RU2574007C2 - Injection solution for treating viral diseases specified in h1n1, h3n2, h5n1 influenza, tick-borne encephalitis and west nile fever - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry and represents an injection solution for treating viral diseases specified in H1N1, H3N2, H5N1 influenza, tick-borne encephalitis and West Nile fever, containing triazavirin in an amount of 0.05-4.0 wt %, additives in an amount of 0.1-30.0 wt % and water up to 100 wt %.

EFFECT: invention provides high metabolic stability and accessibility with maximum efficacy in antiviral action on virus replication.

4 cl, 4 tbl, 4 ex

Description

The invention relates to the pharmaceutical industry and relates to the development of a new injection solution of the antiviral drug Triazavirin - 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 ( 4I ′) - she.

The parenteral infusion solutions used mainly relate to antibiotic solutions and detoxification agents and to provide control over disturbances in water-salt metabolism.

Known infusion solution of metronidazole based on 2- (2-methyl-5-nitro-1H-imidazol-1-yl) ethanol, which refers to nitrogen-containing heterocyclic compounds and therefore the physicochemical properties are closest to 2-methylthio-6 -nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4I ′) - it (triazavirin) and analogues based on 5-methyl-6-nitro-1, 2,4-triazolo [1,5-a] pyrimidin-7-one, dihydrate [V.L. Rusinov, O.N. Chupakhin, E.N. Ulomsky et al. RF Patent 2345080 dated January 27, 2009), as well as 4 - ((Z) -4′-Hydroxybuten-2′-yl) -2-R-6-phenyl-1,2,4-triazolo [5 , 1-s] [1,2,4] triazin-7-ones (O. N. Chupakhin, V. L. Rusinov, E. N. Ulomsky, and others. RF Patent 2376307 dated 12/20/2009; B. L. Rusinov, EN Ulomsky, ON Chupakhin, VN Charushin, Azolo [5.1-c] -1,2,4 triazines - a new class of antiviral compounds. Izvestiya AN, Chemical Series, 2008, No. 5, p. 967-995). 2-R-6-nitro-4-allyloxymethyl-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) also possess antiviral activity against influenza viruses of types A and B ) -ones, R = Н, СН3, SCH3 (V.L. Rusinov, E.N. Ulomsky, S.L. Deev. et al. RF patent 2340614 dated 12/10/2008; V.L. Rusinov, O.N. Chupakhin, SL Deev, TS Shestakova, EN Ulomsky, LI Rusinova, OI Kiselev, EG Deeva “Synthesis and antiviral activity of nucleoside analogues based on 1, 2,4-triazolo [3,2-c] [1,2,4] triazin-7 (4H) -ones ”// Izvestia AN, Chemical Series, 2010, No. 1, pp. 135-142].

Water injection or infusion solutions are known and widely used in medical practice for treating patients in intensive care wards for various terminal conditions: solutions of reambirin, cytoflavin, antibiotics from the group of fluoroquinolones and simple water-salt solutions [Gromova LI, Buryakina A.V. ., Kuzmina Yu.A., Avenirova E.L. Infusion solution to replenish the volume of circulating blood, restore water-electrolyte balance and normalize blood formation processes. 2009149813/15, 12/31/2009] to fill the deficit of fluid in the bloodstream [https://mi.aup.ru/res/79/562949980667179.html)].

The chemical structure of metronidazole is 2- (2-methyl-5-nitro1H-imidazol-1-yl) ethanol and is an inhibitor of the growth of anaerobic infections. This drug has been widely used in medical practice for the treatment of a wide range of bacterial and protozoal infections [Freeman C.D., Klutman N.E., Lamp K.C. Metronidazole. A therapeutic review and update. Drugs 1997 Nov; 54 (5): 679-708, Löfmark S., Edlund C, Nord C.E. Metronidazole is still the drug of choice for treatment of anaerobic infections. Clin Infect Dis. 2010, Vol. 50, Suppl 1: S16-23]. The mechanism of action of this drug is the biochemical recovery of the 5-nitro group of metronidazole by intracellular transport systems of oxidative phosphorylation of anaerobic microorganisms and protozoa. The reduced 5-nitro group of metronidazole interacts with the phosphate groups of DNA cells of microorganisms, inhibiting the synthesis of their nucleic acids, which leads to the death of microorganisms. However, the nitro group, before being reduced to the amino group under aerobic conditions, is capable of exhibiting independent reaction activity, which is typical for nitro derivatives of triazolo-triazines. Regarding the chemical structure and properties of the reaction groups (nitro group in the 5th position of the imidazole ring), it is close to nitro-containing triazolo-triazines, in particular to antiviral drugs based on them [V.L. Rusinov, O.N. Chupakhin, S.L. Deev, T.S. Shestakova, E.N. Ulomsky, L.I. Rusinova, O.I. Kiselev, E.G. Deeva. Synthesis and antiviral activity of nucleoside analogues based on 1,2,4-triazolo [3,2-s] [1,2,4] triazin-7 (4H) -ones. Izvestia AN, Chemical Series, 2010, No. 1, p. 135-142].

As a prototype, an injection or infusion solution of enrofloxacin was selected [RF patent 2147225].

Ciprofloxacin solutions are made on the basis of lactic acid salts with an excess of acid [Bonze G., Hamm M., Müller H.-P.], etc., which can have an adverse effect on metabolism and tissue respiration in severe infectious diseases characterized by acidosis and hypoxia accompanied by the generation of free radicals. These processes play a key role in the pathogenesis of acute viral infections, including influenza caused by highly pathogenic type A influenza viruses and viral hemorrhagic fevers. Therefore, it must be borne in mind that when creating infusion solutions, first of all, the physicochemical properties of the main drug component are taken into account.

Injection and infusion solutions of antiviral drugs in medical practice, in fact, are absent [S.G. Reshetnikov, A.V. Babayants, D.N. Protsenko, B.R. Gelfand. “Infusion therapy in the perioperative period” (literature review). 2008, No. 1b p. 29-44]. Only during the 2009/2011 influenza pandemic were injectable forms of the drug peramivir developed specifically for the treatment of patients in terminal stages with complicated forms of influenza infection [https://www.clinicaltrials.gov/]. The drug peramivir refers to selective neuraminidase inhibitors [Kiselev O.I. Chemotherapy and flu chemotherapy. SPb, Publ. “Rostock", 2012, S. 268]. Later, the use of this group of drugs for the treatment of complicated influenza seems rather doubtful due to the impossibility of manifesting their therapeutic potential. Therefore, the development of new approaches to the creation of highly effective infusion therapy is extremely relevant to reduce mortality from influenza and other acute viral infections. The development of complex infusion solutions that provide not only a specific (etiotropic) effect on the reproduction of infectious agents, but also have a definite effect on the pathological processes associated with infections, is the basis for increasing the effectiveness of treatment of severe conditions in intensive care for infectious diseases. During the 2009/11 flu pandemic. detoxification therapy with traditional infusion solutions showed that in most patients this increased the risk of pulmonary and cerebral edema. As a result, the Ministry of Health of the Russian Federation quickly issued recommendations rejecting such treatment tactics [Kiselev OI, Pokrovsky VI Influenza in pregnancy: a combination of functional and infectious immunosuppression. SPb, Publ. “Print”, 2012. S. 148].

When developing an infusion (injection) solution containing antiviral drugs, it is necessary to take into account the need for the introduction of additional components that affect the metabolism of the drug and pathological changes in tissue respiration, including hypoxia and excessive formation of free oxygen radicals. Under these conditions, significant changes in the metabolism of drugs and their pharmacodynamics are observed, which must be taken into account when developing composite injection and infusion solutions. Additional or auxiliary components of infusion and injection solutions to a certain extent affect both the interstitial distribution and diffusion in the tissues of the pharmacologically active component, and the rate of its excretion in urine (Bonze G., Hamm M., Müller H.-P., etc. Enrofloxacin Injection and Infusion Solution [RF Patent No. 2147225]).

The invention is directed to the development of a medicinal composition for injection or infusion form of the drug Triazavirin and allows for high metabolic stability and availability with maximum efficiency in antiviral effect on virus replication.

The injection and infusion solution according to the invention contains the biologically active substance triazavirin [V.L. Rusinov, O.N. Chupakhin, E.N. Ulomsky et al. RF patent 2345080 from 01/27/2009; Karpenko I., Deev S., Kiselev O., Charushin V. et al. Antiviral Properties, Metabolism, and Pharmacokinetics of a Novel Azolo-1,2,4-Triazine-Derived Inhibitor of Influenza A and B Virus Replication Antimicrob. Agents Chemother. 2010, Vol. 54, p. 2017-2022] in concentrations of preferably 0.05-4%, particularly preferably 0.1-1.0%, based on the total weight of the solution.

As amino acids and their derivatives, arginine, histidine, cysteine, N-acetylcysteine and other components of chemical and biological origin are used. Additional components and, in particular, amino acids may be present in 0.1-5-fold molar excess relative to the amount of triazavirin in solution. An excess of 0.2-2 mol is preferred, an excess of 0.1-1 mol is particularly preferred.

Aqueous solutions for injection may, as a carrier liquid, contain, for example, glycerin, propylene glycol, polyethylene glycol and triethylene glycol as a carrier liquid. Various substances such as tris, salts of ascorbic, succinic, tartaric, lactic, fumaric and gluconic acids can be used to establish the pH value, if possible in the range from 3 to 7.25. The pH of the aqueous compositions according to the invention is from 4.5-7.25, preferably 4.5-7.0. The osmolarity of aqueous compositions is 400-510 m osm / l.

Auxiliaries to the compositions can also be used, such as thickeners (e.g. methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, gelatin, etc.), absorption aids, light stabilizers, crystallization inhibitors, complexing agents (e.g. sodium ethylene diamine tetra, phosphates, nitrates, acetates, citrates, etc.), antioxidants (ascorbic acid, sulfite compounds, L-cysteine, thiodipropionic acid, thiolactic acid Ota, monothioglycerol, propyl gallate, etc.) as well as accessible preservative (chlorobutanol, benzyl alcohol, ethanol, butanol, 1,3-butanediol, chlorohexidine salts, benzoic acid and its salts, sorbic acid, etc.).

In some cases, there is a need to add local anesthetics to the solutions according to the invention, such as procaine hydrochloride, lidocain hydrochloride and others. A combination with acids, for example succinic acid, sugars, including glucose, mannose, alcohols, for example mannitol or sorbitol, is possible. To accelerate dissolution, the solution is heated in a water bath to 50-60 ° C. Immediately after receiving the salt, the remaining components are dissolved in the composition with stirring before or after cooling, or introduced into it. After adding the rest of the solvent, the composition is sterilized by filtration through a bacterial filter and / or by heating.

Indications for use should take into account the diuretic effect of the drug and, therefore, in the case of a disease accompanied by dehydration, the use should be combined with infusion solutions that promote fluid retention. If there is a threat of tissue edema and especially pulmonary and brain edema, use diuretics taking into account the contribution of the infusion of triazavirin solutions.

The combined use of injection and infusion solutions of triazavirin is accompanied by oral and parenteral use of antioxidants such as tempol, quercetin, dihydroquercetin, riboflavin and other known means of reducing the toxicity of reactive oxygen species.

The invention is implemented as follows.

Example 1. Obtaining solutions of triazavirin

In the preparation of injection and infusion solutions of triazavirin, the authors were guided by:

1. The determination of the solubility threshold to achieve the optimal concentration of injection and infusion solution.

2. Determining the conditions for increasing solubility, bioavailability and metabolic stability.

3. Achieving the conditions of combination with other substances required to increase the therapeutic antiviral efficacy of the drug Triazavirin.

Triazavirin solubility study was carried out in two ways.

According to the first method, the anchor of a magnetic stirrer, 100 g of water (by weight), 8 g of the preparation were placed in a suspended glass of 250 ml volume and stirred for 8 hours at room temperature. The measurement limit of the weights was 0.2 g. After stirring, the mixture was filtered in a device consisting of a weighed round-bottom flask with a volume of 250 ml, a transition with a tap and a weighed funnel with a thin section. After filtration, the filtrate and the wet cake were weighed separately. The precipitate was allowed to dry, the filtrate was evaporated from the flask completely on a rotary evaporator at a residual pressure in the device of 10–15 mm Hg, the temperature in the bath was 40–50 ° C and also left to dry overnight at room temperature. It was found that the mass of the solution before evaporation was 104.2 g, the mass of the dry preparation was 4.2 g. Thus, the solubility of triazavirin in water was 4%. In the same way, the solubility of triazavirin in physiological saline was studied.

In the second method for determining the solubility of triazavirin, the following operations were carried out.

100 ml of water, 10 g of triazavirin were placed in a 250 ml round-bottom flask equipped with a water-cooled reflux condenser, the mixture was heated to boiling and boiled for 10 minutes. After boiling, the solution is cooled to room temperature and incubated for 2 hours. The mixture is filtered, the filtrate is placed in a weighed flask with a volume of 500 ml, the weight of the solution is determined by weighing and evaporated completely on a rotary evaporator at a residual pressure in the device of 10-15 mm Hg, temperature bath 40 ÷ 50 ° C. The residue was allowed to dry overnight at room temperature. It was found that the solubility of triazavirin in water was 4%.

Thus, regardless of the dissolution conditions, the solubility of triazavirin in water is 4%, which is sufficient to obtain an injection or infusion solution for intramuscular or intravenous administration.

Example 2. The study of the toxicity of triazavirin solutions with intravenous administration.

In experiments on rabbits and rodents, the acute toxicity of triazavirin substance samples synthesized at the Institute of Organic Synthesis of the Ural Branch of the Russian Academy of Sciences was studied.

According to a study of acute and chronic toxicity of triazavirin in rodents, the average lethal dose of triazavirin with intraperitoneal administration to rats is 1320 (950-1840) mg / kg. The absolute lethal dose with intraperitoneal administration was 2520 mg / kg. When triazavirin was administered to rats inside, animals transferred the drug in doses up to 9000 mg / kg without any visible signs of intoxication. It was not possible to establish the average lethal dose in these experiments because of the administration of large volumes of the drug to animals. According to studies conducted at the Research Institute of Toxicology, when administered orally to rats, LD ₅₀ = 8320 ± 480 mg / kg. Information on the toxicity of the drug are presented in tables 1-2.

In the process of these experiments, a pathomorphological study of the organs of dead mice and rats treated with large doses of triazavirin (intraperitoneally and orally) was carried out. It was found that the nature of the toxic effect is similar in studies in mice and rats. Histologically, the method of drug administration does not affect the type and nature of lesions due to exposure to toxic doses of the drug.

In acute rabbit experiments, the toxicity and tolerance of the triazavirin substance during the intravenous route were studied. A solution of triazavirin for intravenous administration was prepared using a bipolar solvent of dimethyl sulfoxide (DMSO) and a 0.9% solution of sodium chloride, heated to a temperature of 38-39 ° C. The need for DMSO is due to the limited solubility of triazavirin. So, at room temperature, the solubility of triazavirin in water is 4%, at 100 degrees - 39%.

The study of the acute toxicity of triazavirin with the intravenous route of administration was carried out on rabbits with a body weight of 3.5 kg to 4.7 kg contained in the vivarium of the research institute of influenza. Triazavirin was administered in increasing dosages from 700 mg / kg to 1500 mg / kg. The death of animals was not observed. Due to the limited solubility of triazavirin, it was necessary to resort to an increase in the allowable volume of intravenous administration to rabbits. The "Guidelines for the experimental (preclinical) study of new pharmacological substances" limits the volume of solutions administered intravenously to rabbits in a toxicological study of 20 ml. In the experiments, triazavirin was administered intravenously by both a jet and a drop method in a volume of 100 ml, which is 5 times the recommended volume. At the same time, a dose of 1500 mg / kg was administered. Despite the hypervolemic shifts caused by the introduction of excess fluid, the animals did not die. Only when the triazavirin solution was injected in a jet, did the rabbits close their eyes and show no resistance, but the reflexes remained intact. Subsequent observation of animals for 7 days did not allow to reveal changes in their behavior, intensity and nature of motor activity, coordination of movements, food and water consumption.

With a dropwise administration of a solution of triazavirin to rabbits at a dose of 1000 mg / kg 45 minutes after formulation, abundant urination was noted with the diaper stained in bright yellow color, characteristic of a triazavirin solution. At the same time, even minimal signs of intoxication were not observed during the infusion, the animals were active. An increase in the dose of triazavirin to 1500 mg / kg did not lead to the death of the animals and was also accompanied by an increase in diuresis. A further increase in the dose is technically difficult due to limitations in the solubility of the drug and the acceptable volumes of administration. The initiation of diuresis should be attributed to the useful properties of the drug, since with toxic flu the use of diuretics for the prevention of pulmonary edema is mandatory. Therefore, in the compositions of the infusion solutions of triazavirin there is no need for combination with drugs - diuretics.

Thus, the average lethal dose for intravenous administration in rabbits could not be determined. Triazavirin can be classified as practically non-toxic compounds.

* Graduation of the degrees of toxicity during the intravenous route of administration is determined by multiplying the values of standard doses to assess the toxicity of the drug with the oral route of administration by a factor of 0.1.

The injection (infusion) dosage form of the drug Triazavirin should correspond to the concentration of the solution of triazavirin, which in a certain volume of administration will ensure its effective concentration in the blood, namely 0.5-10 μg / ml, preferably 2-4 μg / ml.

The range of effective doses for humans with a parenteral route of administration is 1.5-25 mg / kg. The optimal release form for triazavirin infusion solutions is 100 ml vials with a triazavirin substance content of up to 500 mg / vial or 5 mg / ml. For the treatment of clinically severe forms of influenza with the development of influenza pneumonia and the threat of pulmonary edema, it is recommended that the drug be administered up to 3 times a day in 1 vial with a dose of 500 mg per intravenous administration.

For the treatment of influenza during the 2009/11 pandemic caused by the swine strain of the H1N1 virus, the injection and infusion preparation Peramivir (https://www.biocryst.corn/peramivir) was registered in the United States. The drug is used in an aqueous solution in doses of 6 to 10 mg / kg and is available in powder form in 300 or 600 mg vials to dissolve 0.9 or 0.4% sodium chloride in a solution. The practice of application has shown that the optimal rate of administration is 40 mg / min.

However, this use of an antiviral drug inevitably leads to the need for other infusion solutions that provide detoxification. For these purposes, various solutions and compositions are used. However, the excessive use of known infusion solutions for influenza and influenza pneumonia in large volumes is completely unacceptable due to the threat of pulmonary edema. So, succinic acid-based reambirin, due to the low concentration of the main component, is recommended in doses up to 800 ml / day. The conclusion of the developers about the antioxidant effect of the drug is also controversial: stimulation of tissue respiration under conditions of acidosis can only stimulate the production of reactive oxygen species. Oxygen support in the face of acute respiratory failure becomes fatal.

In this case, Mexidol, which may be compatible with triazavirin infusions, is more adequate in this case in relatively small volumes.

However, the most optimal is the combination of triazavirin with known antioxidants. Therefore, the following examples provide triazavirin infusion solution compositions based on these findings and known recommendations for the treatment of emergency conditions in infectious disease clinics [https://www.drrobertamorgan.com/intravenous-therapies-and-infections.htm].

Example 3. The composition of the injection or infusion solutions of the drug Triazavirin for the treatment of acute viral infections.

No. 1

Triazavirin - 500 mg

Sodium Chloride - 900 mg

Water d./inject. up to 100 ml

pH 5.5-7.0

osmolarity 400-410 m osm / l

Number 2

Triazavirin - 500 mg

Arginine - 1000 mg - 2500 mg

Sodium Chloride - 900 mg

Water d./inject. up to 100 ml

pH 5.5-7.0

osmolarity 400-410 m osm / l

Number 3

Triazavirin - 500 mg

Arginine hydrochloride - 1000 mg

Sodium Chloride - 900 mg

Water d./inject. up to 100 ml

pH - 5.5-6.5

osmolarity 350 m osm / l

Number 4

Triazavirin - 500 mg

Cysteine - 1000 mg

Sodium Chloride - 900 mg

Water d./inject. up to 100 ml

pH 5.5-7.0

osmolarity 400-410 m osm / l

Number 5

Triazavirin - 100 mg - 200 mg

Sodium Chloride - 90 mg

Water for injection up to 10 ml.

pH - from 4.5 to 6.5

Number 6

Triazavirin - 100 mg - 200 mg

Arginine - 100-200 mg

Sodium Chloride - 90 mg

Water for injection up to 10 ml

pH 5.5-6.5

No. 7 - g / 100 ml

Triazavirin - 0.5

Gluconolactone - 4,000

Glutamic acid - 2,670

Benzyl alcohol - 1,400

Sodium sulfite - 0,100

Water for injection - 87.300-100 ml = 105.500

pH 5.2-6.5

Osmolarity 450-500 m osm / l

No. 8 - g / 100 ml

Triazavirin - 0.5

Betaine hydrochloride - 4,000

Glutathione (reinst.) - 2,000

Sodium sulfite - 0,100

Water for injection - 87.300-100 ml = 105.500

pH 5.5-6.5

Osmolarity 410-470 m osm / l

No. 9 - g / 100 ml

Triazavirin - 0.5

Cysteine - 2.0

Glutathione 2.0

Carnosine - 1.0-2.0

Water for injection up to 100 ml

pH 5.0-6.0

Osmolarity 450-500 m osm / l

From the above data it follows that the content of triazavirin in the composition is 0.05-4.0%, based on the total weight of the solution, in the form of salts with bases or amino acids and peptides or their mixtures, and auxiliary substances - 0.1-30.0 the weight.%.

The study of the antiviral activity of injection or infusion solutions obtained in examples No. 1-9 was performed on cell cultures of MDCK and Vero infected with influenza viruses H1N1, H3N2, H5N1, tick-borne encephalitis, and West Nile fever. The screening results are shown in the table in the in vitro system.

Claims (4)

1. Injection solution for the treatment of viral diseases selected from H1N1, H3N2, H5N1 flu, tick-borne encephalitis and West Nile fever, containing triazavirin in an amount of 0.05-4.0 wt.%, Excipients in an amount of 0.1-30, 0 wt.% And water up to 100 wt.%. 2. The injection solution according to claim 1, characterized in that it further comprises an amino acid selected from the group: arginine, arginine hydrochloride, glutamic acid, histidine, cysteine and its derivative N-acetylcysteine, which may be present in 0.1-5 fold molar excess relative to the amount of triazavirin in solution. 3. The injection solution according to claim 1, characterized in that it further comprises a carnosine peptide in an amount of 1.0-2.0 wt.%. 4. The injection solution according to claim 1, characterized in that it can be a solution for infusion.