ml of the image is out gamma glutamylcysteine leads to a fourfold reduction in the number of animals, affected by ulcers, and nine-fold reduction in length educated ulcers 0,310,28 mm against 2,810,90 mm in the control /R<0,02/ (see Fig. 10.1, 10.2).

At the same time, gamma glutamylcysteine does not affect the development of gastric ulcers and duodenal ulcers as a model of acute reserpine gastric ulcers in rats: length ulcers is 19,61,9 mm in experience and 20,43,7 mm in the control, the severity of injuries by-point scale 2,100,23 in the experiment (n=10) and 1,800,33 in control (n=10), and a model of chronic acetate ulcers in rats, the damaged area in square millimeters is 32,18,2 in the experiment (n=6) and 43,113,4 in control (n=6).

Gamma glutamylcysteine not also affects the secretory activity of the gastrointestinal tract of rats: the rate of secretion of gastric contents /μl/h/ equals 48053 in experience and 58151 in control, acidity /mEq/ml/ equals 81,15,7 in experience and 89,94,3 in control, the rate of secretion of H /mkacf/h/ equals 42,58,0 in experience and 52,55,2 in control.

Thus, protection from cysteamine ulcers due to, apparently, not antiulcer action as such, but antiradical action of gamma-glutamylcysteine through a system of cytochromes.

4.3. The reduction in pain intensity on the model chemical intraperitoneal introduction of a 2% solution of acetic acid. Gamma glutamylcysteine physiological solution or saline was administered 30 minutes prior to the introduction of acid. The number of groups of 10 animals. The severity of the protective effect of the drug was assessed by the decrease in the number of "cramps" (percentage of control) within 20 minutes of observation.

It is established that gamma-glutamylcysteine reduces the amount of "cramps" 70% intraperitoneal dose of 20 mg/kg, 50% after intragastric dose of 20 mg/kg, 35% when administered by intramuscular dose of 50 mcg/kg comparison Drug Voltaren reduces the amount of "cramps" 50% after intragastric dose of 25 mg/kg due To the fact that the gamma-glutamylcysteine does not affect neurotransmitter systems (see section 6.4), the effect of anesthesia on the model of "cramps" perhaps explained his protivoradikulitnyy activity.

4.4. The increase under the influence of gamma-glutamylcysteine activity of lysosomal enzymes in the tissues surrounding the area of alteration on the model of myocardial infarction.

Myocardial infarction was reproduced in outbred rats weighing 250-300 g were Bandaged the descending branch of the left coronary artery in the region of the heart, which is 2-3 mm above the border of ear left W the and was about 50%. Gamma glutamylcysteine was administered intravenously daily doses of 100 mg/kg for 2 weeks (5 rats). In the control was also 5 rats.

After a specified time on a cold extracted infarction. It washed, cleaned of connective tissue, removing the atrium, was isolated areas surrounding the zone of necrosis of the left ventricle, and areas of the right ventricle, weighed, crushed with scissors and homogenized in a glass homogenizer (Potter-Alfama with Teflon pestle (clearance 0.21 mm) for 180 seconds at 40 revolutions per minute in ice-cold buffer solution of 0.6 M KCl (H. H.), 0.25 M sucrose (H. H.), 10 mm imidazole ("Serva", Germany), 1 mm EDTA ("Serva", Germany), pH of 7.25. The mass ratio of fabric and environment in the homogenate was 1:10.

In an ultracentrifuge "Beckman" /the USA/ the homogenate was subjected to differential centrifugation. The supernatant (cytosol) was determined by the activity of dissolved lysosomal enzymes. The fraction of sediment resuspendable 1:1 in buffer solution (0.7 M sucrose, 1 mm EDTA, pH 7.0) and divided it into two parts, one of them was determined available for substrate activity of enzymes intact lysosomes, the other part was subjected to freeze-thawing in liquid nitrogen to destroy the lysosomes and is cosaminds conducted with the substrate 4-nitrophenyl-M-acetyl--D-glucosamine (5 mm) in 0.2 M acetate buffer, 0.7 M sucrose, pH 5.5. To 100 μl of the incubation mixture was added 20 µl sample material, incubated 20 minutes at 37 degrees Celsius. The reaction was stopped with 0.5 ml of 0.5 M NaOH solution. Photocolorimetry performed on a spectrophotometer "DH-8B"/"Beckman, USA/ at a wavelength of 405 nm. The specific activity of enzyme was expressed in nanomolar released for 1 minute 4-NITROPHENOL per 1 mg protein. Protein was determined by the method of Lowry.

The results are presented in Fig. 11. It is established that gamma-glutamylcysteine statistically increases the activity of the enzyme in the lysosomes. In periinfarct zone activity in a fraction of the destroyed lysosomes is in the experience 2,640,08 Ed. (the range of variation of 2.26-2,87 Ed.) against 1,40,01 Ed. (1,38-1,42 Ed. in the control, i.e. the activity increased by 89%. 45% increased activity in a fraction of cytosole: 1,130,06 Ed. (0,94-1,23 Ed.) against 0,780,02 Ed. (0,73-0,83 Ed.). 22% increased activity in the test for permeability of membrane to substrate 1,110,05 Ed. (0,98-1,24 Ed.) against 0,91+0,05 Ed. (0,77-1,04 Ed.).

In the right ventricle is also observed changes in the activity of the enzyme. In a fraction of disintegrated lysosomes - an increase of 28%: 2,43 0,20 Ed. (2,07-is 3.08 Ed.) against 1,90,05 Ed. (1,78 e 2.06 Ed.) in the control. Despite this increase, the activity Rast is the Rupp /0,720,03 Ed. (0,63-0,79 Ed.)/. And the permeability of the membranes of lysosomes to the substrate decreased by 39%: 0,520,06 Ed. (0,36-0,65 Ed.) against 0,840,05 Ed. (0,72-0,96 Ed.).

Thus, the data obtained indicate systemic exposure gamma glutamylcysteine lysosomal apparatus, reflected, on the one hand, to increase the capacity of lysosomal hydrolytic apparatus and, on the other hand, in the gain control of this unit from lysosomal membranes.

4.5. The decrease under the influence of gamma-glutamylcysteine mortality of animals, square RAS, suppression of serous effusion, the acceleration of self-cleaning of debris on the model of chemical skin burns.

Research, in General, performed according to the "Methodological recommendations on experimental (preclinical) study of non-steroidal anti-inflammatory pharmacological substances (Official publication)"/. In kN. "Guidance on experimental and clinical study of new drugs (Official edition), Part 6, pages 51-62 / the USSR Ministry of health, Department for introduction of new drugs and medical devices, Pharmacological Committee / M., 1986.

Model chemical skin burns reproduced on white inbre what about were injected intraperitoneally with saline 0.2 ml or hydrochloric acid salt of gamma-glutamylcysteine (19 ág/kg) in 0.2 ml of saline. Next day took into account the number of surviving animals, size of the wound, presence of necrotic masses. In the experience of the ground with a 3% solution of acid used in 14 animals in the experimental and control groups. In the experience of the second with a 2.3% solution of acid used in 16 animals.

The results are shown in Fig. 12.1 - 12.3. In the control group died in the first experiment - 8 animals of 14, in the second experiment - 3 animals from 16. In both experiments, all animals survived, gained gamma glutamylcysteine. In animals that received gamma glutamylcysteine, the average size of wounds decreased in comparison with such for surviving control animals 2.4 times in the first experiment and about 10 times in the second experiment. While in the second experiment, in fact, of the 16 experimental animals wound formed in only 1 animal, although comparable in size with wounds in control animals.

Importantly, in both experiments, animals received gamma glutamylcysteine, within 8 hours after acid burn (first observation), the surface of the RAS has no necrotic masses and fibrinogen plaque; it is pure, pink, glossy in appearance. And this pattern is maintained until complete healing of wounds. In animals that have not received gamma knife is CLASS="ptx2">

4.6. Activation of the growth of granulation tissue in the wound under the influence of gamma-glutamylcysteine.

Model "cotton granuloma reproduced on white inbred rats male. Sterilized in alcohol cotton balls weighing 15 mg implanted subcutaneously. On the eighth day of the granuloma was removed, weighed, dried to constant mass at 55 degrees Celsius and weighed. The mass of newly formed granulation-fibrous tissue was determined as the difference between the masses of dried granuloma and implanted with a cotton ball.

The results are presented in Fig. 13. It is established that gamma-glutamylcysteine in doses of 50 mg/kg and 100 mcg/kg increases the growth of granulomatous tissue at 94% and 74%, respectively, and the comparator drug Ortofen (5 mg/kg), in contrast, reduces the growth of tissue to 41%.

4.7. The acceleration of wound healing of oral mucosa of rats under the influence of gamma-glutamylcysteine.

White outbred rats weighing 200-220 g were caused mechanical damage to the mucosa of the oral cavity. The treatment was carried out daily by irrigation of the wound with a solution of gamma-glutamylcysteine distilled water (1 mg/ml), control animals received the wound was irrigated with distilled water is epithelium.

The results are presented in Fig. 14. It is established that the disappearance of redness and epithelization of wounds of the oral cavity in treated animals comes the third or fifth day, the untreated animals for 7-8 days.

4.8. Conclusions: suppression exquisitely (excessive, extreme) part in non-constructive (destructive) phase of the inflammatory response and activation of constructive (recovery) phase of the inflammatory reaction under the influence of gamma-glutamylcysteine.

Thus, experiments were carried out to study the effects of gamma-glutamylcysteine on deconstructive phase of inflammatory reaction showed that this substance reduces the intensity of certain elements of the deconstructive phase and deconstructive phase, in General:

reduction of acute exudative process of chemical alteration fabrics,

reduction of ulcer formation in chemical alteration of the gastric mucosa,

reducing the number of "cramps" if chemical alteration of the tissues of the abdominal cavity,

reducing mortality of animals and size of wounds in chemical alteration of the skin.

It is possible that the mechanisms involved in the decrease of intensity of destructive reactions in the acts of:

reduction triggered by the release of histamine - the primary biochemical mediator of the inflammatory response,

the increased activity of the enzymes of the P450 system protection from free radical process,

increasing the strength of lysosomal membranes, which should lead to a reduction of secondary damage healthy tissue from the released lysosomal enzymes.

At the same time, the experiments showed that the substance gamma glutamylcysteine leads to an increase in the intensity of the recovery phase of the inflammatory response:

the sharp acceleration of the process of self-cleaning wounds from non-viable cells and fibrin, apparently, due to the increase of the specific activity vnutrilaboratornyj hydrolases,

accelerate the growth of granulation tissue in the treated wound

the increase in the rate of wound healing.

5. Hepatoprotective activity of the substance gamma glutamylcysteine.

Acute toxic damage of the liver of mice was induced by single intraperitoneal introduction of carbon tetrachloride dose DL50. The solution of gamma-glutamylcysteine (50 µg/kg) was administered 1 hour before priming and then after 24 hours. The protective effect was evaluated by an index having survived the Yeni rats caused by daily for 4 days of injection of carbon tetrachloride in single doses of 0.4 ml/100 g body weight in the form of a 50% solution in liquid paraffin. Gamma glutamylcysteine (50 µg/kg) or Essentiale (50 mg/kg) was administered intramuscularly daily for 10 days. On the following day the animals were scored. Investigated the activity of enzymes in the serum spectrophotometric methods. The lipid composition of the liver was studied by thin-layer chromatography in the system hexane:diethyl ether: glacial acetic acid 80:20:2, chromatogram was assessed spectrophotometrically after elution stains lipids, manifested phosphorus molybdenum acid, the results were expressed as the percentage distribution of the fractions. Performed well as histological examination of liver tissues, the results expressed in terms of the index lesion in points.

Found that in acute experiments gamma glutamylcysteine compared with Essentiale increased the survival rate of 17-20% and intragastric, and intramuscular.

In the chronic experiment in the tissues of the liver are observed marked degenerative changes centrolobular vacuolization fatty degeneration with loss of beam structures, events karyolysis of hepatocytes, with small inflammatory infiltrates consisting of lymphocytes, the expansion of the Central veins and sinusoids. the nchima with centrolobular vacuolization dystrophy, the extension sinusoidal.

In the animals treated with gamma glutamylcysteine marked reduction in the severity of fatty degeneration as compared to untreated animals. Three animals were observed moderate fatty degeneration violation of beam structures, with the phenomena of Pinoso and karyolysis individual hepatocytes. Four animal fat droplet degeneration of individual hepatocytes, beamed structure intact, nearly normal, inflammatory infiltrates are absent, sine wave almost normal. Index of liver damage points: intact animals - 0, Tatralandia-tetrachloride - 1,4, Tatralandia + Essentiale - 0,9, Tatralandia + gamma glutamylcysteine - 0,9.

Thus, gamma-glutamylcysteine shows hepatoprotective activity equal to that of Essentiale. But is this activity, presumably through other mechanisms of action that differ from those used for the comparison drug. This is evidenced by the results of the study of biochemical parameters of blood and liver (see table 7).

6. Pharmacological safety of the substance gamma glutamylcysteine.

6.1. Testing of acute toxicity of gamma-glutamylcysteine outbred mice-males.

Guinea pigs (n=5) weighing 70010 g gamma glutamylcysteine physiological solution was introduced at a dose of 814 mg/kg intraperitoneally. In the next few hours after injection and then within two weeks of any changes in condition or behavior of the animals was not observed. Thus, the maximum dose exceeded therapeutic dose 16-33 times.

Mice weighing 25 1.5 g were injected intraperitoneally in 0.1 ml of physiological solution with gamma glutamylcysteine, the dose of which was 100 µg/kg (n= 9), 1000 ág/kg (n=9), 10000 µg/kg (n=5), 20000 mg/kg (n= 10). Nor in the next few hours or within two weeks of any changes in condition or behavior of the animals was not observed. Thus, the maximum dose exceeded in therapeutic 400-800 times.

Mice weighing 201 g were injected intraperitoneally once a solution of gamma-glutamylcysteine physiological solution with the maximum concentration of gamma-glutamylcysteine 60 mg/ml Volumes of injectate was 1 ml, and the maximum volume of 1.2 ml and, accordingly, the maximum dose of 3.2 g/kg (n=10). With the introduction of maximum doses are not marked expressed outward signs of intoxication. Within the first 30 - 40 minutes after administration was observed, prichina 14 days of any deviations are not observed. Thus, the maximum dose exceeded therapeutic dose in 64000-128000 time.

Mice weighing 201 g was administered intragastrically probe solution gamma glutamylcysteine at a concentration of 200 mg/ml physiological solution in two doses of 0.5 ml with an interval of 40 minutes. Maximum dose of gamma glutamylcysteine was 10 g/kg (n=10). Within the first hour after injection was observed associated with the reaction of animals to the introduction into the stomach of a large volume of fluid, the signs of some of excitation: grooming, upright position. Next, within 14 days of any deviations in the condition of the animals was not observed. Thus, the maximum dose exceeded therapeutic dose in 200000-400000 time.

Consequently, the substance gamma glutamylcysteine can be attributed to the drug class V toxicity practically non-toxic substances (fourth group of experiments), or to drugs group VI toxicity is relatively harmless substances (the third group of experiments). Given the magnitude of the proposed therapeutic dose of 5-50 μg per kg of body weight, it is possible to predict a greater breadth of therapeutic action of the substance gamma glutamylcysteine.

6.2. Immunologicals the recommendations for assessment of allergenic properties of pharmacological agents" of the Pharmacological Committee of Ministry of health USSR, 1988 [23].

6.2.1. No sensitizing activity of gamma-glutamylcysteine relative to the intact organism in hypersensitivity reactions of the delayed type /GST/.

The experiments were performed on mice. Gamma glutamylcysteine in doses of 25 mg/kg or 250 mg/kg of a water-soluble emulsion (1:1) incomplete adjuvant's adjuvant in a volume of 60 μl was injected into the tail. On the fifth day after sensitization in pad rear paws were introduced allowing the dose of gamma glutamylcysteine 25 or 250 μg/kg in a volume of 40 µl of physiological solution. Control animals were injected with an adequate number of incomplete adjuvant's adjuvant with saline solution (1:1) and the resolving dose of gamma glutamylcysteine. The severity of the reaction GST account within 24 hours according to the difference between the thickness of the foot control and experimental animals. The number of groups of 10 animals.

It is established that ecometrica indicators (/Mm/ in mm) are in control - 0,15+0,02 (range data range from 0.11 to 0.19), at a dose of 25 mg/kg - 0,180,03 (from 0.16 to 0.24), at a dose of 250 mg/kg - 0,210,02 (from 0.12 to 0.24). The difference between statistically significant.

6.2.2. No sensitizing activity of gamma-GT/.

The experiments were performed on inbred Guinea pigs. The number of groups of 10 animals. Sensitization gamma glutamylcysteine was performed twice with an interval of a week of single doses of 5 mcg in the first group and 50 mcg in the second group. Gamma glutamylcysteine was injected under the skin of the right side. Immediately before the first injection of gamma glutamylcysteine was injected emulsion incomplete adjuvant's adjuvant with saline solution (1:1) in the pads of the hind legs. The control animals at the same time was introduced only incomplete adjuvant's adjuvant (1:1) and solvent. The resolving dose of gamma glutamylcysteine (50 µg) was administered by intracardiac at the twenty-first day after reimmunization.

It is established that the introduction of the resolving dose does not lead to significant changes in the state of sensitized and control animals.

6.2.3. Gamma glutamylcysteine it reinforces the process of sensitization of the intact organism to heterogeneous protein antigen.

Studies performed on Guinea pigs (weighing 300 g) on the model of anaphylactic shock. Animals were senzibilizirani once horse Ig G (100 µg intramuscularly). Additionally, the pads of the hind paw was injected control imputability in 0.2 ml of a mixture of incomplete adjuvant's adjuvant with saline solution (1:1). On the fourteenth day of the experiment was carried out by allowing the injection of antigen (Ig G 0.6 mg intracardiac). The severity of allergies to the protein antigen was estimated by the formula Weigle.

It is established that gamma-glutamylcysteine in the range of doses of 200-250 mg/kg does not affect the process of allergization induced equine Ig G: control - 3.5 units, the experience of 3.5 units.

6.3. Gamma glutamylcysteine not have a cardiotoxic effect.

In the experiments used rabbits breed "chinchilla" weight 2.5-3 kg and white outbred rats weighing 250-300 g

Experiments for the study of Central and peripheral hemodynamics was performed under local anesthesia with 0.5% solution of novocaine. Elektroanaliticheskie determine the maximum and minimum blood pressure in the Central end of the common carotid artery. Then under geksenalovy anesthesia invasive method was determined by the contractile function of the heart ventricles. Open heart in the cavity of the right and left ventricles were introduced cannula and registered elektroanaliticheskie real intraventricular pressure. Then determine the maximum pressure developed by the ventricles in isometric conditions the lateral pressure in a five-minute occlusion of the ascending aorta to the left ventricle and the pulmonary artery to right ventricular ECG was recorded in a standard abstraction and calculated the duration of the phases of systole and diastole. Isoproterenol myocardial damage reproduced by intravenous isoproterenol at a dose of 15 mg/kg and after 5-6 minutes, injected with a solution of gamma-glutamylcysteine.

Ischemic myocardial infarction was created in rats by ligation of the descending branch of the left coronary artery in the region of the heart, which is 2-3 mm above the boundary loops of the left ventricle. Mortality of animals after the creation of ischemic necrosis was about 50%. Gamma glutamylcysteine was injected intravenously in physiological solution.

Tissue myocardial infarction were selected for biochemical study (see section 4.4) and for electron microscopic examination. In the latter case, the heart was perfesional a 2.5% solution of glutaric dialdehyde under pressure 80-120 mm Hg for 8-10 minutes, followed by excision of the plots in infarction and periinfarct areas. The fixation of these sections was performed in a 2.5% solution of glutaric dialdehyde and the final fixation in 1% buffered solution of camerahouse OS. The material is then poured into araldit and examined using electron microscope Tesla BS-540".

The influence of gamma-glutamylcysteine on the excitability of cardiomyocytes was studied on the preparation of izolirovani of 7.2 to 7.4 at room temperature. Optionally, the bath was added gamma glutamylcysteine. Microelectrodes with resistance 5-15 Mω were filled with a solution of 3-molar potassium chloride and injected into the ventricular fibers.

Found that in rabbits intact or suffering from acute or chronic isoproterenol myocarditis and intact rats or suffering from myocardial infarction gamma glutamylcysteine in acute or chronic introduction does not cause significant changes both peripheral and intracardiac hemodynamics (see tables 1-3).

Elektronno-microscopic picture infarction and periinfarct zones in animals treated with gamma glutamylcysteine within two weeks, do not differ from those in animals not treated with the substance. So, when the two-week observation infarction in the areas of experimental and control animals showed extensive areas of MOLISA, pronounced intra - and extracellular edema, homogenization and leaching of the mitochondrial matrix, periinfarct zones revealed a mosaic pattern: alternating regions expressed destructive changes with good plots.

The results of the study excitability of cardiomyocytes shown in table 4, where VCG does not affect the performance of excitability. Changes in excitability observed at doses of 300 μg/kg, 700 μg/kg, i.e. at doses 6-28 times the effective therapeutic dose for mammals. However, the absence of change of the slew rate of the rising phase of the action potential indicates that gamma glutamylcysteine not affect the density of fast, directed into the cell current of sodium ions and does not change the rate of membrane depolarization. Perhaps, registered the increase of the amplitude of the spike and overshoot is due to the increase of fast incoming current of calcium ions (at the end of phase 0). The lengthening of the plateau of the action potential under the action of large doses shows the potentiation of slow calcium channels and an increase in the density of the inward flow of ionized calcium and the reduction of the steepness of the descending phase of the action potential phases of repolarization is about weakening facing potassium flows. At the same time, no reduction in the level of the resting potential may indicate the absence of membranopovrezhdayuschih properties of the substance gamma glutamylcysteine.

6.4. The lack of influence of substances gamma-glutamylcysteine on receptor systems.

6.4.1. Tests on skin painful chuvstvovany light beam. Statistical processing of the results using student's criterion.

The Mature white rats swelling of the feet caused subplanetary the introduction of 0.1 ml of 1% solution carragenine /n=10/. The research was done on analgesiometer "Ugo Basil" /Italy/. It is established that gamma-glutamylcysteine (50-100 µg/kg, intramuscularly) does not affect the pain threshold (see table 5).

In the test of "hot plate" (55 degrees Celsius) in adult albino rats apparatus Ugo Basil" the duration of the latent period of painful reaction (licking paws, jumping) did not change within 90 minutes after intramuscular injection of a solution of gamma-glutamylcysteine at doses of 50 and 100 mcg/kg (see table 5).

Antinociceptive properties investigated in mice male BALB weighing 18-22 g using analgesiometer "812" company "Hugo Sachs Electronic KG /Germany/. As thermal stimulus used a focused beam of heat rays of not longer than 7 seconds when the initial reaction 2-4 seconds. The experiments were carried out on a series of animals in one week during days 11-14 o'clock in the afternoon.

The results of the study antinociceptive properties are presented in table 6. As wit on the latent period nociceptive reactions.

6.4.2. The lack of effect on the airway smooth muscle of isolated organs.

The experiments were performed according to the guide Blatter, M. and et al. [24]. Used organs Mature inbred animals. The day before the experiment, animals were deprived of food. Guinea pigs males were killed by the displacement of the cervical vertebrae, male and female rats - blow on the head and perform phlebotomy. Substance gamma glutamylcysteine and substance comparison was dissolved in distilled water. Use the following neurotransmitters and their analogues: serotonin, histamine, bradykinin, oxytocin, karbaholina agonist m - and n-cholinergic receptors, pyrilamine antagonist H1-receptors, cimetidine antagonist H-receptors cyproheptadine at - blocker of serotonin receptors, opioid peptide dalargin, blocking the release of acetylcholine from presynaptic nerve endings, mezaton, izadrin.

6.4.2.1. The ileum of the Guinea pig in isotonic conditions of registration contractile activity.

A segment of ileum length of about 1 cm were placed in a continuously aerated in a glass cell, 10 ml hypocalcaemia (0.9 mmol of calcium chloride) solution Tirade, pH 7,2-7,4, with temperatures of 35 degrees Celsius. Segm the SE). Signal through bridgeable amplifier "301" HSE taken to the Registrar "R-64" ("Rikadenki Kooyo" /Japan/). The initial elongation of the segment amounted to 0.5 cm After a period of equilibration (30-40 minutes) in a cell made of aqueous solutions of the investigated substances in amounts of 5-25 ál. Each subsequent introduction was made after 3-4 times the money laundering authority for 6-9 minutes. Quantitative changes in activity were expressed as mean values of changes in the amplitude of contractions relative to the maximum response at the appropriate neurotransmitter.

When the quantitative assessment found that the amplitude of contractions on the karbaholina (3/100000000 mol/l) is 506% of the maximum response and almost no changes to the background prior (two minutes) added gamma glutamylcysteine in concentrations 1/1000000000 mol/l and 1/100000 mol/l: 528% 488%, respectively. The response to histamine (3/10000000 mol/l) is 519% of the maximum and does not change on the background of the action of gamma-glutamylcysteine in concentrations 1/1000000000 mol/l and 1/100000 mol/l: 489% 547%, respectively. Response to serotonin (1/1000000 mol/l) is 557% of the maximum, and almost no changes to the background of the action of gamma-glutamylcysteine in concentrations 1/1000000000 mol/l and 1/100000 mol/l: 436% and contractile activity.

A segment of ileum was placed in conditions similar to the above, with the following exceptions: a modified Krebs solution, the sensor isometric registration "To 30" HSE, the initial tension of the body was equal to 1, the Electrical stimulation of the segment was performed using plate electrodes "F 2H" HSE and stimulator "215" HSE single rectangular pulses with a duration of 1 MS and a frequency of 0.1 Hz at a voltage of 80 C. changes in the amplitude of contractions was expressed in percentage to the original level, taken as 100%. The change in the amplitude of contractions characterizes the direct or indirect effects of substances on the release of acetylcholine from presynaptic nerve endings or by blocking acetylcholine receptors postsynaptic localization.

When the quantitative assessment found that the amplitude of contractions on the karbaholina (3/100000000 mol/l) is 506% of the maximum response and almost no changes to the background prior (two minutes) added gamma glutamylcysteine in concentrations 1/1000000000 mol/l and 1/100000 mol/l: 528% 488%, respectively. The response to histamine (3/10000000 mol/l) is 519% of the maximum and does not change on the background of the action of gamma-glutamylcysteine in concentrations 1/1000000000 not changed on the background of the action of gamma-glutamylcysteine in concentrations 1/1000000000 mol/l and 1/100000 mol/l: 436% 461%, respectively.

6.4.2.3. Duodenum Guinea pigs in conditions of isometric registration of contractile activity.

0.5 cm of the colon was placed in a modified Krebs solution at an initial tension of 0.5, it is Shown that gamma glutamylcysteine in the concentration range 1/1000000000000 - 1/10000000 mol/l does not affect the contractility of the body, including the airway, stimulated by histamine.

6.4.2.4. The uterine horn of the rat under conditions of isometric registration of contractile activity.

1 cm uterine horns were placed in hypocalcaemia (0.18 mmol of calcium chloride) solution of Tyrode or in a modified Krebs solution with normal calcium. The initial tension of 1 g

It is shown that the body does not respond to gamma glutamylcysteine (1/10000000 - 1/1000000 mol/l). The amplitude of the responses to serotonin (3/1000000 mol/l) is 729% and does not change the background gamma glutamylcysteine in concentrations 1/1000000 mol/l and 1/100000 mol/l 738% and 7110%, respectively. Responses to bradykinin (1/100000000 mol/l) are 6014% and do not change the background gamma glutamylcysteine concentration 1/1000000 mol/l 589%. Responses to oxytocin (0.75 mcme) are 629% of your max and do not change the background gamma glutamylcysteine in concentrations of 1/motri retains the ability to spontaneous rhythmic contractions, gamma glutamylcysteine in concentrations of up to 1/1000000 mol/l does not affect the frequency and amplitude of contractions, as well as the tone of the myometrium. On the background of the action of oxytocin (0,025 ME) gamma glutamylcysteine concentration 1/100000 mol/l causes a decrease in the tone of the myometrium without significant effect on its rhythmic activity.

6.4.2.5. Ring rat trachea

The segment of the rat trachea length of about 0.5 cm was placed in a modified Krebs solution Check the reductions were carried out in an isometric mode, the Initial tension of 1 g

It is shown that the drug does not respond to gamma glutamylcysteine in concentrations 1/1000000000000 - 1/100000 or histamine at concentrations 1/1000000 - 1/100000 Karbaholin clearly relaxes the trachea

6.4.2.6. Ring of rat aorta

The segment of the thoracic aorta, the rats were placed in a modified Krebs solution. Contractile activity was assessed in isometric mode. The initial tension of 1 g

It is shown that the drug is insensitive to gamma glutamylcysteine in the concentration range 1/100000000000 - 1/100000 mol/l, including background tonic action Mezatona (1/1000000 mol/l). Izadrin in these conditions has a relaxing effect.

6.4.2.7. The atrium of the rat.

It is shown that the drug is insensitive to gamma glutamylcysteine in concentrations 1/1000000000000 - 1/100000 mol/l Karbaholin (1/100000 mol/l) causes suppression of the amplitude of contractions of the body.

6.4.2.8. The strip of rat stomach.

Strip the fundus of the stomach of a width of 3 mm and a length of 10 mm were placed in a modified Krebs solution and record the reduction in isometric mode. The initial tension of 1 g

It is shown that the drug is insensitive to gamma glutamylcysteine in the concentration range 1/1000000000000 - 1/100000 mol/l Gamma-glutamylcysteine (1/100000 mol/l) no effect also on the contractile responses induced by histamine (1/100000 mol/l). Blocker H1-receptor - Pyrilamine - eliminates the action of histamine.

6.4.2.9. The ductus deferens of the rat.

1 cm VAS duct was placed in a modified Krebs solution. Reductions were recorded in isometric mode or in the background electrical stimulation (1 MS, 0.1 Hz) or without electrical stimulation, in which the reduction caused by the release of endogenous norepinephrine. The initial tension of 0.5 g

It is shown that gamma glutamylcysteine in kuliana mezatona on the tone of the duct as when electrical stimulation, and without it.

Thus, gamma-glutamylcysteine does not affect neurotransmitter systems, if the systems are in "normal" conditions as in tests in vivo and in vitro tests. In particular, in tests in vitro, if the bodies are in labor or in hypocalcaemia environment and basal tone or initiated airway are controlled by internal or external neurotransmitters.

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1. Method of regulating the activity of protective body systems: immune, inflammatory, free radical reigister injected in doses of 0.01-250 mcg per kg of body weight per day.

3. The method according to PP. 1 and 2, characterized in that the gamma glutamylcysteine enter through the mouth, parenteral application, by water or dipsomania-emulsion irrigations.

4. The method according to PP. 1-3, characterized in that the gamma glutamylcysteine enter into the composition of tablets, capsules, pills, supplements, water amouliani solutions, liposomal emulsion slurries.

5. The method according to PP. 1-4, characterized in that the gamma glutamylcysteine injected in the form of its pharmacologically acceptable salts.

 

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6 cl, 7 dwg, 2 tbl, 7 ex

FIELD: medicine, experimental cardiopharmacology.

SUBSTANCE: in an experiment one should intragastrically introduce APP inhibitor analapryl for laboratory animal at endothelial dysfunction, at the background of its modeling, at the dosage of 0.5 mg/kg and intraperitoneally - L-arginine at the dosage of 200 mg/kg once daily. This enables to activate correction of endothelial dysfunction due to enhancing enzymatic NO formation, L-carnitine being its donator.

EFFECT: higher efficiency of correction.

1 ex, 2 tbl

FIELD: medicine, endocrinology, pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to nateglynide-containing preparation used in treatment of diabetes mellitus that comprises nateglynide as an active component and a carrier wherein nateglynide in amorphous form and indicated carrier represents hydrophilic material. Amorphous property of crystalline nateglynide is provided by the following methods: 1) by dissolving nateglynide crystals in pharmacologically acceptable solvent in common with hydrophilic materials taken among the group consisting of water-soluble polymers, water-swelling polymers, sugar alcohols and salts followed by granulation in fluidized layer, granulation by stirring at high rate, drying by spraying and process for coat applying for granulation of amorphous nateglynide; 2) by mixing nateglynide crystals with hydrophilic materials taken among the group of water-soluble polymers, water-swelling polymers, sugar alcohols and salts and the following application of the high shift force to the prepared mixture; 3) by mixing nateglynide crystals with hydrophilic materials taken among the group of water-soluble polymers, water-swelling polymers, sugar alcohols and salts and the following plasticizing the prepared mixture in melt by heating and milling at cooling; 4) by dissolving nateglynide crystals in pharmacologically acceptable liquid additives wherein liquid additives represent water-soluble polymers that are liquid at 37°C. Using amorphous nateglynide allows preparing the nateglynide preparation with immediate release wherein the dissolving rate of medicinal agents is high and without crystalline transition during preparing or preserving preparations.

EFFECT: valuable pharmaceutical properties of preparation.

6 cl, 3 tbl, 9 dwg

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to creation of therapeutical and prophylactic balsams, which can be used as generally restorative and tonic agents to increase resistance of organism to reduced levels of atmosphere oxygen (increase of hypoxia resistance) as well as to prevention and correction of functional disorders associated with stress and exhausting environmental factors affecting organism. Invention proposes two variants of therapeutical-prophylactic balsam producing generally restorative and tonic effects, each containing vegetable oils, vitamins, amino acids, hepatic implant cells (Hepatosan), adaptogen (Astragerm), antioxidant, odorant, and thickener.

EFFECT: achieved normalization of vitality in exhausted organism.

6 cl

FIELD: medicine.

SUBSTANCE: method involves administering Nateglynid and Glytazon in pharmaceutically permissible carrier. The combination is optionally administered as combined preparation or pharmaceutical composition. The pharmaceutical composition (the second version) has Nateglynid and Methformin as the second anti-diabetic agent enclosed into the pharmaceutical carrier.

EFFECT: enhanced effectiveness of synergetic preparations action upon diabetes II; prolonged therapeutic drug action time; postponed insulin administration.

13 cl, 1 tbl

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