RU2033157C1 - Drug possessing interferon-inducing and immunomodulating (immunostimulating) activity - Google Patents

Abstract

FIELD: medicine, particularly, pharmacology. SUBSTANCE: 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5- oxy-6-bromindole hydrochloride monohydrate (conventional name "arbidol") for new object, that is, as interferon-inducing and immunomodulating (immunostimulating) remedy. EFFECT: pronounced interferonogenic and immunostimulating effect without undesired side effects. 11 tbl

Description

 The invention relates to medicine, namely to a group of drugs with interferonogenic and immunomodulatory effects, and relates to the well-known drug arbidol.

 Currently, much attention is paid to the search and study of drugs that can modulate (stimulate or suppress) the immune response of the body-immunomodulators.

Immunostimulants increase the overall resistance of the body, affecting specific immune responses and nonspecific defense factors, including the synthesis of endogenous interferon. They find application for the treatment of diseases in the pathogenesis of which an important role is played by violations of the body's immune status of primary and secondary immunodeficiency states, including malignant neoplasms, as well as chronic and recurrent viral infections [1]
Among the few synthetic chemicals used as immunostimulants, the most active and widely used in medical practice is levamisole 2,3,5,6-tetrahydro-6-phenylimidazo [2,1-b] thiazole hydrochloride [2] Initially, this the drug was used as an anthelmintic. As an immunostimulant, levamisole is used in the complex treatment of various diseases associated with immunodeficiency states. The use of levamisole in the treatment of severe recurrent forms of herpes virus infection is known [3]
Levamisole, like some other immunomodulators, stimulates the synthesis of serum interferon in the body. The titers of interferon synthesized by levamisole do not exceed 80-160 u / ml in cell culture; 160-320 units / ml in the blood serum of mice and 40-80 units / ml in the blood serum of humans [4,5] However, levamisole is highly toxic and can cause various side effects: headache, sleep disturbances, fever, change in taste sensations, dyspeptic symptoms, olfactory hallucinations (change in odors), allergic skin reactions, urticaria, flu-like and neurological symptoms. The most dangerous side effect that can occur during therapy with levamisole as an immunostimulant is agranulocytosis, a decrease in neutrophilic granulocytes below 25%. Therefore, blood tests should be systematically performed during the treatment with levamisole.

 Levamisole supplies Hungary with the USSR (production of the chemical plant Gedeon Richter AO Budapest).

 The purpose of the invention is a drug with a pronounced interferonogenic and immunostimulating effect and without undesirable side effects.

по новому назначению, а именно в качестве интерфероноиндуцирующего и иммуномодулирующего (иммуностимулирующего) средства.This goal is achieved by the use of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole hydrochloride, a monohydrate of the formula (I) (conventionally arbidol)

for a new purpose, namely as an interferon-inducing and immunomodulating (immunostimulating) agent.

Arbidol underwent a clinical study and was recommended by the FC of the Ministry of Health of the USSR for medical use as a treatment for influenza in the form of tablets for oral administration [6,7]
Arbidol is non-toxic, well tolerated and has no side effects in both healthy and flu patients.

 Interferon-inducing and immunostimulating activity of arbidol was studied in experimental studies on animals and partially on people (on volunteers in a clinic).

 The study of interferon-inducing activity of arbidol.

 The effect of arbidol on non-specific defense factors, in particular on its ability to induce interferon, was studied.

 The study of the interferon-inducing activity of arbidol was carried out in in vitro experiments (on a primary trypsinized culture of chicken embryo FEC fibroblast cells), in in vivo experiments on 340 non-inbred white mice weighing 18-20 g and 25 volunteers (healthy young people of both sexes aged from 18 to 30 years old).

In order to induce interferon, various doses of arbidol with 2% DEAE dextran in a 1: 4 ratio of 0.2 ml per tube with a monolayer of cells were introduced into the FEC cell culture. Incubation of treated cells was carried out at ³⁷ ° C for 1 hour. The cells were then washed from preparation 199 and N medium 1 was poured 199 ml of N medium without addition of serum of cattle. After 8, 24 and 48 hours of incubation at ³⁷ ° C, the culture fluid was collected and titrated for the presence of interferon in cell culture FEC Venezuelan encephalomyelitis virus by the standard procedure [8]
The titration results are presented in table 1.

When studying the interferon-inducing activity of arbidol, a highly active interferon inducer ds RNA of phage RF ₂ (double-stranded ribonucleic acid of phage RF ₂ ) was used as a standard comparison drug [4]
The results presented in table 1, allow us to conclude that arbidol is a highly active inducer of interferon in cell culture FEC. At a concentration of 20 μg / ml, arbidol induces interferon after 8 hours (titer 640-1280 units / ml, the maximum titer of 2560 units / ml is determined after 24 hours, by 48 hours the titer of interferon is reduced to 320 units / ml).

 The dose-dependent effect of induction of interferon in the cell culture of FEC under the influence of arbidol has been established. A decrease in the concentration of arbidol by 2 times (up to 10 μg / ml) leads to a decrease in the induction of interferon by about 8 times, but the dynamics remain the same.

A comparative study of interferon induction under the influence of arbidol and the reference highly active inducer of interferon ds RNA of phage RF ₂ revealed similar dynamics of induction and close titers of interferon, however, ds RNA of phage RF ₂ required 20 times more (400 μg / ml), concentration 100 μg / ml caused a weak induction of interferon.

 In the next series of experiments, the ability of arbidol to induce interferon in the blood serum of experimental animals was studied.

 Arbidol in doses of 250, 125 and 62.5 mg / kg was administered to mice per os once (3 groups of mice, 100 pieces each) and after 16, 24, 48 and 72 hours received blood serum, which was used to titrate interferon in a transplanted mouse cell line L in relation to the test virus of vesicular stomatitis. In addition, a group of 20 mice received arbidol intraperitoneally (i.v.) at a dose of 10 mg / kg once and after 24 hours blood serum was obtained, in which the titer of interferon was also determined.

 The results are presented in table.2.

 The results presented in table. 2, indicate that under the influence of arbidol, administered per os, induction of interferon in the blood serum of mice occurs. The effect is characterized by dose dependence and is most pronounced in the dose range from 250 to 62.5 mg / kg of animal body weight. High titers of interferon (640 units / ml) are detected in the blood serum of mice after 16 hours and are observed up to 48 hours. Arbidol stimulates the induction of interferon in the blood serum of mice also with intraperitoneal administration (see Table 2).

 With repeated administration of arbidol to mice in order to induce interferon in mice, a state of hyporeactivity occurs, which is expressed in a decrease in titers of interferon in the blood serum. The state of hyporeactivity is a characteristic phenomenon for interferon inducers [9] Table 3 summarizes the results of experiments conducted on 400 mice to study the state of hyporeactivity after repeated administration of arbidol.

 Based on the data obtained (Table 3), it can be concluded that with repeated administration of arbidol in mice, a state of hyporeactivity occurs, which is manifested in a decrease in titers of interferon in the blood serum, especially sharp after 3-4 consecutive injections of the drug: from 320-640 to 20-0 units / ml.

Arbidol as an inducer of interferon has a prophylactic effect in experimental viral infections. The prophylactic activity of arbidol was studied in influenza pneumonia in mice caused by intranasal infection of animals with the influenza A / Bethesda / 63 virus (H2N2), and in generalized herpes simplex in mice caused by intranasal infection of herpes simplex virus type 1 herpes simplex strain L ₂ .

 The results are presented in table 4.

 Prophylactic administration of arbidol 24 and 6 hours before infection with per os in doses from 31.2 to 125 mg / kg reduces the death of mice from influenza pneumonia by 40-50% compared with the control. The introduction of arbidol at a dose of 30 mg / kg per os once 24 hours before infection protects 40% of animals with a generalized herpetic infection from death.

 Interferon-inducing activity of arbidol has also been studied in humans. The blood serum of people who received per os tablets of arbidol (0.1 g) according to various schemes was examined for the presence of serum interferon. Serum titration was carried out according to the standard method using a culture of human diploid cells M-22 and mouse encephalomyocarditis test virus. The results are presented in table.5.

 It was established that a single dose of arbidol at a dose of 100 mg per os causes the induction of serum interferon in titers of 40-80 units / ml in 8 out of 13 (61.5%) volunteers.

 A 3-fold intake of 1 tablet of arbidol (300 mg) during the day led to a sharp increase in the induction of interferon: interferon was determined in the blood serum of all 12 volunteers (100%) in titers reaching 160-320 units / ml. With a further increase in the dose to 600 mg for 2 days (3 tablets per day), a sharp decrease in the induction of interferon was observed: the titer of interferon in the blood serum did not exceed 40-80 u / ml.

 The results obtained indicate a pronounced interferon-inducing activity of arbidol in humans when taken per os. The maximum amount of interferon in human serum is determined after taking 300 mg of arbidol (100 mg 3 times a day). An increase in the dose and duration of taking arbidol led to the development of a state of hyporeactivity in people, which was characterized by a sharp decrease in serum interferon titers.

 The study of immunomodulatory (immunostimulating) activity of arbidol.

 The effect of arbidol on the functions of the immune system was studied.

 The function of phagocytic cells under the influence of arbidol was studied in sexually mature female mice of a hybrid (CBA / C 57 BL6) F. Arbidol was administered per os at a dose of 125 mg / kg once and for 5 days, 1 time per day. As a comparison drug, levamisole was used, which was administered according to the same scheme at a dose of 50 μg / mouse. At different times after drug administration, peritoneal macrophages were isolated in mice, the absorbance of which was determined by the absorption of neutral red and quantitatively determined by a calibration curve spectrophotometrically at 530 nm.

 The results are presented in table.6.

 The introduction of arbidol or levamisole did not affect the number of macrophages washed out from the abdominal cavity, but stimulated their absorption capacity. So, by 2-3 days after a single injection, the absorption capacity of macrophages in mice treated with arbidol increased to 155% and in mice treated with levamisole to 135% (p <0.05).

 After daily administration of the drugs in the same doses for 5 days, the absorption capacity of macrophages remained at the same level of 164 ± 13% (p <0.05) and 13.8 ± 11% (p <0.05), respectively.

 Thus, in vivo administration of arbidol stimulates the absorption capacity of macrophages to a greater extent than levamisole administration.

The effect of arbidol on antibody production has been studied in comparison with levamisole and ds RNA F ₂ . Two lines of mice were used: CBA / C 57 BL ₆ ) F, as well as tumor-bearing animals (breast carcinoma grafted subcutaneously in the thigh), intact and irradiated with a total of 2 Gy.

Antitelogenesis was studied in relation to sheep erythrocytes during immunization intraperitoneally at a dose of 1 x 10 ⁸ cells. 5 days after immunization, the animals were killed by decapitation and the content of antibody-forming cells (AOK) in the spleen was determined according to the Cunningheim method. The results of the experiments are summarized in table.7.

Arbidol increased the amount of AOK under various administration schemes to 167-246% levamisole and ds RNA of phage RF ₂ in the range of 143-170%
Only arbidol had a pronounced immunostimulating effect in relation to the irradiated animals, and to the greatest extent when administered 3 days before irradiation: the immune response immediately after irradiation was 230% with respect to the control. Levamisole in these animals is ineffective; ds RNA of phage RF _{2 is} ineffective.

 Arbidol also had an immunostimulating effect in relation to tumor-bearing animals (7-17 days of tumor growth) after a single (immunization after 3 days) or five times daily administration. Levamisole and DS RNA are ineffective in these experiments.

 Arbidol is active in totally irradiated tumor-bearing animals and had a pronounced immunostimulating effect even when the level of antibody production was reduced by about 50 times compared with intact animals. Levamisole also had an immunostimulating effect in relation to these animals; ds RNA was ineffective.

Thus, arbidol has the ability to stimulate antibody production in all studied mouse strains, both intact and irradiated, and is superior in effectiveness to comparing levamisole and ds RNA of phage RF ₂ .

The effect of arbidol on cellular immunity reactions tested by the graft versus host (GVHD) reaction was studied by a known method [10]
Mice hybrids (CBA x C 57 BL / 6) F ₁ males aged 3 months were irradiated with 5 Gy (apparatus "Ray", the dose rate of about 0.8 Gy / min) for stimulating endogenous spleen colony formation, surviving after irradiation hematopoietic colony forming units (CFU-C).

 1 day after irradiation, he was injected intravenously with lymphocytes obtained from mesenteric lymph nodes of the parental CBA line at the age of 3 months at concentrations of 1.25, 2.5 and 5.0 million karyocytes. In the experimental groups, immediately after intravenous administration of lymphocytes, arbidol was administered orally at the rate of 100 mg / kg body weight or levamisole at the rate of 5 mg / kg in a 2% starch solution. There were 15 mice in each group. Eight days after irradiation, mice were sacrificed, spleens were fixed, and the number of colonies with a diameter of at least 0.2 mm was counted.

The data obtained are summarized in table 8. The introduction of allogeneic lymphocytes in large doses (2.5 and 5 million) in the control leads to a decrease in the yield of splenic colonies, and at the lowest dose used (1.25–10 ⁶ ) it slightly increases the output of splenic colonies. Administration of arbidol reduces both observed deviations to the control value, unless the dose of introduced cells is the largest (5–10 ⁶ ). Levamisole has a similar, but less pronounced effect. In addition, in the case when allogeneic lymphocytes are not introduced, levamisole somewhat reduces the yield of splenic colonies, and arbidol does not have such an effect.

 Thus, the administration of arbidol under conditions of induced GVHD normalizes the proliferative activity of “target cells” and their ability to produce a viable colony both in case of inhibition (at high doses of lymphocytes) and in case of stimulation (lower doses of lymphocytes). In its effectiveness, arbidol is superior to the standard drug levamisole.

 As an immunostimulating agent, the effect of arbidol on the growth of a transplanted tumor and thermal damage to the skin was studied.

 We studied the effect of arbidol on the growth of sarcoma-45, grafted subcutaneously in the right thigh to sexually mature rats of the male Wistar strain. Arbidol was administered intragastrically as a suspension in a 1% starch solution at a dose of 125 mg / kg for 5 days daily two weeks after tumor inoculation. Only starch solution was administered to control animals. Tumor growth was monitored by intravital measurement of its volume.

 Results. When observed for two months, inhibition of tumor growth was detected in the group (10 animals) treated with arbidol (Table 9).

 Thus, arbidol has a depressing effect on the growth of a solid transplantable experimental tumor, possibly indirectly through the stimulation of immunological reactivity.

 Due to the fact that arbidol is intended to be used not only for purely radiation exposure to the tumor, but also for thermal radiation therapy, it was of interest to evaluate the effect of this drug on the heat sensitivity of the skin of mice. Previous studies have shown that the reference drug levamisole did not have any effect on the heat sensitivity of the skin.

The experiments were conducted on white nonlinear male mice at the age of 3 months. In animals anesthetized (Nembutal 70mg / kg body weight) in a water bath heated tail at ⁴⁵ ° C for various periods of time (11-23 min). Then, within 2 weeks, the incidence of necrosis was taken into account. In each group there were 25-70 mice. Arbidol was administered the day before hyperthermic exposure orally in a 2% starch solution at the rate of 100 mg / kg body weight.

 The results of the experiment are given in table 10. Arbidol has a low thermoprotective effect, and the differences for the majority of exposure durations studied are statistically significant.

 Thus, we can recommend the use of arbidol in the thermal radiation treatment of tumors on the skin, since it has a heat-shielding effect.

 The effect of arbidol on the immunological status of people was studied.

 Healthy young people (volunteers) from 18 to 30 years old (25 people) were monitored.

 The state of immunological status was judged on the basis of a study of T-cell immunity: the state of lymphocytes in the peripheral blood, the absolute and relative content of T cells (according to the Jondal method), and the state of stability of receptors on the surface membrane of lymphocytes.

 Under the influence of various doses of arbidol, the immunological status of healthy young people without preliminary antigenic stimulation and vaccine influenza infection was studied, which was created by vaccination with a commercial influenza vaccine type A, manufactured by the Odessa company for the production of vaccine preparations of the Ministry of Health of the USSR, intranasally at a 1: 1 dilution of 10: 1 ml

 The study was carried out using the double blind control method, along with 100 mg arbidol tablets, a similar placebo was used. Results are processed statistically.

 The data obtained indicate that arbidol with a single dose of 100 mg by mouth and a daily dose of 300 mg (100 mg 3 times a day) does not change the size of the T population, but leads to a change in the connection of receptors with the surface membrane of lymphocytes, which persists for 14 days (observation period).

 Vaccination carried out a day after the administration of the drug did not affect the nature of the changes in the connection of receptors with the membrane of lymphocytes.

 The change in the surface properties of the lymphocyte membrane under the action of arbidol is likely to affect the change in the functional properties of these cells, which leads to an increase in the function of the immune system, as evidenced by the observed protective effect of arbidol in relation to the development of influenza infection. Arbidol prevents the development of post-influenza complications and significantly reduces the frequency of exacerbations of chronic diseases in people with influenza.

 Experimental and clinical studies indicate that arbidol is an active interferonogen and a very promising immunostimulant.

 Arbidol stimulates the synthesis of interferon both in vitro (in cell culture) and in vivo (animals, humans).

In terms of the severity of interferon-inducing activity in the experiment, arbidol is superior to levamisole and is close to the known interferonogen ds RNA of phage RF ₂ [4] It should be noted that the presence of interferon in human blood serum for ds RNA of phage RF _{2 has} not been established (see Table 11).

 The study of arbidol as an immunomodulator showed that it is able to stimulate various functions of the immune system, which increases the body's resistance to the growth of transplanted tumor. The effect of arbidol is also manifested in the irradiated body, as shown by the example of the effect of the drug on the manifestations of GVHD. In most of the tests studied, arbidol is superior to, or at least not inferior to, levamisole (or ds RNA) used as reference compounds. In addition, it was found that arbidol is able to slightly reduce thermal damage to the skin. All these data indicate the promise of conducting clinical trials of arbidol as an immunostimulant in radiation or thermo-radiation treatment of cancer patients.

 It is known that such treatment currently has a significant immunosuppressive effect. Therefore, against the background of a pronounced primary regression of the tumor, relapses and distant metastases may develop in the future. Modern ideas about the treatment strategy for cancer patients suggest the mandatory correction of the immunological functions of the tumor-bearing organism after (or in the process) intensive cytostatic and cytotoxic therapy. In this regard, the use of arbidol seems to be very promising. It should be noted that the presence of pronounced interferonogenic activity in arbidol also indicates the prospects of its inclusion in the treatment regimen of cancer patients. At present, certain experience has been accumulated in the use of interferons in the treatment of cancer, and the promise of combining interferon with chemotherapy drugs, hyperthermia, and ionizing radiation has been shown.

 In a clinical study of the effectiveness of arbidol in influenza, it was shown that the drug is practically non-toxic, well tolerated by healthy and flu-infected people. This is a great advantage of arbidol over levamisole, which is a highly toxic drug.

To develop optimal schemes for the use of arbidol as an immunostimulant, a synthesis and study of labeled arbidol ¹⁴ C was carried out. The results show that the drug is rapidly excreted, which allows not to fear its possible accumulation in the body.

 The totality of the data obtained allows us to consider arbidol as an effective interferonogen and immunostimulant that does not have toxic and side effects on the body. Arbidol can be recommended for clinical studies as an immunostimulant in secondary immunodeficiency states, including radiation and thermo-beam treatment of cancer patients, as well as chronic and recurrent viral infections. Literature. 1. Petrov R.V. Immunology. M. Medicine, 1987, p. 379-389.

 2. Mashkovsky M. D. Medicines, part 2. M. Medicine, 1984, p. 169-171.

 3. Kasparov A.A. et al. Inducers of interferon. M. Medicine, 1982, p. 155-160.

 5. Ershov F.I. and Zhdanov V.M. Interferon Inductors. M. Medicine, 1982, p. 7-18.

 5. Hadden J.W. Yopez C. O'Reily R.J.O. Hadden E. In: Third Conference on Antiviral Substances. Annals of the New York Academy of Sciences, 1977, 284, p. 139-152.

 6. Application VNIHFI N 2.078.859 / 23-4, 1974.

 7. Application VNIHFI N 4106938/14, 1986.

 8. Fomina A. N. et al. Biological activity of a new domestic natural inducer of double-stranded RNA. Antibiotics, 1980, N 1, p. 28-32.

 9. Ershov F.I. and Novokhatsky A.S. Interferon and its inducers. M. Medicine, 1980, pp. 40-41.

 10. Petrov R.V. and other Radiobiology, 1970, T. 70, N 4, s.532-535.

Claims (1)

Means possessing an interferon-inducing and immunomodulating (immunomodulating) activity, characterized in that it is 1-methyl-2-phenylthiomethyl-3-carbetoxy-4-dimethylaminomethyl-6-oxo-hydromide-5-oxoindromide

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