EA014434B1 - Pharmaceutical composition with immunosuppressive and anti-tumor action and a method of preparation thereof - Google Patents

Abstract

The invention relates to medicine, in particular to preparation of novel pharmaceutical composition and a method for producing thereof. The pharmaceutical composition having an immunosuppressive and anti-tumor action comprises an effective amount of tris-(2-hydroxyethyl)ammonium salt of 1-benzylindolyl-3-thioacetic acid and auxiliaries (potato starch, lactose, magnesium stearate and methyl cellulose). A method for preparing a pharmaceutical composition comprises mixing the tris-(2-hydroxyethyl)ammonium salt of 1-benzylindolyl-3-thioacetic acid with lactose, potato starch and moistening the obtained mixture by a binding solution (2% methylcellulose solution), wet granulation, drying and dry granulation. The composition is made as capsules for internal administration, is low-toxic and has high bioavailability.

Description

The invention relates to medicine, specifically to the creation of a pharmaceutical composition having an immunosuppressive and antitumor effect, containing as an active substance a synthetic chemical compound - tris- (2-hydroxyethyl) ammonium salt (1 benzylindolyl-3-thio) acetic acid (hereinafter - A) .

It is known that A, being a biologically active substance with immunosuppressive and antitumor properties, can be used in medicine and pharmacology to create drugs [1, 2].

Pharmaceutical compositions and finished dosage forms obtained using A are not described in the literature.

Known synthetic immunosuppressant azathioprine, used in modern medical practice in the form of tablets of 0.05 g [3, 4].

This drug causes undesirable side reactions in patients - nausea, vomiting, loss of appetite, allergic conditions, toxic hepatitis, etc. It is contraindicated in case of severe inhibition of hematopoiesis, leukopenia, severe liver diseases, pregnancy.

The objective of the present invention is to expand the range of synthetic drugs with immunosuppressive and antitumor effects, as well as characterized by good tolerance and a minimum of side effects, a new pharmaceutical composition is proposed.

The objective of the invention is the creation of a pharmaceutical composition having immunosuppressive and antitumor activity, having optimal bioavailability and effectiveness, containing tris (2-hydroxyethyl) ammonium salt (1 benzylindolyl-3-thio) acetic acid as an active substance, and also the development of a method for its preparation.

The solution of this problem is achieved by the fact that the proposed pharmaceutical composition contains as active ingredient tris- (2-hydroxyethyl) ammonium salt (1-benzylindolyl3-thio) acetic acid and expertly selected auxiliary substances (approved for use in the pharmaceutical industry).

The pharmaceutical composition (finished dosage form) is a hard gelatin capsule that contains the active substance A in a therapeutically effective dose of 0.1 g, as well as excipients: potato starch - 0.05 g, lactose - 0.0963 g, magnesium stearic acid - 0.0025 g and methyl cellulose - 0.0012 g (total 0.250 g).

The pharmaceutical composition and finished dosage forms obtained based on the use of A have not been previously described in the literature.

The claimed ratios of the components provide the necessary technological properties of the granular mass for filling capsules and the pharmacopoeial quality of the finished dosage form.

A method for preparing a composition is also proposed, including mixing tris- (2-hydroxyethyl) ammonium salt (1-benzylindolyl-3-thio) acetic acid with lactose and potato starch, moistening the resulting mixture with a binder solution (aqueous methylcellulose solution), wet granulation, drying and dry granulation.

Due to the fact that the substance of the active substance does not have the technological properties necessary to obtain a granular mass (lack of flowability, large bulk density, electrostaticity), the composition of the mass was obtained by wet granulation. Using the direct mixing method A with excipients does not give positive results, since the resulting mass has no flowability, and it has a large bulk weight, which prevents the capsules from filling with the necessary amount of active substance.

The use of these auxiliary substances and their experimentally selected ratios made it possible to obtain a mass with good flowability for filling capsules. Capsules have the necessary disintegration, optimal release of the active substance, stable during storage.

Example 1

A method of obtaining a pharmaceutical composition (composition, wt.%: Compound A - 40; potato starch - 20; lactose - 38.5; magnesium stearic acid - 1.0; methyl cellulose - 0.5).

50.0 g of potato starch, 96.25 g of lactose and 100 g of tris- (2-hydroxyethyl) ammonium salt (1-benzylindolyl-3-thio) acetic acid are loaded into the mixer; the mass is mixed until smooth. Then, a previously prepared 2% aqueous solution of methylcellulose in the amount of 62.5 ml is loaded into the mixer, mixed, subjected to wet granulation on a granulator and transferred to drying to a residual moisture content (1 ± 0.5%). Dry granulation is carried out manually on a metal sieve, then granulate is dusted with magnesium stearic acid - 2.5 g. 247 g of granular mass is obtained (yield 98.5%), which is filled with hard gelatin capsules.

Disintegration: not more than 20 minutes.

Dissolution: at least 80% in 30 minutes.

Example 2. The study of the General toxic properties of the pharmaceutical composition

Nonlinear rats of both sexes were used to study acute toxicity.

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The contents of the capsules were dissolved in distilled water and administered to the animals intragastrically using a probe at doses of 500, 1000, 2000, 3000 and 5000 mg / kg. There were 6 rats in each group. The animals were observed for two weeks. Studied the death of rats (L ₅₀ ) and the clinical picture of poisoning. It was found that according to the indications of acute toxicity, the composition containing A and excipients refers to low toxicity drugs.

To study chronic toxicity, rabbits of both sexes were used, for which capsules containing A and excipients were administered intragastrically for 6 months (daily doses of the composition were 40 and 80 mg / kg, which is several times higher than the expected therapeutic dose for humans). There were 10 animals in each group. It was established that the composition (finished dosage form) does not have a toxic effect on the organism of experimental animals: the behavior, appearance, body weight, and rectal temperature did not change. Separately observed fluctuations in biochemical parameters were not associated with the toxic effect of the composition and were within the physiological norm. There were no significant changes in hematological parameters, indicating violations in the leukoerythrocytic composition of the blood.

When pathomorphological study of tissues of internal organs is not established pathological changes associated with the toxic effect of the drug.

Intragastric six-month administration of capsules (containing the composition) to rabbits does not have a local irritant effect on the gastrointestinal tract.

Example 3. Determination of the bioavailability of the composition.

In experiments on rabbits, the bioavailability of a composition containing A and excipients was determined.

To determine the absolute bioavailability, one capsule of the composition was administered orally to rabbits. The content of the active substance in the blood plasma of animals was determined (HPLC method), the pharmacokinetic parameters characterizing the rate and degree of absorption of the active substance, its elimination were calculated, the absolute bioavailability of the capsule formulations was determined.

It is shown that, after the composition has been administered inside, the active substance A is well released from the capsules and is rapidly absorbed from the gastrointestinal tract. The active substance A is long in the blood (24 hours) and is slowly excreted from the vascular bed. The absolute bioavailability for the composition is 0.71.

Example 4. The study of the specific pharmacological (immunosuppressive and antitumor) activity of the composition.

Research methods

Animals.

Healthy sexually mature animals were used in the work — CBA, С / 2, C57BB / 6 mice, hybrids mice (C57BB / 6xEVA / 2) P1 (Ό6Ό2 )1) mice of both sexes, 8–10 weeks old, body weight 1820 g. initial body weight does not exceed ± 10%. Control and experimental animals of the same age were obtained simultaneously from one cattery (Dawn, Tomsk).

Before and during the experiments, control and experimental animals were kept in a vivarium under the same conditions — standard plastic cages with small wood chips (no more than 10 individuals) on a standard diet.

All studies were carried out at the same time of the day (in the morning).

The experiments were carried out in accordance with the rules adopted by the European Convention for the Protection of Animals (Strasbourg, 1986) and approved by the Committee on Biomedical Ethics of the Research Institute of Clinical Immunology SB RAMS (Novosibirsk). The study of the pharmaceutical composition was carried out in several series of experiments, respectively, each series of experiments had its own control.

Determination of the amount of 1dM antibody-forming cells ΐη νίνο

The pharmaceutical composition (capsule contents dissolved in culture medium No. 1640) was administered to mice intragastrically at a dose of 25 mg / kg (corresponding to a dose of 10 mg / kg of pure substance A) in a volume of 0.5 ml daily once a day on the day of immunization and the next 3 days (inductive / productive phases of the formation of a humoral immune response). The control animals were injected with a solvent of the same volume and mode (culture medium 1640). Animals were immunized intravenously with ram erythrocytes (EB) at a dose of 10 / mouse once. The amount of 1dM AOK in the spleen of mice was evaluated at the peak of the immune response (5th day after immunization) by the number of local hemolysis zones in a semi-liquid medium by the modified method [5]. The results were expressed in the absolute amount of 1dM AOK in the spleen. In each study group, there were 10 animals.

A model of lupus-like immunocomplex glomerulonephritis in mice was carried out by transferring Β6Ό2Ε1 female lymphoid cells of the ΌΒΑ / 2 parental line to females [6]. Cells of lymph nodes, thymus and spleen were introduced in a ratio of 1: 3: 6 (5x10 ⁶ cells of lymph nodes, 15x10 ⁶ thymus cells, 30x10 ⁶ spleen cells, respectively), isolated externally, in a sterile medium of ΚΡΜΙ-1640. Each recipient mouse received 50x10 ⁶ cells by intravenous injection in the needles

- 2 014434 stasis vein in a volume of 0.5 ml of medium twice with an interval of five days. For control, intact animals of the same genotype, sex, age as in the experiment were used. Moreover, recipient mice by the 3rd month develop lupus-like kidney damage of autoimmune origin.

Kidney damage was tested by the level of protein in the urine. The protein content in the urine was determined calorimetrically with the dye Kish8a1 LrShap! Lye (Loba Rethysete) with the help of ThiePes MiSzkap, wavelength λ 570 pt. The reagent was prepared as follows: 10 mg of Coomassie was dissolved in 5 ml of C ₂ H ₅ OH. After complete dissolution of the dye, 11.2 ml of 70% H ₃ PO _{4 was} added. The total volume was adjusted to 100 ml, filtered. To 5 μl of urine diluted 5 times in PBS, 150 μl of Coomassie dye was added. The calibration curve was built according to the IDL (100-1000 μg / ml).

The acute GVHD model (graft versus host reaction) was carried out by intravenous transfer to females of Β6Ό2Ρ1 100x10 ⁶ spleen cells of the parent line C57BB / 6 (C57B1 / 6 ^ B6I2P1). The severity of acute GVHD was evaluated by the number of live mice 21 days and 28 days after the induction of the reaction.

To determine the antitumor properties of the composition, a model of metastasis of hepatoma G27 cells to the lungs in mice was used.

Research results

1. The study of the immunosuppressive properties of the composition in the model of the primary humoral immune response.

The effect of the active substance A and the pharmaceutical composition on the inductive / productive phases of the primary humoral immune response was studied: the contents of the capsules at a dose of 25 mg / kg (and substance A at a dose of 10 mg / kg) were administered to mice simultaneously with the antigen and then for 3 days daily. Several series of experiments were carried out. The results are presented in table. one.

Table 1. The effect of A and composition on the primary humoral immune response in mice Β6Ό2Ρ1

Groups of animals 1§M KLA / spleen The control 22484 BUT 14521 (-36%) * Composition 15133 (-33%) *

* percent suppression

Thus, the obtained results indicate that the composition is characterized by a pronounced reliable immunosuppressive (inhibitory) effect with respect to the formation of a humoral immune response, comparable to a pure substance (with the same dose of the active substance).

2. The study of the immunosuppressive properties of the composition in the model of acute GVHD.

On recipient mice Β6Ό2Ρ1 on the day of transfer of spleen cells from the parent C57B1 / 6, the composition was administered intragastrically at a dose of 25 mg / kg (at a dose of 10 mg / kg of the active substance) and then daily for 12 days. The number of live mice was recorded on days 21 and 28 from the time of GVHD induction. The data are presented in table. 2.

Table 2. The effect of the composition on the development of acute GVHD

Groups of animals

GVHD ________

GVHD + A *

day

5/17 *

11/16 * day 4/17 10/16 in the numerator is the number of living mice, in the denominator is the number of mice in the experiment.

As can be seen from the table. 2 data, the composition significantly inhibits the development of acute GVHD and increases animal survival by more than 2 times.

3. The study of the immunosuppressive properties of the composition in a model of autoimmune disease.

Mice with immunocomplex glomerulonephritis were daily administered an intragastric composition at a dose of 25 mg / kg (at a dose of 10 mg / kg of active ingredient), the course was 13 administrations. In the same mode, control mice were administered azathioprine (at a dose of 10 mg / kg of active ingredient). The urine protein level was determined before drug administration, 7 days after the start of administration and at the end of the course. The data are presented in table. 3 and 4.

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Table 3. The effect of the composition on the protein level in urine in mice with glomerulonephritis

Pharmaceutical composition Protein in the urine (mg / ml) before treatment 8 day from the start treatment 19 day from the start treatment one 2 3 5.3 4.8 4.2 6.6 one 5.2 4.1 5.8 6.0 <l 5.9 2 2.5 3.8 5.8 5.2 c about α.ζ. 4.5 3 2.9 4.0 3.1 3.1 L £ 6.7 6.1 5.8 3.5 1.5 3.0 3.6 3.2 3.3 M * = 5.3 M * = 4.4 M * = 3.7

- mean

Table 4. The effect of azathioprine on urine protein levels in mice with glomerulonephritis

Azathioprine Protein in the urine (mg / ml) before treatment 8 day from the start treatment 19 day from the start treatment 4.4 3.8 4.0 5.0 4.5 4.4 5.6 4.5 4.3 5.8 4.7 3.3 M * = 5.2 M * = 4.4 M * = 4.0

* - mean

As can be seen from the data table. 3 and 4, the composition has an immunosuppressive effect in mice with immunocomplex glomerulonephritis comparable to the well-known drug azathioprine.

4. The study of the antitumor properties of the composition

CBA mice were injected intravenously with G27 hepatoma cells at a dose of 100x10 / ml. Metastases in the lungs were taken into account on day 15 after tumor inoculation. The composition was administered in various doses intragastrically (14 administrations once a day). The results are presented in table. 5.

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Table 5. The effect of the composition on the process of metastasis of hepatoma G27 cells in the lungs of CBA mice

Groups M18 * Metastasis frequency Number of metastatic mice / total number of mice % inhibition The control 5,4 4/5 Composition 25 mg / kg 1,6 3/5 3.4 times 50 mg / kg 2.6 3/5 2.0 times

M18 * - the average number of metastases in the lungs

As can be seen from the data table. 5, the composition effectively (reliably) inhibits the process of metastasis of tumor cells (G27 hepatoma) to the lungs in mice.

Thus, the results obtained indicate that a new pharmaceutical composition has been developed containing as an active substance an effective amount of tris- (2hydroxyethyl) ammonium salt (1-benzylindolyl-3-thio) acetic acid. The composition is low toxic and has a fairly high absolute bioavailability (0.71). The composition may be in the form of capsules. Capsules have the necessary disintegration and release of the active substance, are stable during storage. Shelf life 2 years.

A method for producing a composition that can be implemented in industry has also been developed.

In experiments on mice, the immunosuppressive and antitumor properties of the composition were studied. It was shown that it exhibits a pronounced immunosuppressive effect in mice in a model for the formation of a humoral immune response, in a model of immunocomplex glomerulonephritis, as well as in a model of acute GVHD. The composition has an antitumor effect, significantly suppressing the process of metastasis of G27 hepatoma cells in the lungs of mice. Currently known drugs with similar properties are used in medical practice to treat autoimmune and tumor diseases, as well as to prevent rejection of transplanted organs and tissues [3].

List of references

1. Pat. 2228178 RF A61K 31/405, A61P 37/06, declared 12/23/2002, publ. 05/10/2004.

2. Pat. 2240793 RF A61K 31/404, A61P 35/00, declared 12/9. 2003, publ. 11/27/2004.

3. Mashkovsky M.D. // Medicines, 14th edition, 2001, vol. 2, p. 197.

4. Seei Tekhoook ο1 Furniture, 18Sebyb, 1988, p. 584 apb 1095.

5. Sipptkat A.1., Sentente A. // 1tipododu, 1968, νοί. 14 (4), P.599-600.

6. K1tiga M., Oyeytapp E..// C1t. 1ttypo1. Apb 1ttiporaFo1., 1987 ^ o1.43, Ho.1.-R.97-109.

Claims (2)

1. A pharmaceutical composition having immunosuppressive and antitumor properties, characterized in that it is made in the form of a capsule and contains, as an active ingredient, tris (2-hydroxyethyl) ammonium salt (1-benzylindolyl-3-thio) acetic acid and excipients in the amount of wt.%: active ingredient - 40 and excipients - 60, while as excipients it includes in wt.%: potato starch - 20, lactose - 38.5, magnesium stearic acid - 1 and methyl cellulose - 0.5. 2. The method of obtaining the pharmaceutical composition according to claim 1, characterized in that the active substance of tris (2-hydroxyethyl) ammonium salt (1-benzylindolyl-3-thio) acetic acid is mixed with lactose and potato starch, then the resulting mixture is moistened with a binder solution (2% methylcellulose solution), then wet granulation is carried out successively, drying and dry granulation, the granulate is dusted with magnesium stearic acid. α Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2