RU2355415C2 - Method of treating mucosal lesions of gastrointestinal tract - Google Patents

Abstract

FIELD: medicine; gastroenterology.

SUBSTANCE: invention relates to method of treating mucosal (M) lesions of gastroenterological tract (GIT). For this purpose water or physiological solution of interleukin -1 (IL-1), containing 5×10^-7-5×10^-5 wt % of IL-1 is introduced perorally.

EFFECT: getting high medicinal concentrations of preparation directly in lesion focus, stimulation of local immunity with no side effects.

6 cl, 6 tbl, 4 ex

Description

The invention relates to medicine, namely to methods for treating diseases of the gastrointestinal tract (GIT), namely to treating injuries of the gastrointestinal mucosa (CO) of the gastrointestinal tract using interleukin-1 (IL-1) preparations.

As a rule, the treatment of lesions of the mucous membrane (CO) of the digestive tract is carried out by a complex effect. To stimulate CO regeneration processes, the complex of therapeutic measures includes solcoseryl, methyluracil, aloe, vitamins, autohemotherapy. In cases of scarring ulcers, hyperbaric oxygenation is used, local administration of drugs to the area of the ulcer defect through the endoscope, and a helium-neon laser is used. In the presence of perforation or perforation of a stomach ulcer or duodenal ulcer, as well as with malignancy of the ulcer, surgery is performed. Eliminate the damaging effect of the acid-peptic factor, normalize the motor and evacuation functions of the stomach and duodenum, stimulate the formation of mucus, improve blood supply to the mucous membrane and others (Brief Medical Encyclopedia. / Ed. By Academician B.V. Petrovsky, M., “ Soviet Encyclopedia ”, 1990, pp. 437-439). The disadvantages of complex methods are the complexity, long periods of treatment, the presence of a significant number of contraindications and the possibility of complications.

One of the promising areas is the use of cytokines, in particular, interleukin-1 (IL-1) for the treatment of gastrointestinal diseases, which allows monotherapy. It has now been established that IL-1 preparations are effective for the prevention of gastrointestinal tract damage with indomethacin. The preparations IL-1α and IL-1β are administered intraperitoneally 90 minutes before the introduction of the damaging factor (Wallace J.L. et al., Gastroenterology, 1992, v. 102, p. 1176).

The disadvantage of this method of exposure is the limited scope and technology of drug administration, worked out only for use in animal experiments and not intended for the treatment of people.

Closest to the claimed invention is a method of treating patients with gastric ulcer by intravenous administration of a drug based on IL-1 beta - Betaleikin (Dolgushina A.I., Abstract of Cand. Diss., Chelyabinsk, 2003). The course of treatment consists of 3 daily intravenous drip infusions at a dose of 5 ng / kg body weight. After the use of IL-1, acceleration of the healing of ulcerative defects of the gastric and duodenal mucosa was shown. However, this method of using IL-1 has several disadvantages, in particular, the relatively low efficiency and the presence of undesirable side effects, such as a febrile reaction after infusion and a negative reaction from systemic immunity.

The technical challenge faced by the authors is to develop a more effective way to treat gastrointestinal tract injuries using IL-1 preparations.

The technical result is achieved by administering to the patient an oral preparation of IL-1 in the form of an aqueous or physiological solution containing 5 × 10 ^-7 -5 × 10 ^-5 wt.% IL-1 in an amount of 25-2500 ng per 1 kg of weight for 1-5 days depending on the characteristics of the disease.

Studies have shown that the use of IL-1 significantly enhances the healing of ulcerative lesions of the coolant in various models.

As the preparation of IL-1, recombinant or natural IL-1, as well as IL-1 alpha and IL-1 beta, for example, Betaleikin, which is a pharmacopoeial preparation of recombinant human IL-1β, can be used. The use of IL-1 in a dose of less than 10 ^{-7 is} ineffective, its use in a dose of more than 10 ^-5 wt.% Is possible when used in certain complicated pathologies, but, as a rule, is not economically feasible. The choice of a specific interleukin-1 and the duration of use is determined mainly by the characteristics of the patient's disease.

The industrial applicability of the method and its effectiveness are illustrated by the following examples.

Example 1. Obtaining a natural preparation of IL-1 was carried out by stimulation of human cells. Cell isolation was performed as follows (Boyum A., Scand. J. Clin. Invest., 1968, V.21, S.97, P.77-89). To 10 ml of fresh venous blood taken on heparin (20 IU / ml) in a sterile tube, 1 ml of a 10% gelatin solution was added, mixed and incubated at 37 ° C for 30 min. A density gradient centrifugation Histopaque ("Sigma") was then performed. For this, 6 ml of the obtained leukomes suspension was layered on a 3 ml density gradient and centrifuged for 40 minutes at 450 g at + 4 ° С. The “ring” of mononuclear cells formed in the interphase was pipetted, and the resulting cell suspension was washed three times with 0.9% NaC solution. Cell viability, which was determined by the method of staining with trypan blue, was at least 95%. Cells were resuspended at a concentration of 5 × 10 ^-6 cells / ml in RPMI 1640 medium supplemented with 2 mM glutamine and 80 μg / ml gentamicin. To prepare a working solution of prodigiosan, 2.4 ml of RPMI 1640 medium and 800 μl of a 0.005% solution of prodigiosan were mixed, while 100 μl of the resulting solution contained 1.0 μg of prodigiosan. An inducer of 100 μl per well was added to a 96-well cell culture plate ("COSTAR"), then 100 μl of diluted cells were added to all wells. Cultivation was carried out at 37 ° C in 5% CO ₂ for 24 hours, after which supernatants were carefully selected.

In the obtained supernatant, the content of bioactive IL-1 was determined in the mouse thymocyte proliferation test according to the following procedure. CBA mice were sacrificed by cervical dislocation. Under aseptic conditions, thymuses were removed from them, which were then homogenized in the Needle medium using needles of various diameters. The resulting thymocyte suspension was resuspended in 1 ml of Eagle’s medium and counted in the Goryaev’s chamber. An Eagle medium containing 4% fetal serum was adjusted to a cell concentration of 10 million / ml. Test wells, concanavalin A solution, and 100 μl of previously prepared thymocyte suspension were added to the wells of a 96-well culture plate. At the same time, the necessary controls were placed in the same tablet:

- control of cell suspension - to 100 μl of culture medium was added 100 μl of cell suspension;

- control of the action of a suboptimal dose of concanavalin A: 50 μl of a working solution of concanavalin A and 100 μl of a suspension of thymocytes were added to 50 μl of culture medium;

- control of the effect of the optimal dose of concanavalin A: 100 μl of thymocytic suspension was added to 100 μl of the working solution of concanavalin A.

Cultivation was carried out for 72 hours in a CO ₂ incubator. 18 hours before the end of cultivation, 40 μg of 3H-thymidine in a volume of 10 μl was added to all wells of the plate. At the end of the cultivation, thymocytic cultures from all wells of the plate were transferred using a harvester to filters, which were then dried. Filters were placed in vials with scintillation fluid and 3H-thymidine incorporation by thymocytes of each culture was determined using a scintillation counter (RACKBETA 1217). Data for each three parallel cultures were averaged. For a standard sample, a graph of the dependence of the level of 3H-thymidine incorporation on drug dilution was constructed. In order to calculate the activity of the test sample, the average value of 3H-thymidine incorporation in each dilution was found and the last dilution was determined, in which the sample gives proliferation enhancement that fits the standard chart.

The calculation of the activity of the test sample was carried out as follows: on the graph constructed for the standard sample, we found the point corresponding to the minimum proliferation gain, expressed in pulses per minute, which gives the test sample in any dilution and which fits on the straight part of the standard graph, then, dropping the perpendicular to the abscissa axis, we found the dilution of the standard, causing the same increase in proliferation. Thus, it was found that the percent increase in proliferation of the standard sample and the test sample are equal and are not necessary for calculating activity. The content of IL-1 was adjusted to the desired concentration by dilution in sterile saline.

In the experiment, female Wistar rats weighing approx. 150 g. 24 hours before the experiment, rats were not fed, leaving free access to water. Mucosal damage was caused by intragastric administration of 1 ml of 96 ° ethanol (Wallace J.L., Eur. J. Pharmacol., 1990, v.186, p.279). 1 hour after the lesion, 1 ml of IL-1 solution or placebo in the same volume was administered intragastrically through a probe. Five animals were examined at each point. Animals were slaughtered 3 hours, one day, and three days after the administration of IL-1. The stomachs were removed, fixed with 10% formalin, cut along a large curvature, pinned to a wax platform and photographed with a digital camera, after which the lesion area was measured using the Scion Image program. The percentage of lesions was calculated as the ratio of the affected areas to the total area of the gastric mucosa. The results are presented in table 1.

Table 1 The effect of IL-1 on the area of lesions of the coolant Study period The concentration of IL-1 beta 5 ng / ml (5 × 10 ^-7 %) 50 ng / ml (5 × 10 ^-6 %) 500 ng / ml (5 × 10 ^-5 %) Placebo The area affected by the coolant,% 3 hours 10.8 ± 2.34 12.8 + 5.5 9.0 ± 1.5 10.4 ± 1.6 24 hours 5.3 ± 1.55 5.2 ± 1.4 * 4.9 ± 1.9 14.5 ± 3.3 72 hours 3.8 ± 0.8 3.4 ± 1.8 2.8 ± 1.8 6.8 ± 1.5

Number of observations: n = 5 at each point.

The data showed that, starting from a period of 24 hours, the use of IL-1 1.5-3 times reduced the area of damage in comparison with the control group.

Example 2. In the example, for the treatment of lesions of the stomach used the drug "Betaleikin". The ampoule of the preparation containing 1 μg of recombinant human IL-1β was diluted in 1 ml of sterile saline and mixed by pipetting. Thus, a solution with a concentration of IL-1β of 100 ng / ml was obtained. The solution was used immediately after preparation.

In the experiments, female Wistar rats weighing 150-200 g were used. Gastric ulcer was induced in rats by introducing acetic acid into the gastric cavity (Tsukimi Y., et al., Gastroenterology Hepatology, 1994, v.9, p.60). On the 3rd day after the operation, the formation of a ulcerative defect occurred, which was confirmed by histological examination, with two ulcers forming in each animal. On days 4 and 5 after the operation, the animals of the experimental groups were injected Betaleukin at a dose of 100 ng / ml in 1 ml of solution through a tube into the stomach. Animals of the control group received 1 ml nat. solution. On day 8 of the experiment, blood was taken from the tail vein, after which the animals were sacrificed, the stomach was opened, and the surface area of the ulcers was measured. Calculation of the absolute number of leukocytes was carried out in the Goryaev chamber using a Turk solution. Statistical processing was performed according to student criterion. The results of measuring the area of ulcers are presented in table 2.

table 2 The effect of Betaleikin on the area of coolant ulcers The concentration of IL-1β The ulcer area, mm ² % reduction from control 100 ng / ml - 1 × 10 ^-8 % (n = 8) 18.56 ± 2.00 36.5 Placebo (control) (n = 6) 29.25 + 4.40 0

As follows from the data presented, there was a decrease in the area of ulcers by more than 36% after treatment with Betaleikin compared with the control.

Table 3 The number of blood leukocytes in rats treated with "Betaleikin" Groups of animals IL-1β, 100 ng / ml n = 3 Placebo n = 3 The number of leukocytes, thousand / mm ³ 8.50 ± 1.44 10.70 ± 1.20

The results of calculating the absolute number of leukocytes showed the absence of significant differences between the control and experimental groups (Table 3). This allows us to judge the absence of significant systemic effects of the drug when it is introduced into the stomach.

Example 3. 10 μl of the substance IL-1β (at a concentration of 2 mg / ml in FSB pH 7.2) containing 20 μg of IL-1β was diluted in 980 μl of sterile 0.9% sodium chloride solution (physiological solution), and received Thus, a solution with a concentration of IL-1β of 20 μg / ml. Then 5 μl of the resulting solution was added to 995 μl of sterile saline and mixed by pipetting. The solution was used immediately after preparation. A gastric ulcer was induced in the same manner as described in Example 2. On days 4, 5, and 6 after the operation, the animals of the experimental groups were injected with a recombinant IL-1β in 1 ml of 0.9% sterile physiological saline at a concentration of 100 ng / ml into the stomach through a tube.

On the 8th day of the experiment, blood was taken from the tail vein, after which the animals were sacrificed, the stomach was opened and ulcers were excised for histological examination. After standard histological wiring, sections were made with a thickness of 6 μm, which were stained with hematoxylin-eosin. The preparations were photographed using a microscope with a digital camera (“Leica”), after which the perimeter of ulcers was measured using the Scion Image program. Calculation of the absolute number of leukocytes was carried out in the Goryaev chamber using a Turk solution. Statistical processing was performed according to student criterion. The results of morphometric measurements of ulcers are presented in table 4.

Table 4 The influence of rivers. IL-1β on the size of coolant ulcers River concentration. IL-1β The width of the ulcer, microns % reduction from control 100 ng / ml - 1 × 10 ^-7 % (n = 5) 3440.0 ± 349.0 29.5 Placebo (control) (n = 5) 4880.0 ± 589.0 0

According to histological and morphometric studies, a clear tendency is shown to decrease the perimeter of the ulcer (on average about 30%) in animals treated with recombinant human IL-1β.

Example 4. Recombinant human IL-1α was diluted in sterile saline at a concentration of 100 ng / ml and mixed by pipetting. The solution was used immediately after preparation.

A gastric ulcer was induced as described in Example 2. On days 4, 5, and 6 after the operation, the animals of the experimental groups were injected with a prepared solution of IL-1α rivers in a volume of 1 ml through a tube into the stomach. Animals of the control group received 1 ml nat. solution. On the 8th day of the experiment, blood was taken from the tail vein, after which the animals were sacrificed, the stomach was opened, and the surface area of the ulcers was measured. Calculation of the absolute number of leukocytes was carried out in the Goryaev chamber using a Turk solution. Statistical processing was performed according to student criterion. The results of measurements of the area of ulcers are presented in table 5.

Table 5. The effect of recombinant IL-1α on the size of coolant ulcers River concentration. IL-1β The ulcer area, mm ² % reduction from control 100 ng / ml - 1 × 10 ^-7 % (n = 5) 14.88 ± 2.42 24.03 Placebo (control) (n = 5) 19.58 ± 1.83 0

According to table 5 shows a clear tendency to decrease the perimeter of ulcers (on average by a quarter) in animals treated with rivers. IL-1α.

Table 6. The number of white blood cells in rats treated with rivers. IL-1α Groups of animals IL-1α, 100 ng / ml n = 3 Placebo n = 3 The number of leukocytes, thousand / mm ³ 8.83 ± 1.69 8.83 ± 1.09

The results of the calculation of the absolute number of leukocytes showed the absence of differences between the control and experimental groups (table 6). This also allows us to say that there is no significant systemic effect of IL-1 in local use.

During the experiments, it was shown that when the IL-1 solution is introduced into the stomach, it has a pronounced wound healing effect, stimulates the local mucosal immunity and does not have negative side effects. Local use of IL-1 allows you to achieve high therapeutic concentrations of the drug directly in the lesion and to avoid undesirable systemic effects.

The drug showed efficacy, harmlessness and its promise for the treatment of inflammatory, infectious and stressful lesions, mechanical damage and wounds of the mucous membrane of the esophagus, stomach and duodenum, stomach ulcers.

Claims (6)

1. A method of treating damage to the mucous membrane of the gastrointestinal tract using interleukin-1 preparations, characterized in that the interleukin-1 preparation is administered orally in the form of an aqueous solution containing
5 × 10 ^-7 -5 × 10 ^-5 wt.% IL-1. 2. The method, characterized in that the preparation of interleukin-1 is administered at a dose of 25-2500 ng / kg of body weight for 1-5 days. 3. The method according to claim 1, characterized in that as the preparation of interleukin-1 use natural interleukin-1. 4. The method according to claim 2, characterized in that the preparation "Betaleikin" is used as the preparation of interleukin-1. 5. The method according to claim 3, characterized in that as a preparation of interleukin-1 use recombinant interleukin-1 beta. 6. The method according to claim 4, characterized in that as a preparation using recombinant interleukin-1 alpha.