RU2361606C2 - Composition for treatment of damages of gi tract mucosa - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine and can be used for treatment of damages of a GI tract mucosa. The invention represents a composition on the basis of interleukin-1 and polyvinylpyrolidone in a water containing solution, which in addition contains sodium bicarbonate, and composition components are in a certain mass parity.

EFFECT: provision of massive wound-healthing action, stimulation of local immunity of mucosa without system action.

5 ex, 6 tbl, 4 cl

Description

The invention relates to medicine, namely to create a composition for the treatment of diseases of the gastrointestinal tract (GIT), namely a composition containing preparations of interleukin-1 (IL-1) for the treatment of injuries of the mucous membrane (CO) of the gastrointestinal tract.

Treatment of lesions of the mucous membrane (CO) of the digestive tract, as a rule, is carried out by a complex effect, which includes drugs and techniques aimed at eliminating the damaging effect of the acid-peptic factor, normalizing the motor-evacuation functions of the stomach and duodenum, stimulating the regeneration of epithelial cells of the mucosa shells and the formation of mucus, improved blood supply to the mucous membrane and more. To stimulate CO regeneration processes, the complex of therapeutic measures includes solcoseryl, methyluracil, aloe, vitamins, autohemotherapy and hyperbaric oxygenation (Brief Medical Encyclopedia, edited by Acad. B.V. Petrovsky - M., Soviet Encyclopedia, 1990, p. 437- 439).

The prophylactic use of interleukins for interperitoneal or intravenous administration in inflammatory processes of the gastrointestinal tract in the experiment is known (Wallace JL et al., Gastroenterology, 1992, V.102, p. 1176; Wallace JL, Eur. J. Pharmacol, 1990, V.186, p .279).

The disadvantage of these methods is the relatively low efficiency, the presence of a significant number of contraindications and complications.

Closest to the claimed invention is the preparation developed by the authors of IL-1β "Betaleikin" (RU 2128706; P No. 000222 / 01-2001, 01/25/2001), containing IL-1β, human albumin and polyvinylpyrrolidone. The course of treatment using Betaleikin consists of 3 daily intravenous drip infusions at a dose of 5 ng / kg body weight (Dolgushina A.I., Abstract of Cand. Diss., Chelyabinsk, 2003). After the use of IL-1, acceleration of the healing of ulcerative defects of the gastric and duodenal mucosa was shown. However, this composition of IL-1 has several disadvantages, in particular lack of effectiveness and the appearance of undesirable side effects, such as a febrile reaction after infusion and a reaction from the systemic immunity.

The technical challenge faced by the authors was the development of a more effective and safe therapeutic composition for the treatment of gastrointestinal tract injuries based on Betaleikin.

The technical result was achieved by creating a composition in the form of an aqueous solution containing 5 × 10 ^-7 -5 × 10 ^-5 wt.% IL-1, 1-10 wt.% Sodium bicarbonate (BKN) and 1 × 10 ^-4 -1 × 10 ^-2 wt.% Polyvinylpyrrolidone (PVP).

The drug is administered daily at a dose of 25-2500 ng per 1 kg of body weight. The course of treatment, depending on the characteristics of the disease, is from 1 to 10 days.

The basis of the use of the proposed method was the assumption that charged molecules of polyvinylpyrrolidone and sodium bicarbonate are able, on the one hand, to protect the most vulnerable fragments of the IL-1 molecule from enzymatic and acid biodegradation, increasing the effectiveness of the drug, and on the other hand, can weaken the interaction of IL-1 with immunoglobulins and eliminate the negative immune response to the introduction of a foreign protein.

As studies have shown, the use of this composition of IL-1 both significantly enhances the healing of ulcerative lesions of the gastric mucosa (coolant) in various models, and minimizes the negative effect of the introduction of IL-1 on the immune system, which was not previously noted in the literature.

The use of IL-1 in a dose of less than 5 × 10 ^{-7 is} ineffective, its use in a dose of more than 5 × 10 ^-5 wt.% Is possible when used in certain complicated pathologies, but, as a rule, it is not economically feasible. The use of a solution with a sodium bicarbonate concentration of less than 1% and polyvinylpyrrolidone in a dose of less than 1 × 10 ^-4 does not provide reliable protection of the protein from degradation when it enters the stomach, or does not eliminate negative side effects. The use of solutions containing large amounts of protective additives does not increase the effectiveness of the method.

As the drug IL-1 can be used natural IL-1, as well as recombinant IL-1ά or IL-1β. 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. 10 μl of a solution of the substance IL-1, containing 20 μg of IL-1β, was diluted in 980 μl of a sterile 4% solution of sodium bicarbonate and, thus, a solution with a concentration of IL-1 of 20 μg / ml was obtained. Then 50 μl of the resulting solution was added to 9950 μl of a sterile 4% baking soda solution, mixed by pipetting and a solution with a concentration of IL-1β of 100 ng / ml was obtained. Polyvinylpyrrolidone (PBP) was added at a dose of 1 ml of a 0.1 mg PBP solution. After this, the samples were lyophilized.

To test the biological activity of the composition when administered orally, female Wistar rats weighing about 150 g were used. 24 hours before the experiment, the 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 a substance containing IL-1β in doses of 0.005-0.5 μg / ml and polyvinylpyrrolidone in 4% baking soda 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. A planimetric measurement of the areas of coolant lesions was carried out and their percentage was calculated relative to the total area of the secretory part of the stomach, which is a widely used method. In our study, we took into account the most pronounced lesions of the coolant, which had clear contours, the percentage of their area was the declared values relative to the areas of the secretory part of the stomach. The percentage of lesions was calculated as the ratio of the affected areas to the total area of the gastric mucosa. Statistical processing was performed according to student criterion. The results are presented in table 1.

Table 1
The effect of the claimed drug and recombinant IL-1 beta and Beta-leukin on the area of the lesion of the coolant (% of the lesion area of the total area of the secretory part of the stomach) Study period Drug used The concentration of IL-1 beta 5 ng / ml (5 × 10 ⁷ %) 50 ng / ml IL-1β 500 ng / ml (5 × 10 ⁵ %) Placebo 3 hours claimed 10.66 ± 2.56 14.84 ± 5.0 8.98 ± 1.5 11.48 ± 1.76 Betaleikin 11.8 ± 2.40 14.8 ± 2.5 11.0 ± 1.55 24 hours claimed 4.2 ± 1.62 * 5.62 ± 1.24 * 4.09 ± 1.39 * 15.5 ± 3.83 Betaleikin 6.5 ± 2.55 5.8 ± 1.5 * 5.9 ± 3.9 72 hours claimed 2.74 ± 0.77 ** 3.54 ± 1.18 2.78 ± 1.28 * 6.98 ± 1.05 Betaleikin 4.9 ± 0.8 4,5 ± 1,8 3.8 ± 1.8 Notes: * p <0.05, ** p <0.01; differences with placebo are significant. Number of observations: n = 5 at each point.

The results showed that the use of the IL-1β rivers in the composition in a dose range of two to three times statistically significantly reduced the lesion area in the doses used at the study time of 24 and 72 hours compared with the control group. The use of the Betaleikin preparation also led to positive effects, so, starting from the period of 24 hours, the use of IL-1 1.5-3 times reduced the lesion area compared with the control group. However, the effect of the IL-1β rivers in the composition was more pronounced, which was manifested in significant differences with placebo at 24 and 72 hours in almost the entire dose range, and Betaleikin significantly reduced the area of lesions by 24 hours at a dose of 50 ng / ml .

Example 2. In the conditions of example 1, a study was conducted on the effect of the claimed composition, including natural IL-1 beta, or the drug "Betaleikin" on the healing of acid stomach ulcers.

table 2
The effect of natural IL-1 beta or Betaleukin on the area of coolant ulcers Groups of animals The ulcer area, mm ² IL-1 beta 100 ng / ml - 1 × 10 ^-8 % - the claimed drug (n = 10) 5.65 ± 0.65 ** Betaleikin (n = 6) 11.10 ± 2.13 Placebo (n = 8) 16.56 ± 4.47 ** p <0.01 differences with the control group are significant.

As follows from the data presented, when applying the claimed composition, including natural IL-1 beta, there was a significant decrease in the area of ulcers in the experimental group compared with the control (more than 3 times), which was statistically significant. The use of Betaleikin also reduced the area of ulcers on average by a third, but no significant differences from placebo were observed.

Example 3. Obtaining a natural preparation of IL-1 was carried out by stimulation of human cells. Cell isolation was carried out as follows (Boyum A., Scand. 1. 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 and +40 ° C. The “ring” of mononuclear cells formed in the interphase was pipetted, and the resulting cell suspension was washed three times with 0.9% NaCl 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 and the content of IL-1 was adjusted to the desired concentration.

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 (Tsuki-mi U., 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. Four to five animals were examined at each point, with each ulcer forming two ulcers. On 4 and 5 days after surgery, 1 ml of a 6% bicarbonate solution containing IL-1 at a dose of 100 ng / ml and PVP at a dose of 0.1 mg / ml were injected into the stomach in the experimental groups through a tube. Animals of the control group received placebo for the same periods. 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. The absolute number of leukocytes was calculated in the Goryaev chamber, and the blood formula was calculated on smears stained according to the Romanovsky-Giemsa method. Statistical processing was performed according to student criterion. The results of measuring the area of ulcers are presented in table 3.

Table 3
The effect of IL-1 concentration on the area of ulcers The concentration of IL-1 The ulcer area, mm ² % reduction The claimed - 1 × 10 ^-7 % (n = 10) 5.56 ± 0.65 ** 66,4 Placebo (control) (n = 8) 16.56 ± 4.47 0 ** p <0.01 differences with the control group are significant.

As follows from the data presented, there was a significant decrease in the area of ulcers in the experimental group compared with the control (more than 3 p ~ a), which was statistically significant.

Table 4
White blood cell count and blood count in rats treated with IL-1 Blood counts IL-1 10 ng / ml IL-1 100 ng / ml Betaleikin Placebo The number of leukocytes, thousand / mm ³ 12.0 ± 1.2 11.5 ± 3.3 11.5 ± 3.3 11.5 ± 3.3 Blood formula% Neutrophils 29.6 ± 5.8 20.2 ± 5.9 24.2 ± 5.5 29.1 ± 8.1 Lymphocytes 66.3 ± 5.4 72.4 ± 5.3 64.2 ± 6.1 66.3 ± 7.2 Monocytes 2.6 ± 0.7 4.4 ± 1.4 3.1 ± 1.6 3.2 ± 1.1 Eosinophils 1.8 ± 0.6 1.8 ± 0.4 1.8 ± 0.4 2.0 ± 0.9

The results of calculating the absolute and relative number of leukocytes showed the absence of significant differences between the control and experimental groups (table 4). This allows us to judge the absence of significant systemic effects of IL-1 when it is introduced into the stomach, which confirms the increased safety of the new composition.

Example 4. A gastric ulcer was induced as described in Example 3. On days 4, 5, and 6 after the operation, the animals of the experimental groups were injected into the stomach with various compositions containing a recombinant IL-1a preparation at a dose of 100 ng / ml. Animals of the control group received 1 ml of placebo at the same time. Five animals were examined at each point, with two ulcers forming in each animal. On the 8th day of the experiment, blood was drawn from the tail vein, after which the animals were sacrificed, the stomach was opened, and the surface area of the ulcers was measured. The calculation of the absolute number of leukocytes was carried out in the Goryaev’s chamber. Statistical processing was performed according to student criterion. The results of measuring the area of ulcers are presented in table 5.

Table 5
The effect of composition on the area of coolant ulcers The composition, wt.% The area of the ulcer, mm % reduction from control IL-1a PVP BKN 1 × 10 ^-7 0 0 21.5 ± 2.0 * 21.5 1 × 10 ^-7 0 four 19.2 ± 2.6 * 29.9 1 × 10 ^-7 1 × 10 ^-3 0 20.2 ± 2.8 * 26.3 1 × 10 ^-7 1 × 10 ^-3 four 14.8 ± 2.4 ** 46.0 1 × 10 ^-7 1 × 10 ^-4 one 18.6 ± 2.4 ** 32.1 1 × 10 ^-7 1 × 10 ^-2 10 14.2 ± 2.6 ** 48,2 placebo 27.4 ± 2.8

The best results were achieved with a PVP content of 1 × 10 ^-2 -1 × 10 ^-3 %, sodium bicarbonate - 4-10 wt.%.

Example 5. 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 through a tube into the stomach with a composition containing a recombinant IL-1a preparation at a dose of 100 ng / ml, PVP - 10 ^-3 wt.%, Sodium bicarbonate - 2 wt.%. Animals of the control group received 1 ml of placebo at the same time. Five to eight animals were examined at each point, with two ulcers forming in each animal. On the 8th day of the experiment, blood was drawn from the tail vein, after which the animals were sacrificed, the stomach was opened, and the surface area of the ulcers was measured. The calculation of the absolute number of leukocytes was carried out in the Goryaev’s chamber. Statistical processing was performed according to student criterion. The results of measuring the area of ulcers are presented in table 5.

Table 6
The effect of IL-1a on the area of coolant ulcers Groups of animals The area of the ulcer, mm IL-1a - 1 × 10 ^-7 % (n = 16) 18.2 ± 1.02 ** Placebo (n = 10) 25.2 ± 2.63 ** p <0.01 differences with the control group are significant.

As follows from the data presented, three-course administration of IL-1a into the stomach led to a significant, statistically significant reduction in the area of ulcers compared with the control.

Table 5
The number of leukocytes in rats during treatment with IL-1A Group of animals IL-1a - 1 × 10 ^-7 % Placebo The number of leukocytes, thousand / mm ³ 10.10 ± 1.71 9.25 ± 1.05

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-1a with local use.

Thus, during the experiments it was shown that oral administration of a composition containing IL-1 has a pronounced wound healing effect, stimulates local mucosal immunity and does not have a systemic effect.

The composition has shown efficacy, harmlessness and its promise for the treatment of inflammatory, infectious and stressful lesions, mechanical damage and wounds of the gastrointestinal mucosa.

Claims (4)

1. The composition for the treatment of injuries of the mucous membrane of the gastrointestinal tract based on interleukin-1 and polyvinylpyrrolidone in an aqueous solution, characterized in that it additionally contains sodium bicarbonate in the following ratio of ingredients, wt.%:
interleukin-1 5 · 10 ^-7 -5 · 10 ^-5 bicarbonate of soda 1-10 polyvinylpyrrolidone 1 · 10 ^-4 -1 · 10 ^-2 water rest 2. The composition according to claim 1, characterized in that as interleukin-1 it contains recombinant interleukin-1 beta. 3. The composition according to claim 1, characterized in that as interleukin-1 it contains natural interleukin-1. 4. The composition according to claim 1, characterized in that as interleukin-1 it contains recombinant interleukin-1 alpha.