RU2202547C2 - Antiviral immunomodulating agent - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention relates to N-acridone acetic acid lithium salt eliciting an antiviral and immunomodulating property. Indicated compound enhances effectiveness of effect in respect to DNA-containing viruses and it is able to induce the death of infected cells. EFFECT: enhanced effect of agent. 1 dwg, 14 tbl, 6 ex

Description

 The invention relates to the field of medicine and veterinary medicine, namely to anti-infectious, including antiviral, agents based on hydrophobic lithium salts of acridone having a combined mechanism of action (direct to the microbial causative agent located intracellularly and indirectly through activated factors of cellular immunity), and may find application as a specific antiviral drug for emergency prophylaxis and treatment of viral infections of various localization, as well as infections associated with imm odefitsitnymi states and, as a consequence, the emergence of competing pathology of a different nature.

 Lithium salt-based drugs used in antiviral therapy are known (see Skinner GRB, Hartley C., Buchan A. at al. The effect of lithium chlorid on the replication of Herpes simplex virus. Med. Microbiol. Immunol., 1980, 168 , 139-148), for example lithium chloride, in which lithium ions have an antiviral effect, regardless of which salt they are in. These drugs give a specific prophylactic and therapeutic effect when used in the form of tablets, ointment-based or other pharmaceutically acceptable carriers. However, with relatively low toxicity, these drugs do not have antiviral activity against RNA-containing viruses (for example, influenza viruses, arborviruses, etc.).

 Immunomodulating drugs based on interferon inducers are known for use in antiviral therapy (see US Pat. No. 4,314,061, MKI C 07 D 413/14). This tool also has a certain prophylactic and therapeutic effect and it is used dissolved in water, on an ointment base or in other pharmaceutically acceptable carriers.

 The closest in essence to the claimed invention and the mechanism of action on the cells of the immune system is a low molecular weight inducer of interferon based on the sodium salt of 10-acridonoacetic acid (see RF patent 2020941). With high efficiency and low toxicity, the drug is hydrophilic, unstable during storage, difficult and expensive to manufacture. In addition, it is not effective against DNA viruses. Efficiency is ensured only by increasing non-specific resistance (induction of interferon alpha / beta), there is no effectiveness when used by mouth, difficulty in obtaining, instability when stored in solutions.

 The invention is directed to the synthesis of a low molecular weight chemical compound, the biological effect of which would be a combined, two-sided activity: on the one hand, the drug must have inhibitory activity against certain stages of virus reproduction, and on the other, the ability to induce the death and removal of infected cells from the body or other effector functions that ensure the inactivation and elimination of the pathogen without cytopathic effects on the infected cell.

 The technical result from the use of the invention is to significantly increase the effectiveness of the drug against DNA-containing viruses (several thousand times), the combination of non-specific effects due to the induction of interferon with direct antiviral effect on the reproduction of the virus in an infected cell, ensuring the effectiveness of the drug when administered by mouth, simplification of technology and production costs.

 The structural formula of the synthesized preparation (hydrophobic lithium salt of N-acridonoacetic acid) is shown in the drawing.

The gross formula is C ₁₅ H ₁₀ NO ₃ Li. Molecular mass: 259.19. Purity not less than 98%. Solubility: the cast salt of acridonoacetic acid is slightly soluble in water (0.2-0.3%), solubility increases slightly in hot water, practically insoluble in alcohols, ether, acetone, chloroform, DMAA, DMF, sparingly soluble in hot (90- 110 ^o C) dimethyl sulfoxide.

 The drug is obtained, for example, as follows. A mixture of N-acridonoacetic acid in an aqueous solution of isopropanol or N-acridonoacetic ester with an equivalent amount of an aqueous lithium hydride solution is refluxed in an oil bath, then cooled, filtered, washed with isopropyl alcohol and dried in vacuo. As a result of such a simple technology, a lithium salt powder of N-acridonoacetic acid is prepared, ready for use in a dosage form.

 The invention differs from the prototype in that significant changes are made to the known antiviral agent, namely, instead of the sodium salt of N-acridonoacetic acid, the lithium salt of N-acridonoacetic acid is used.

 The above selection is based on the results of studies conducted by the authors of the present invention, which discovered new physicochemical properties of the lithium salt of N-acridonoacetic acid, the main of which are hydrophobic properties, new pharmacological activity and new pharmacological properties.

 In contrast to the previously known action of lithium salts (lithium chloride, lithium succinate, etc.) on DNA-containing viruses, where lithium ion has an antiviral effect, in the present invention, the lithium salt of acridonoacetic acid is effective not only against DNA, but also RNA- containing viruses, and this action extends not only to viruses of the herpes, smallpox, adenovirus groups related to DNA genomic viruses, but also to viruses such as influenza A and B viruses, Japanese encephalitis and Venezuelan encephalomyelitis, and fever virus and Rift valleys related to RNA genomic viruses.

 Unlike the prototype, the proposed tool is effective not only for the parenteral route of administration - against the same viruses and, possibly, other intracellular parasites (bacteria, protozoa, eukaryotic microorganisms). The intracellular cycle of pathogen development in the sensitive host organism is crucial in predicting the inhibitory effect of the proposed drug.

 The relatively low toxicity (therapeutic index of more than 10) and the low price of the drug will allow it to be widely used as the drug itself for the treatment and prevention of infectious diseases, as well as in combination with other drugs to enhance the effects of the latter, reduce or eliminate the resistance of the microorganism to them, etc. d. It is also possible the combined use of the claimed drug in order to reduce to subtoxic concentrations of another drug while maintaining its effectiveness due to the synergistic action with a lithium derivative.

 The obtained new agent has immunomodulating properties (in vivo effect on cellular and humoral factors of immunity), and, on the other hand, an expanded spectrum of antiviral activity due to the introduction of lithium ion instead of sodium ion in the molecule.

 The structural formula of the drug, proven by known methods, is shown in the drawing, and the physicochemical constants are in the essence of the invention section.

The synthesized preparation is called "virolite". In our opinion, it is an inducer of the synthesis and secretion of cytokines, mainly proinflammatory cytokines, activating the functional activity of natural killers and macrophages, thereby providing a lytic effect on cells that carry foreign genetic information or other effector functions of specific cytoxic CD8 (+) T cells , providing non-cytolytic inactivation and elimination of the pathogen from the infected cell. On the other hand, the drug exhibits an inhibitory effect on the reproduction of viruses, having the ability to specifically interact with the viral target. The molecular weight of virolite is 269.19. The synthesis of the substance is carried out using practically non-toxic components widely used in medicine. Properties do not change when stored in a dark place at room temperature for 1.5 years (observation period). Acute toxicity to mice is LD ₅₀ - 713 mg / kg when administered intraperitoneally, does not cause allergic complications and adverse reactions. Contraindications for use in experiments on laboratory animals have not been established, side effects during use have not been identified. It can be used in conjunction with antibiotics, sulfonamides, hormones, serum proteins. The use of virolite does not prevent the use of traditional pathogenetic and symptomatic therapy. Effective therapeutic doses for mice are 50-100 mg / kg body weight, the therapeutic index is more than 10. After intramuscular injection, the virolite quickly penetrates the blood, reaching a maximum level after 2 hours unchanged. It does not bind to plasma proteins. It is excreted unchanged from the body in the urine and after 24 hours in the blood and urine is not detected.

 The drug has a wide range of biological effects: it is highly active against influenza A and B viruses, Venezuelan encephalomyelitis virus, Rift Valley fever virus, herpes simplex viruses, possibly causative agents of certain invasive infections caused by bacteria, fungi and protozoa. The carcinostatic effect of the drug is assumed.

 Example 1. Synthesis of lithium salt of N-acridonoacetic acid.

Ten grams of N-acridonoacetic acid was added to a 250 ml round bottom flask containing 100 ml of 40% aqueous isopropanol. To the resulting mixture was added 25 ml of a warm solution containing 1.42 g of LiOH • 1/2 H ₂ O in H ₂ O (1.08 eq). The suspension is boiled for 40 minutes in an oil bath under reflux. It is cooled to 50 ^o C, filtered, washed with alcohol (isopropyl), dried in vacuum at 90-95 ^o C. Obtain 9.2 g of lithium salt of acridonoacetic acid.

 Example 2. Synthesis of the lithium salt of N-acridonoacetic acid.

Ten grams of N-acridonoacetic acid ethyl ester was added to a 250 ml round bottom flask equipped with a reflux condenser, 1.35 g LiOH • 1 / 2H ₂ O in 100 ml H ₂ O was added. The mixture was boiled for 1.3 hours ( 80 minutes), cooled to 5-7 ^° C, filtered, washed with 3 • 50 ethanol, dried in vacuo at 90-95 ^° C. 7.9 g of lithium salt of N-acridonoacetic acid are obtained.

 Next, we present the test results of the properties of the claimed drug (virolite). As a comparison drug is the prototype of the invention is the sodium salt of N-acridonoacetic acid (neovir).

 Example 3. The antiviral effect of virolite against herpes simplex virus type 1 serotype (HSV-1, strain L2) in cell culture (Vero).

 The smallest dilution of the drug, causing cell degeneration, was taken as the minimum toxic dose (MTD). In the experiment, a concentration gradient was used, starting with 1/2 MTD.

 Test the prophylactic effect of the drug.

The growth medium was removed from the tubes with a monolayer of cells and 0.8 ml of the support medium containing 1/2 MTD of the preparation were introduced into the test tubes, and 0.8 ml of the support medium in the control tubes. After 2 hours of contact at 37 ^° C, 0.2 ml of HSV-1 were introduced into experimental and control tubes at doses of 10, 100, 1000 ITD (infectious tissue dose). For each dilution of the virus (in the experiment and control) used 9 tubes. The tubes were shaken and placed in a thermostat at 37 ^o C.

Additional controls:
a) culture control - 1.0 ml of the supporting medium were added to 4 tubes
b) drug control - 0.8 ml of the working dilution of the drug were added to 4 tubes and after 2 hours of contact at 37 ^° C, 0.2 ml of the supporting medium was added.

 Testing the therapeutic effect of the drug.

The growth medium was removed from the tubes with a monolayer of cells and 0.2 ml of HSV-1 containing 10, 100, 1000 ITD viruses were added. The contact of the virus with the cells was 1.5 hours at 37 ^° C. After this, the virus was removed from the tubes, the infected cultures were thoroughly washed with Hanks solution, then 1.0 ml of the maintenance medium containing 1/2 MTD of the preparation was added to the control series - 1.0 ml of the supporting medium. For each dilution of the virus, 9 tubes were used. The results were evaluated by suppressing the CPD (cytopathic effect) of the virus (daily microscopy for 3 days) and by suppressing the production of the virus in cell culture in the presence of a working dose of the drug. For this, on 1, 2 and 3 days after infection from control and experimental cultures infected with different doses of the virus, 3 tubes of experimental and control cultures were taken.

 Virus-containing fluid from every 3 tubes was combined and titrated with virus on a Vero cell culture with a dilution factor of 10 according to the generally accepted scheme. The results were taken into account according to the JRC on the 5th day of incubation.

 Results. Daily microscopy of tissue culture for 3 days showed that the toxicity of virolite at a concentration of 20 and 40 mm was manifested in wrinkling of cells and complete destruction of the cell layer on day 1-2 of observation. In the main experiment, 1/2 MTD was used, i.e. working dilution of virolite was 10 mm. Comparison drug - the prototype was used in the same initial concentration of 10 mm.

 Antiviral activity of virolite and drug comparison.

 a) Prophylactic effect of drugs. The prophylactic efficacy of drugs, determined by the difference in infectious activity in experimental and control cultures, under conditions of different multiplicity of infection on days 1, 2 and 3, is presented in Table. 1 and 2. The effective inhibitory effect of virolite (a decrease in the titer of the virus from 2.5 to 6.0 lg) was observed at all periods of observation and after infection with various doses of the virus (from 10 to 1000 ITD). Accordingly, a significant suppression of CPD in experimental cultures was observed compared with the control. The effectiveness of the comparison drug was significantly lower and ranged from 1.5 to 2.5 lg.

 b) The therapeutic effect of drugs. Similar results of the inhibitory effect of virolite when administered at a working dose after infection of the tissue culture of HSV-1 are presented in Tables 3 and 4. With a multiplicity of infections of 1000, 100 and 10 ITD, a pronounced inhibitory effect of virolite was observed at all observation periods: inhibition of virus reproduction was observed from 2.0-2.5 lg on the 1st day to 4.0-4.5 lg on 2-3 days. The inhibitory effect of the prototype drug was significantly lower and ranged from 1.5 to 3.0 lg depending on the duration of observation and the multiplicity of infection.

 A rather high degree of inhibition of virus reproduction was also shown when using lower concentrations of virolite (5 mM and 2.5 mM): suppression of HSV-1 reproduction with a prophylactic effect on cell culture was 4.3 and 3.5 lg, respectively, with treatment - 3 , 0 and 2.7 lg (Table 5).

Thus, the presented results indicate the effectiveness of the antiviral effect of virolite against HSV-1 and, in particular, its prophylactic and therapeutic activity. A clear dose-dependent effect of the drug was also established. A high degree of inhibition of viral reproduction allows attributing it to highly active antiherpetic drugs. The preliminary data obtained on the basis of screening of acute toxicity and specific antiviral activity, as well as the established therapeutic index (LD ₅₀ / ED ₅₀ ), which is more than 10, allow us to consider the proposed drug as potentially promising for use in medicine.

 Example 4. Virus-inhibiting activity of virolite in a culture of the surviving chorionallantoic membrane on the model of influenza virus type A (H3N2) and B.

 Materials and methods. Virus: an allantoic culture of influenza type A viruses (A / Mississipp 1/85 / H3N2) and B (B / Yamanashi / 166/98). Preparations: virolite in the form of 10, 5 and 2.5 mM solutions and the comparison drug is a prototype in the same concentrations. The culture of the surviving chorionallantoic membrane (HAO) was prepared according to the standard method; the contact of the preparations with the culture of HAO before the virus was 2 hours.

Results. When a 10 mM virolite solution was applied to the KLA cell culture in all experiments, a decrease in the EID ₅₀ (50% embryonic infectious dose) of type A influenza virus by 2.0–4.5 lg was recorded. When using a 5.0 mM solution, the decrease in EID ₅₀ ranged from 1.5-3.5 lg, and with a 2.5 mM solution, the infectious activity of the virus decreased by 1.0-1.25 lg. On average, the decrease was 3.4-2.1-1.1 lg, respectively (Table 6).

 Comparison drug - prototype (neovir) showed a slightly less pronounced virus-inhibiting activity. The revealed wide range of fluctuations in virus-inhibiting activity within the same concentration could be due to the nonspecific erythrocyte agglutination detected in some experiments in the control of drugs (drugs in the CAO culture without the virus), which led to an underestimation of their activity indicators.

 When influenza B virus culture was used to infect cells of the KAO culture of influenza B virus (B / Yamanashi / 166/98), a decrease in the infectious activity during the application of the mentioned preparations was recorded at all concentrations used. The pronounced virus-inhibiting effect of virolite and neovir against type B influenza virus was detected even at a solution concentration of 2.5 mM (Table 7).

 Thus, in experiments on the KLA culture, a pronounced virus-inhibiting effect of virolite at a concentration of 10.0-5.0 mM against influenza A (H3N2) and B viruses was revealed.

 Example 5. Comparative evaluation of the antiviral activity of virolite and prototype in experimental models of actual viral infection.

 Materials and methods. The study was carried out on the basis of the Research Institute of Military Medicine of the Ministry of Defense of the Russian Federation.

 Animals: white inbred male mice weighing 16-22 g, were delivered from the Rappolovo nursery RAMS (Leningrad region).

 Viruses: causative agent of Rift Valley fever - strain 8-87 from the museum of the Research Institute of Military Medicine; inoculum - a suspension of the brain of newborn mice previously infected with the virus; the causative agent of Venezuelan horse encephalomyelitis (VEL) - strain "Trinidad" from the Museum of the Research Institute of Military Medicine; the virus for infection was accumulated in the yolk sacs of developing chicken embryos; the causative agent of herpes simplex type 1 (HSV-1) is strain L-1 from the museum of the research institute of military medicine; for infection of animals, newborn mice were accumulated in the brain.

 Drugs: comparison drug - prototype, substance; new drug - virolite, a substance. Drugs were administered subcutaneously, and virolite additionally through the mouth.

 Evaluation of the effectiveness of drugs: the level of survival, life expectancy (days) of animals of the experimental and control groups.

The results of the study. In preliminary experiments, the express method according to V. B. Prozorovsky et al. On white mice determined the acute toxicity of a new drug virolite with intraperitoneal administration. The administered dose was contained in 0.5 ml. The drug was diluted in saline with a small addition of DMSO. It was found that with this route of administration, LD ₅₀ for white mice is 713 ± 86 mg / kg.

Venezuelan horse encephalomyelitis
Experiment 1: subcutaneous administration of virolite. We compared the activity of virolite and the reference drug, the prototype, when administered subcutaneously twice at a dose of 100 mg / kg according to the scheme: 4 hours before infection and 48 hours after infection with the virus. The results are presented in table 8, from which it follows that virolite after infection of white mice with lethal doses of the VEL virus has a high protective effect.

 Experience 2: the introduction of virolite per os. Scheme of administration - 2-fold administration of per os in a single dose of 200 mg / kg 4 hours before infection and 48 hours after infection with the VEL virus. The results are presented in table.9, from which it follows that the appointment of virolite had a positive protective effect and contributed to an increase in resistance of 20% of animals to the pathogen VEL. The antiviral activity of virolite and prototype when administered subcutaneously at a dose of 100 mg / kg on a model of VEL in white mice is shown in table 10.

Rift Valley Fever (LDR)
Experience 1. Comparative evaluation of the antiviral effect of virolite and drug comparison - prototype. The drug was administered according to two schemes. 1st scheme: a single sc administration 4 hours before infection and 48 hours after infection. As follows from table 10, virolite has protective efficacy against the causative agent of LDR, increasing the survival of experimental animals and increasing their life expectancy. The dose-dependent effect was revealed.

 The following experiment showed that virolite had a protective effect both with subcutaneous and oral administration (Table 11).

Herpes
4 hours before infection, test preparations were administered to the animals of the experimental groups (subcutaneously or by mouth), 2 hours before infection, the suspension of hydrocortisone (0.1 ml or 125 mg / kg intramuscularly) was administered to all animals of the experimental or control groups. Infection was performed intraperitoneally, observation was carried out for 14 days. From the data obtained (Table 12), it can be concluded that with generalized herpetic infection in white mice, virolite has a protective effect. The less pronounced protective antiherpetic effect in comparison with the efficiency with respect to RNA-containing viruses is apparently explained by the inadequacy of the deadly infection model itself, since it was initiated as a result of exposure to the immunosuppressant. Undoubtedly, these data should be considered as preliminary.

 Example 6. The study of the effect of virolite on the immune system - on cellular and humoral immunity.

 To assess the effect of virolite on cellular and humoral immunity, you can use, for example, a delayed-type hypersensitivity reaction (HRT) - to assess the effect on cellular immunity and the titer of circulating (serum) antibodies - to assess the effect on humoral immunity. For these purposes, standard methods recommended for such studies are used by the Pharmacological Committee of the Ministry of Health of the Russian Federation.

 The immunomodulatory activity of the lithium salt of 10-acridine acetic acid is confirmed by the following example.

Non-inbred white mice weighing 21-23 g sourced from the Rappolovo nursery RAMS were immunized with fresh sheep red blood cells. The red blood cells were washed from the preservative with a 0.9% sodium chloride solution, and a suspension of washed red blood cells in a 0.9% sodium chloride solution in a volume of 0.5 ml was used to immunize animals. Red blood cells were administered intraperitoneally to white mice at a dose of 5 • 10 ⁵ cells. A solution of virolite at a dose of 100 mg / kg was administered once subcutaneously during immunization.

,
где ИР - индекс реакции, h_о - вес опытных образцов, h_k - вес контрольных образцов.Production of HRT. The test was set 14 days after immunization of animals. For this purpose, animals of the experimental and control groups under the corn tubercle of the 4th finger of the right hind limb were injected with 0.05 ml of a suspension of red blood cells in a dose of 5 • 10 ⁸ cells, and the same volume of a 0.9% sodium chloride solution was introduced into the control limb. The reaction was taken into account after 24 hours, measuring the mass of the feet of the hind limbs, for which the animals had their legs cut off at the level of the ankle joint after slaughter and weighed on an analytical balance. The reaction index was calculated by the formula

,
where IR is the reaction index, h _o is the weight of the test samples, h _k is the weight of the control samples.

The mice that received the injection of red blood cells without the virolite preparation served as controls — K ₁ , K ₂ -intact mice that were not immunized, which were given a resolving dose of red blood cells on the day of HRT placement.

Determination of the level of antibodies. Antibodies to sheep erythrocytes (EB) were determined by titration of blood sera by the conventional method. Serum was diluted in a volume of 0.5 ml in plastic tablets, red blood cells were used at a concentration of 1%. Accounting for the reaction - after 16-18 hours of incubation (1 hour in the thermostat at 37 ^o C, and then at room temperature) according to the 4-cross system, the reaction to 2+ was considered positive.

 Statement of the reaction. Experimental animals were divided into groups of 8-10 mice.

 The results obtained are presented in table.13.

 As follows from these data, virolite increased the response of HRT of white mice to sheep erythrocytes: in the first experiment - by about 35%, in the second - by 17% compared with the control.

 The effect of virolite on the humoral immunity was evaluated by the percentage of seroconversions and titer of antibodies to sheep erythrocytes. Antibodies were determined 14 days after immunization of animals. The data obtained are given in table. 14.

 Thus, virolite has immunomodulating properties, affecting the cellular and humoral parts of the immune system. When administered at a dose of 100 mg / kg with ramogen erythrocytes, erythrocytes at a dose of 100 mg / kg stimulated the cellular immune response of white mice, increasing the HRT reaction indices by 17-35%, while at the same time exerting a depressing effect on humoral immunity. A more accurate assessment of the immunomodulatory properties of virolite can be obtained using linear oppositely reacting mice.

 The developed new original drug virolite has a wide spectrum of antiviral activity and is highly effective against both RNA and DNA viruses, which is shown on specific models of virus reproduction on various biological systems. Along with inhibiting the reproduction of viruses, virolite has the ability to activate specific and nonspecific factors that are functionally aimed at the lysis of infected or transformed cells and the elimination of the pathogen. The activity of the developed drug is equally important both with the parenteral and the enteral route of administration, which opens up wide possibilities for creating various dosage forms.

Claims (1)

The lithium salt of N-acridonoacetic acid of the structural formula

exhibiting antiviral, immunomodulatory property.

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