RU2847587C2 - Use of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide as antimicrobial agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is using 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-iodide in the patients for reducing infectious activity of meningitis viral agents.

EFFECT: invention provides the biological activity of the declared compound with respect to viral meningitis agents.

1 cl, 12 dwg, 9 tbl, 6 ex

Description

The prior art discloses a substance known as enisamium iodide, which is a derivative of isonicotinic acid.

Enisamium iodide is an endogenous interferon inducer. It binds the hemagglutinins of the influenza A virus, rendering the virions unable to bind to target cells for replication.

Enisamium iodide stabilizes lysosomal and cellular membranes, inhibits basophil degranulation, normalizes prostaglandin levels, has an antioxidant effect, and attenuates vascular reactions in inflammatory sites. Enisamium iodide is a component of several medications registered in Russia and internationally with the INN "enisamium iodide."

In addition, the specified substance is included in the recommendations for doctors as a drug with a direct antiviral effect (see “Drug therapy for acute respiratory viral infections (ARVI) in outpatient practice during the COVID-19 epidemic”, version 2 (16.04.2020), hereinafter - [1].

Information on a method for producing enisamium iodide is known from the prior art.

The specified method includes several stages; the raw material for obtaining enisamium iodide is isonicotinic acid (see RU 2689775, 05/29/2019, hereinafter referred to as [2]).

At the same time, the closest analogues to the proposed group of inventions [1] and [2] do not disclose information about the new substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide, its activity in relation to human diseases of various etiologies.

Furthermore, the method according to the patent document [2] is aimed at obtaining enisamium iodide, and not the new substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

Therefore, the technical task of the claimed group of inventions is to study the biological activity of the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

The technical result of the claimed group of inventions is:

- Biological activity of the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide against viral and non-viral infections in patients;

- Biological activity of the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in relation to neuroinfections in patients;

- Penetration of the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide through the patient’s blood-brain barrier;

- Achieving high tissue availability of the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in patients;

- Entry of the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide into the patient’s systemic bloodstream;

- The ability of the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide to be distributed into the organs and tissues of patients.

The use of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in an effective amount as an antimicrobial agent against S. pneumoniae bacteria, influenza viruses, parainfluenza, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, ECHO and Coxsackie enteroviruses is proposed.

The use of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in an effective amount for the treatment of neuroinfections in patients is proposed. The essence of the invention is illustrated by graphic materials.

Table 1. A number of metabolites of enisamium iodide.

Table 2. Prediction of permeability of enisamium iodide metabolites through the blood-brain barrier.

Table 3. Results of experiments on obtaining 4-(benzylcarbamoyl)-3-hydroxy-1-methyl pyridin-1-ium iodide.

Table 4. Content of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain of rats after a single intragastric administration of the test object at a dose of 100 mg/kg.

Table 5. Main indicators of penetration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide into the brain of rats after a single intragastric administration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg.

Table 6. Content of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain of rats after repeated intragastric administration of the test 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg, μg/ml.

Table 7. Main parameters of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide penetration into the rat brain after repeated intragastric administration of the tested 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg.

Table 8. The effect of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide on the reproduction of S. pneumoniae serotype 19A, influenza viruses, parainfluenza viruses, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, enteroviruses ECHO and Coxsackie.

Table 9. The effect of enisamium iodide on the infectious activity of S. pneumoniae serotype 19A, influenza viruses, parainfluenza viruses, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, enteroviruses ECHO and Coxsackie.

Figure 1. Concentration-time curve of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the rat brain after a single intragastric administration of the test object 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg in linear coordinates

Figure 2. Concentration-time curve of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the rat brain after a single intragastric administration of the test 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg in semilogarithmic coordinates

Figure 3. Concentration-time curve of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the rat brain after single and multiple intragastric administration of the test 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg in linear coordinates

Figure 4. Concentration-time curve of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain of rats after single and multiple intragastric administration of the test 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg in semilogarithmic coordinates

Figure 5. Tissue availability (ft) of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in tissues and organs of rats after single and repeated intragastric administration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg.

Figure 6. Chromatograms of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide and sodium iodide samples.

Figure 7. ¹ H NMR spectrum of ethyl 3-hydroxyisonicotinate. Figure 8. Mass spectrum of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide. Figure 9. ¹ H NMR spectrum of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

Figure 10. Chromatogram of the compound 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

Figure 11. Effect of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide on the reproduction of S. pneumoniae virus serotype 19A.

Figure 12. Reduction of infectious activity of S. pneumoniae serotype 19A bacteria, viral meningitis (influenza viruses, parainfluenza viruses, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, ECHO and Coxsackie enteroviruses) in the presence of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

The essence of the declared group of solutions

The PASS (Prediction of Activity Spectra for Substances) computer program was used to predict the biological activity profiles of enisamium iodide, its analogues, metabolites and derivatives.

The development of the PASS computer program, which predicts the biological activity spectra of organic molecules, began more than 30 years ago as part of the State System for Registration of New Chemical Compounds Synthesized in the USSR.

This program, based on the structural formula of a drug-like organic compound, predicts its biological activity profile.

The biological activity profile includes several thousand types of biological activities, based on the analysis of structure-activity relationships, containing over 1 million biologically active organic compounds.

In the PASS program, the biological activity spectrum of an organic compound refers to the many different types of biological activity that reflect the results of its interaction with various biological objects. The biological activity spectrum reflects the "intrinsic" properties inherent to a given compound, which depend solely on the structure of its molecules.

This is a qualitative characteristic property of a substance, dependent solely on its molecular structure, which enables the integration of large data sets from various information sources into a single training set. Computer-aided evaluation of the biological activity profiles of enisamium iodide, its analogs, derivatives, and metabolites using the PASS program was performed for the first time. It should be noted that the structural formula of enisamium iodide includes a charged tetravalent nitrogen with substituents other than hydrogen. The charge of the enisamium molecule cannot be neutralized, and no basic form corresponds to its salts.

Consequently, enisamium iodide and its analogs do not meet generally accepted criteria for preparing training sets for constructing structure-property relationships. Therefore, to obtain results for enisamium iodide in the biological activity spectra prediction program, the program's algorithm was modified to handle compounds whose main structural component has a net charge different from zero (for cases containing tetravalent nitrogen). As a result of this study, a number of enisamium iodide metabolites were identified. The list of metabolites is presented in Table 1.

In addition, the applicant predicted the permeability of enisamium iodide and its metabolites through the blood-brain barrier using the GUSAR program.

This work revealed that all of them possess the ability to cross the blood-brain barrier. The results are presented in Table 2.

According to the data presented in Table 1, among the metabolites of enisamium iodide, the most promising in terms of biological activity are Metabolite 5 (4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridinium iodide), Metabolite 6 (N-(cyclohexa-1,3-dien-1-ylmethyl)isonicotinamide) and metabolite 9 (N-benzyl-2-oxo-1,2-dihydropyridine-4-carboxamide).

These compounds are calculated to have activity equal to or greater than that of enisamium iodide itself.

Metabolite 5 is more than 50 times more active than enisamium iodide in terms of predicted activity, and metabolites 6 and 9 are more than 40 times more active.

Moreover, from the point of view of searching for approaches to practical production, Metabolite 5, which has accessible chemical blocks in its composition, is significantly superior to Metabolites 6 and 9, the synthesis of which is impossible to carry out from simple accessible precursors.

Thus, Metabolite 5 (4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridinium iodide) was selected as the most promising for synthesis, study of biological activity in vitro, in vivo and further study as a potential drug.

The structural formula of the metabolite 5-4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide is shown below.

The gross formula of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide is C ₁₄ H ₁₅ IN ₂ O ₂ .

The substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide has not been previously synthesized.

From the closest analogue [2] to the proposed method for obtaining 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide, it is known that isonicotinic acid is used as a raw material for its production.

The following synthesis scheme was used as a basis for obtaining the compound 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide:

It is known from the prior art that the acylation of benzylamine 2 can be carried out using either the carboxylic acid itself or its derivatives, esters, acid halides, etc.

Moreover, the acylating ability of different derivatives differs, and the higher it is, the better the formation of the target amide.

Thus, it is known that esters have a greater acylating capacity compared to carboxylic acids, and acid chlorides have an even greater capacity.

The use of various types of cross-linking reagents is based on the same principle; a compound with a higher acylating capacity is formed.

The applicant conducted a series of experiments to obtain amide 3 using various acylating agents. However, not all of them were successful.

The experimental results are shown in Table 3.

First, conditions similar to those used for the preparation of enisamium iodide in the patent document [2] were reproduced (see in [2], Table 1, experiment 1).

However, even after additional heating of the reaction mass, the distillation of water and the formation of the product did not occur.

To facilitate the process of separating water from the rather viscous reaction mixture, solvents were added.

The purpose of adding solvents was to ensure the possibility of azeotropic distillation of water (see Table 3, experiments 2 and 3).

However, even in this case, the final product was not obtained.

Having failed to achieve a result, it was decided to use various cross-linking agents to obtain the amide (see Table 3, experiments 4-7).

The main purpose of using crosslinking agents was to utilize their ability to form intermediates with carboxylic acid that have greater acylating ability.

The following types of cross-linking reagents were used:

- Carbodiimides;

- Carbonyldiimidazole;

- Uronium salt TBTU, which is an analogue of HBTU and HATU.

For each type of crosslinking agent, appropriate temperature and time conditions were selected.

However, the use of cross-linking reagents in the synthesis also did not lead to a positive result - obtaining the final product.

This was likely due to the presence of a hydroxyl group in the starting acid. The resulting intermediates with cross-linking agents (at the carboxyl group) are reactive enough to undergo intra- and intermolecular reactions at the hydroxyl group instead of forming the target amide.

Moreover, according to 1H NMR spectroscopy and TLC data, a substance similar to the target amide 3 was observed in small quantities in the reaction mixtures.

However, the resulting mixtures were too complex and the amide content too small to isolate it in pure form.

Subsequently, it was decided to abandon the use of acid 1 as the starting reagent.

The acid chloride of the parent acid was tested as an acylating agent.

As a result, the desired amide 3 was obtained (see Table 3, experiment 8).

However, its yield was only 22%, which is a very low figure.

Then, the ethyl ester of acid 1 was used as the starting material.

As a result of the synthesis, ethyl ester was obtained with a yield of 87%.

Structural formula of ethyl 3-hydroxyisonicotinate. In experiment 9, the ethyl ester was heated to 90°C for 3 hours.

However, the yield of the resulting amide product was low, 8% (see Table 3, experiment 9). A solvent was then added to the process to reduce the excessive viscosity of the reaction mixture (see Table 3, experiment 10). However, the addition of the solvent did not increase the yield.

Then it was decided to increase the reaction time and temperature to 120°C and 6 hours.

This solution made it possible to obtain the target amide with a yield of 55%, which can be called satisfactory (see Table 3, experiment 11).

After obtaining the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide. The following spectral instruments were used to identify the substance. NMR spectra were recorded on a Varian MercuryPlus 400 spectrometer (operating frequency 400.45 MHz for the 1H nucleus and 100.70 MHz for the 13C nucleus) at a temperature of 323 K. The signal of residual solvent protons (7.26 ppm for CDC13 and 2.48 ppm for d6-DMSO) was used as an internal standard.

Chemical shifts are given in ppm on the δ scale relative to TMS.

Mass spectral analysis was carried out on the following instruments:

1) Surveyor MSQ (Thermo Fisher Scientific). Mass spectral sensor: ionization method - APCI, registration of positive and negative ions. Injection volume is 2 µl. Analysis time is 4.5 min.

2) Shimadzu, consisting of two LC-30AD pumps, a SIL-30AC autosampler, a CTO10-AS column thermostat, an SPD-M20A diode array detector, an SVM-20A communication module, and an LCMS-8040 mass spectrometric detector. Mass spectral sensor: ionization method - ESI, registration of positive and negative ions.

The injected sample volume is 20 µl. Analysis time is 60 min.

Determination of the purity of the obtained compound.

The purity of the obtained compound according to NMR spectroscopy and mass spectrometry data was higher than 95%.

However, we encountered the formation of an additional peak when using the HPLC-MS method.

This peak was observed using the acetonitrile-water solvent combination, but it "disappeared" when replacing the water with a 0.1% formic acid solution. After analyzing the literature, we concluded that this peak is formed due to the presence of the iodide anion in the molecule.

We tested this hypothesis by comparing the chromatograms of the product we obtained and sodium iodide (see Figure 6).

Our assumption was also confirmed by the fact that in negative ions the main peak was the iodine anion with a mass of m/z=127.

Thus, the development of a fine synthesis of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide was completed, and the compound was obtained. Let us consider in detail the steps for obtaining 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

1. Obtaining N'-benzyl-3-hydroxyisonicotinamide

For the acylation of benzylamine, both carboxylic acid and its derivatives—esters, acid halides, etc.—are used.

However, the acylating ability of the above derivatives will be different.

Moreover, the higher the acylating capacity of the substance, the better the formation of the target amide.

It is well known that esters and acid chlorides have greater acylating capacity than carboxylic acids.

In the process of developing the synthesis of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide, a series of experiments were carried out using various acylating and cross-linking agents.

Thus, several types of cross-linking agents were used: Carbodiimides (DCC, EDC), Carbonyldiimidazole (CDI); Uronium salt (TBTU), which is an analogue of HBTU and HATU.

Ethyl ester of the acid was used as the starting material.

As a result, the ethyl ester of 3-hydroxyisonicotinic acid was obtained in the synthesis process with a yield of 87%.

With the following structural formula:

Ethyl ester of 3-hydroxyisonicotinic acid

The experimental results are shown in Table 3.

Obtaining ethyl ester of 3-hydroxyisonicotinic acid

Scheme for obtaining ethyl ester of 3-hydroxyisonicotinic acid In a 500 ml two-necked round-bottomed flask equipped with a magnetic stirrer, heating bath, thermometer and reflux condenser with a calcium chloride tube, 6.00 g (43.1 mmol) of 3-hydroxyisonicotinic acid 1, 240 ml of ethanol and 6 ml of concentrated sulfuric acid are placed.

The resulting mixture is heated to boiling (Tbath = 90°C, TPM = 78°C) and until the acid is completely dissolved. The mixture is refluxed with stirring for 48 hours.

After completion of the RM reaction, the mixture is cooled to room temperature, the solvent is evaporated to dryness on the RPI.

The resulting residue is dissolved in 60 ml of water.

NaHCO ₃ was added to the resulting solution in portions while stirring until gas evolution ceased (the soda requirement was 10.75 g). The target ester was extracted with DCM (3×60 ml).

The combined organic extracts were dried over Na ₂ SO ₄ and filtered. The solvent was evaporated to dryness using RPI.

After evaporation, 6.29 g (37.6 mmol, 87%) of the product is obtained in the form of a mobile oil, which upon crystallization forms beige-brown crystals.

Figure 7 shows the H NMR spectrum of ethyl 3-hydroxyisonicotinic acid.

Obtaining N'-benzyl-3-hydroxyisonicotinamide.

Scheme for obtaining N'-benzyl-3-hydroxyisonicotinamide

In a 100 ml round-bottomed flask equipped with a heating bath, magnetic stirrer and reflux condenser with a calcium chloride tube, 4.19 g (25.1 mmol) of ethyl 3-hydroxyisonicotinic acid ester 1a and 16.12 g (6 equiv., 150.4 mmol, 16.43 ml) of benzylamine 2 are placed.

After which the ethyl ether swells and becomes lighter.

Next, the resulting mixture is heated with stirring to Tbath = 130°C, resulting in its RM homogenization.

The mixture is then heated for 6 hours.

After completion of the RM reaction, it is evaporated.

The synthesis results in a viscous residue, which, upon cooling, hardens into a solid glassy substance.

The product is separated using column chromatography (Vsilik.=200 ml, 1=14.7 cm, d=4.0 cm), eluent: DCM - methanol 4:1.

As a result, 3.15 g (13.7 mmol, 55%) of N'-beuyl-3-hydroxyisonicosinamide3 are obtained. Figure 8 shows the mass spectrum of N'-beuyl-3-hydroxyisonicosinamide 3 in positive ions of methyl-(III-indol-3-yl) pyrimidin-2-yl]benzene-1,2,4-triamine.

2. Obtaining a compound with the structural formula

As a result of the alkylation reaction, the target compound is obtained with a yield of 55%.

The purity of the obtained compound according to NMR spectroscopy and mass spectrometry is above 95%.

However, when using the HPLC-MS method, the formation of an additional peak was detected in the chromatogram (see Figure 6).

This peak was observed only when using the acetonitrile-water solvent combination.

When replacing water with a 0.1% solution of formic acid, the peak was absent.

The formation of the additional peak is due to the presence of the iodide anion in the molecule. When comparing the chromatograms of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide and sodium iodide, it is clear that the main peak in the negative ions was the iodine anion with a mass of m/z = 127.

Obtaining a compound with the structural formula 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide

Scheme for the preparation of a compound with the structural formula 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide: 2.40 g (10.5 mmol) of N'-benzyl-3-hydroxyisonicotinamide 3 and 14.2 ml of acetone are placed in a two-necked round-bottomed flask equipped with a heating bath, magnetic stirrer, thermometer, and reflux condenser with a calcium chloride tube. The resulting mixture is heated to TPM = 50°C and stirred until the amide is completely dissolved.

After complete dissolution, 2.24 g (15.8 mmol, 982 µl) of methyl iodide 4 are added to the RM.

Stir the mixture for 5 hours. Observe the formation of a precipitate.

After completion of the reaction, cool the reaction mixture to 10°C while stirring.

The resulting precipitate is filtered and washed with cold acetone (5 ml).

Dry the sediment in a stream of air for 1 hour, and then on a RPI at 30°C for another 3 hours.

The target product is obtained in the form of a beige-yellowish powder, in an amount of 3.28 g (8.85 mmol, 84%) with a purity of 97.9% according to HPLC.

Figure 9 shows the H NMR spectrum of the compound 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

Figure 10 shows the chromatogram of the compound 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

The characteristics of the semi-finished products and the target product 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide were determined using physicochemical analysis methods: ¹ H NMR, HPLC-MS, etc.

3. Study of the activity of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in vivo, in vitro

Example 1. In vivo studies of the biological activity of the compound 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide

To perform the preclinical in vivo study, sexually mature male Wistar rats were used.

These animals are the standard animal species used for pharmacokinetic studies where tissue and organ distribution assessment is required.

The rats submitted for the study were divided into two groups of 50 animals. Each group included ten subgroups (based on the number of time points), with five animals in each subgroup.

Rats without signs of abnormalities in appearance were selected for the experimental groups, so that the individual weight value did not deviate from the average value for all animals by more than 20%.

The distribution of rats into subgroups was performed using the modified block randomization method.

For this purpose, all animals transferred to the study were randomly placed into cells of the randomization block (the number of cells of the randomization block is a multiple of the number of subgroups in the experiment).

Next, using a random number generator, we obtained a list of data containing the numbers of cells with rats and the corresponding numbers of subgroups where the animals were subsequently placed.

Each animal included in the study was assigned an individual number. Before the first (Group 1) and last (Group 2) administration of the test substance, the rats were deprived of food for 16 hours.

The animals were given purified water standardized for organoleptic properties, pH, dry matter, reducing substances, carbon dioxide, nitrates and nitrites, ammonia, chlorides, sulfates, calcium, and heavy metals. The test substance was administered to the experimental animals intragastrically, once and multiple times (5 days), as the original preparation is intended for oral use in humans.

To study the pharmacokinetics of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide, a dose of 16.7 mg/kg was used for humans (similar to the unchanged active ingredient enisamium iodide).

According to the instructions for medical use, the drug Nobazit®, film-coated tablets, 250 mg, (owner of the RU JSC Avexima, Russia) is taken orally, after meals, 0.5 g 3 times a day.

The maximum single dose is 1 g, daily dose is 2 g. The recommended course of treatment is 5 to 7 days.

Therefore, the maximum single dose (MSD) of enisamium iodide will be: 1000 mg/60 kg (average human body weight) = 16.7 mg/kg.

When converting doses taking into account interspecies coefficients, the equivalent dose for rats will be: 16.7 × 6.2 (conversion factor) = 103.54 mg/kg ≈ 100 mg/kg. Thus, in the pharmacokinetic study, 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide was administered to rats intragastrically at a dose of 100 mg/kg (maximum single dose, MSD).

In this pharmacokinetic study, the test 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide was administered to animals intragastrically at a dose of 100 mg/kg (1 MDI) once and repeatedly (for 5 days, 1 time per day) as a solution in an inert carrier.

The selected dose made it possible to evaluate the main pharmacokinetic parameters (Cmax, Tmax, AUC, MRT, T1/2) of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide after single and multiple intragastric administration and the distribution of the test object in the internal organs.

Based on the results of the pharmacokinetic study of enisamium iodide, the following sampling time points were selected for the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide: 0 (before administration of the test object), 0.5, 1, 2, 3, 4, 6, 8, 10 and 24 hours after administration.

In case of multiple administration, blood and organ samples were taken immediately before the last administration (point 0) and 0.5, 1, 2, 3, 4, 6, 8, 10 and 24 hours after the last administration of the test object.

When selecting organs and tissues for assessing distribution into internal organs, tissues differing in the degree of vascularization (highly, moderately, and poorly vascularized), target organs, and organs responsible for elimination were selected.

To conduct the study of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide, the following organs and tissues were selected: liver, kidneys (highly vascularized organs responsible for elimination); lungs, heart, and brain. The test object - the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide - was administered to rats as a solution in an inert carrier (purified water), selected based on the results of test sample preparation, intragastrically using a probe. The substance was administered single (group 1) and multiple times (group 2).

The drug was administered sequentially to animals of different subgroups with the shortest possible interval between administrations to individual animals within a subgroup. Administration to rats of a single subgroup took no more than 1-2 minutes. The results of studies confirming the activity of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide after single and multiple administrations to rats are shown in Figures 1-5.

Example 2. Study of the pharmacokinetics of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide after a single intragastric administration to rats

Data on the change in the concentration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain of rats after a single administration of the test object are presented in Table 4, the average pharmacokinetic curves in linear and semi-logarithmic coordinates are shown in Figures 1 and 2.

In this case, chromatography of each obtained biological sample was performed in two replicates.

Then, based on the obtained data, the analyte content in each sample was calculated.

To calculate the pharmacokinetic parameters, the average values of the analyte content presented in Table 4 were used.

The analyzed substance in the studied brain samples was determined within 6-8 hours.

The maximum concentration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide was observed 0.5-2 hours after administration of the test object.

Next, a gradual decrease in the concentration of the test substance in the brain tissue of rats was observed (Figures 1 and 2).

The average content of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain 6 hours after its administration to the test subject was approximately 4.2% of its maximum concentration.

Eight hours after administration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide, the analyte was detected in brain tissue in only one animal. Then, 10 hours and beyond after administration of the test substance, the concentration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in all samples was less than the LLOQ of the bioanalytical method used.

The main parameters characterizing the degree of penetration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide into the brain are given in Table 5.

The tissue availability of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide for the brain was 0.12 (see Figure 5).

Thus, after a single administration of the test object, intensive intake and prolonged presence of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the tissues of the liver, kidneys, heart, and lungs, as well as penetration through the blood-brain barrier, were noted.

Example 3. Study of the pharmacokinetics of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide after repeated intragastric administration to rats

Data on the change in the concentration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain of rats after repeated administration of the test object are presented in Table 6, the average pharmacokinetic curves in linear and semi-logarithmic coordinates are shown in Figures 3 and 4.

The analyzed substance in the studied brain samples was determined within 4-6 hours.

The maximum concentration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide was observed 0.5-2 hours after administration of the test object. A gradual decrease in the concentration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the rat brain was then observed (Figures 3 and 4).

The average content of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain 6 hours after its administration was about 3% of its maximum concentration.

Eight hours after the introduction of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide, the analyte was detected in brain tissues only in one animal; after 10 hours and further after the introduction of the test object, the concentration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in all samples was less than the LLOQ of the bioanalytical method used.

The main parameters characterizing the degree of penetration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide into the brain are given in Table 7.

The tissue availability of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide (ft) for the brain was 0.11.

The obtained data allowed us to assume the absence of accumulation of the active substance in the brain with repeated intragastric administration of the test object to rats.

Summary data on the tissue availability of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in rat tissues and organs after single and repeated intragastric administration of the test object are presented in Figure 5. Intensive uptake and prolonged presence of the test substance in the tissues of the liver, kidneys, heart and lungs, as well as its penetration through the blood-brain barrier, were noted.

For liver, kidney and heart tissues, the tissue availability of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide after repeated administration was higher than after a single administration, which confirms the assumption of more intensive penetration of the test substance into organs and tissues after its repeated administration.

Example 4. Study of the antimicrobial activity of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in in vitro experiments

It is well known that pneumococcal meningitis is associated with the highest mortality rate among bacterial meningitis. Furthermore, pneumococcal meningitis often leads to neurological complications, despite the use of antibiotic therapy. Currently, the increasing resistance of Streptococcus pneumoniae to antibiotics is a widespread problem.

The main reasons for the ever-increasing resistance of pneumococci are the unjustified use of powerful antibiotics in outpatient practice, the prescription of drugs without taking into account the spectrum of antimicrobial activity and the biological properties of the pathogen, errors in the choice of dosage and duration of treatment, and the use of broad-spectrum antibiotics to prevent complications in viral infections.

Therefore, at present, most strains of Streptococcus pneumoniae are resistant to antibacterial drugs such as macrolides, tetracycline, chloramphenicol, and sulfonamides, but remain highly sensitive to third-generation cephalosporins.

Moreover, according to recommendations for the diagnosis and treatment of bacterial purulent meningitis in children, the drugs of choice for pneumococcal meningitis are third-generation cephalosporins.

The aim of the study was to clarify the antimicrobial activity of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide against the human strain of Streptococcus pneumoniae.

Cefotaxime/ceftriaxone was used as the reference drug. The studies were conducted in accordance with the following documents: "Rules of Laboratory Practice in the Russian Federation" (Order of the Ministry of Health of the Russian Federation No. 708n dated August 23, 2010) and "Guidelines for the Conduct of Preclinical Studies of Medicinal Products" Part One//edited by A.N. Mironov (2012).

The studies were conducted in accordance with the technical specifications approved in the contract. The study design and organization were aimed at achieving the stated objective and were based on the general principles of conducting studies assessing the antimicrobial activity of drugs.

The study used S. pneumoniae serotype 19A bacteria with typical morphological and cultural properties, tested with a complete set of monospecific K-sera.

The strains were cultured in broth with the addition of cattle blood for 22-24 hours, then on brain heart agar.

The strains were then transferred to brain-heart or semi-synthetic (with the addition of aminopeptide) media.

Identification of S. pneumoniae bacteria was carried out based on the assessment of cultural properties, the results of microscopy with Gram staining, sensitivity to optochin and bile, and the presence of latex agglutination (Slidex pneumo-Kit).

Bacterial cultures identified as S. pneumoniae were subjected to PCR typing.

The results of the study showed that 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide exhibits activity comparable to the reference drug.

Example 5. Study of the reproduction of S. pneumoniae serotype 19A bacteria under conditions of using 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide

From the data presented in Table 8 and Figure 11 it follows that the substance 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide exhibits activity in the cells of S. pneumoniae serotype 19A bacteria.

Example 6. Study of the effect of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide on the infectious activity of viral pathogens of meningitis: influenza viruses, parainfluenza viruses, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, enteroviruses ECHO and Coxsackie

Viral meningitis is an inflammation of the membranes that cover the brain (meningitis) caused by viral invasion. Viral meningitis is an infectious disease. It can be caused by various viruses that enter the meninges through the hematogenous, lymphogenous, or perineural routes. Depending on the virus type, viral meningitis can occur through contact or airborne transmission. Once in the body, the viruses penetrate the subarachnoid space and infect the arachnoid and pia mater. Isolating the virus is challenging because it is present in small quantities in the cerebrospinal fluid, and can be present in other sources (blood, urine, stool, nasopharyngeal swabs) during carriage or after an infection without developing viral meningitis. Thus, the diagnosis of viral meningitis is very difficult, and specific and non-specific antiviral therapy for viral meningitis is necessary, especially in patients with a weakened immune system and in infants.

The aim of the study was to determine the antiviral activity of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide against viral pathogens that cause meningitis: influenza, parainfluenza, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, enteroviruses, ECHO, and Coxsackieviruses. Immunoglobulin was used as a reference drug.

The studies were carried out in accordance with the following documents: “Rules of laboratory practice in the Russian Federation” (Order of the Ministry of Health of the Russian Federation No. 708n dated August 23, 2010) and “Guidelines for conducting preclinical studies of drugs” Part One // edited by A.N. Mironov (2012).

The studies were conducted in accordance with the technical specifications approved in the contract. The study design and organization were aimed at achieving the stated objective and were based on the general principles of conducting studies assessing the antimicrobial activity of drugs.

The study utilized viruses from the viral strain collection of the Pasteur St. Petersburg Research Institute of Epidemiology and Microbiology: influenza virus A/California/07/09 (H1N1)pdm09, human parainfluenza virus type 3, human adenovirus type 6, herpes simplex virus type 2 (HSV-2), tick-borne encephalitis virus, Nipah henipavirus, ECHO virus, and Coxsackievirus B3. Viruses were accumulated in permissive cell lines: MDCK, Vero, MA-104, A549, L-41, and HEp-2.

Permissive cell lines were seeded in 24-well plates and incubated for 16-24 h until a monolayer was formed. 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide was added to the wells of the plates with a cell monolayer in a range of concentrations in a volume of 0.5 ml. The plates with cells were incubated in an atmosphere of 5% CO2 at 36 °C for 1 h. After this, 0.5 ml of the corresponding virus (m.o.i. 0.01) in the alpha-MEM medium were added to the wells and incubated for 1 h. After this, the liquid from the wells was collected, the cells were washed and 1 ml of fresh medium with 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the appropriate concentrations was added to the wells. The plates were incubated in a 5% CO2 atmosphere at 36°C for 48-120 hours, depending on the virus (Table 1). The infectious titer of the virus was then determined in the culture fluid. The titer was calculated using the Reed and Muench method and expressed as logarithms of the 50% infectious dose.

As can be seen from the data presented in Table 9 and Figure 12, a decrease in the activity of the studied virus in the direct CPE reduction test was observed at relatively high concentrations of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide.

A number of metabolites of enisamium iodide

Prediction of the permeability of enisamium iodide metabolites across the blood-brain barrier

Results of experiments on obtaining 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide

The content of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain of rats after a single intragastric administration of the test object at a dose of 100 mg/kg

The main indicators of penetration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide into the brain of rats after a single intragastric administration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg

The content of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in the brain of rats after repeated intragastric administration of the test 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg

The main parameters of penetration of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide into the brain of rats after repeated intragastric administration of the tested 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide at a dose of 100 mg/kg

The effect of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide on the reproduction of S. pneumoniae serotype 19A, influenza viruses, parainfluenza viruses, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, enteroviruses ECHO and Coxsackie

The effect of enisamium iodide on the infectious activity of S. pneumoniae 19A bacteria, influenza viruses, parainfluenza viruses, adenoviruses, herpes viruses, arboviruses, paramyxoviruses, enteroviruses ECHO and Coxsackie

Claims (1)

The use of 4-(benzylcarbamoyl)-3-hydroxy-1-methylpyridin-1-ium iodide in patients to reduce the infectious activity of viral pathogens of meningitis.