RU2185822C2 - Preparation for treatment and prophylaxis of viral diseases - Google Patents

Abstract

FIELD: medicine, virology, pharmaceutical industry. SUBSTANCE: preparation comprises remantadine fixed on polymer and 20-60% sugar syrup or sorbite syrup. The preparation is prepared by mixing remantadine, sodium alginate and indicated syrup in the following ratio of parent components, wt.-%: rematadine, 0.05-5.0; sodium alginate, 0.05-5.0; 20-60% sugar syrup or sorbite syrup, the balance. Preferable ratio remantadine to sodium alginate is 1:1. EFFECT: high bioavailability and effectiveness of preparation. 2 cl, 5 tbl, 3 ex

Description

 The invention relates to medicine, more specifically to pharmacy, and can be used in the manufacture of a drug for the treatment and prevention of influenza, acute respiratory infections and encephalitis, as well as other viral diseases in children and adults.

 The problem of treating viral pathology is extremely relevant in view of the increase in the number of such patients. Means for the prevention and treatment of viral diseases are actively developed by the pharmaceutical industry.

 In the patent fund of Russia and the USSR, inventions devoted to this problem are widely represented.

 So, in the patent RU 2135187 C1, A 61 K 31/765, 08.27.99 described the use of a copolymer of styrene with anhydride for the treatment of herpetic keratitis. Treatment of lip herpes with polymethacrylic acid is described in patent RU 2111751 Cl, A 61 K 31/78, 05.27.98. The treatment of herpetic eye damage with polyacrylamide is described in patent SU 628924, A 61 K 31/78, 10.25.78. Treatment of herpes zoster with the use of a 0.1% mixture of glycosaminoglycans isolated from human placenta is described in patent RU 2140786 Cl, A 61 K 31/725, 10.11.99.

 Means for treating acute respiratory infections are known from patents RU 2127116 C1, A 61 K 35/80, 03/10/99, SU 1336301 Al, A 61 K 35/78, 03/22/74. Strengthening the properties of the well-known antiviral drug acyclovir is described in the copyright certificate SU 1375113 A3, A 61 K 9/107, 07/18/80. The prevalence and severity of diseases requires the creation and prevention, first of all, of diseases such as acute respiratory infections, influenza, herpes, tick-borne encephalitis.

 Antiviral drugs are known containing remantadine as an active principle.

Механизм действия препарата в настоящее время хорошо изучен. В институте Гриппа детально исследованы все стадии фармакологического действия препарата. Ремантадин эффективно взаимодействует с клеточными мембранами и, в частности, с отрицательно заряженными на поверхности клеточных мембран фосфолипидами. Гидрофобное ядро адамантана интеркалирует внутрь мембран между остатками жирных кислот. Поэтому ремантадин относится к мембранотропным препаратам. Высокая атрактивность к мембранам позволяет ремантадину быстро достигнуть белка-мишени. Таким белком является белок М2 вируса гриппа. Вирус гриппа кодирует в 7 фрагменте два мембранных белка: Ml и М2. Белок М2 организован в вирусной и клеточной мембране в виде тетрамера, который выполняет функции ионного канала - протонной помпы. Именно поэтому вирус гриппа является зависимым от рН, то есть кислотности окружающей среды. Ремантадин, связываясь с мембранами инфицированных, увлекается потоком протонов внутрь полого канала тетрамера белка М2. Положительно заряженная аминогруппа ремантадина взаимодействует с гидроксилом Серина в 31 положении белка М2, что приводит к практически полной блокаде протонного насоса и, следовательно, блокирует инфекционную активность вируса на уровне лизосомального этапа декапсидации вирусных частиц. Фактически это блокирует развитие инфекции. К этому следует добавить, что ремантадин адамантановым ядром прочно связывается с так называемым адамантан-связывающим сайтом белка М2, составляющим часть трансмембранного домена: 25-Про-Лей-Вал-Вал-Але-Але-Сер-31. Эта связь необратимая, потому ремантадин можно отнести к суицидным для белка М2 ингибиторам.Remantadine - aminoethyladamantane, is a framework compound having a three-dimensional structure in the form of a hydrophobic adamantate core. The pharmacological activity of remantadine is largely determined by the presence of a positively charged amino group at α-1C:

The mechanism of action of the drug is currently well understood. At the Influenza Institute, all stages of the pharmacological action of the drug are studied in detail. Remantadine effectively interacts with cell membranes and, in particular, with phospholipids negatively charged on the surface of cell membranes. The hydrophobic core of adamantane intercalates into the membranes between residues of fatty acids. Therefore, remantadine refers to membranotropic drugs. High membrane attractiveness allows remantadine to quickly reach the target protein. Such a protein is the influenza virus M2 protein. The influenza virus encodes two membrane proteins in fragment 7: Ml and M2. Protein M2 is organized in the viral and cell membranes in the form of a tetramer, which serves as the ion channel - the proton pump. That is why the influenza virus is dependent on pH, i.e. the acidity of the environment. Remantadin, binding to infected membranes, is carried away by the flow of protons into the hollow channel of the M2 protein tetramer. The positively charged amino group of remantadine interacts with Serin hydroxyl at the 31 position of the M2 protein, which leads to almost complete blockage of the proton pump and, therefore, blocks the infectious activity of the virus at the level of the lysosomal stage of decapsidation of viral particles. In fact, this blocks the development of infection. It should be added that rimantadine by the adamantane core is strongly bound to the so-called adamantane-binding site of the M2 protein, which forms part of the transmembrane domain: 25-Pro-Lei-Val-Val-Ale-Ale-Ser-31. This connection is irreversible, because remantadine can be attributed to protein M2 suicidal inhibitors.

 This explains the high specificity of remantadine against influenza A viruses and its highest effectiveness in the treatment of influenza infection. Remantadine does not act on influenza B and other respiratory viruses. But it should be borne in mind that during the epidemic period, only 15% of the incidence of influenza is attributable to influenza B and parainfluenza viruses. This justifies the widespread use of remantadine in the epidemic period (Ershov F.I., Chizhov EP, Tazulakhova E.B. Antiviral agents. St. Petersburg, 1993, p. L9; Amer. J.Med. - 1987, Y. 82, 6A, P. 35-41; Pershiy G.N., Bogdanova N.S. Chemotherapy of viral infections. M: Medicine, 1973, p. 73).

It should be noted that remantadine in the standard dosage form has a number of significant drawbacks that limit its use in high dosages for adults and preschool children. These disadvantages include:
- the appearance of side effects with prolonged use (more than 5 days) of nausea, headaches (in 10% of cases);
- the rapid emergence of strains resistant to the drug;
- rapid elimination from the body within 1.5-4.5 hours;
- lack of effect on neurotoxicosis in case of complicated influenza and acute respiratory infections, in case of development of neurological symptoms;
- insufficient activity in the treatment of encephalitis;
- change in the acidity of the stomach with low acidity due to the main properties of remantadine.

 A compound of remantadine with a copolymer of vinyl alcohol and N-vinylimidosuccinic acid was synthesized by the formation of a CO bond under the name Polymer (see patent RU 2071323, A 61 K 31/785, 01/10/97). This tool has several advantages over native remantadine, because has a more pronounced antiviral effect, has an interferonogenic effect, is harmless.

 This object was selected as the closest analogue of the claimed invention. However, the known drug has side effects (including a change in the acidity of the stomach), insufficient immunomodulatory properties and a short period of elimination from the blood.

 The technical result of the invention is to reduce toxicity, providing a therapeutic effect on the frequency of isolation of rimantadine-resistant influenza viruses, tick-borne encephalitis viruses while increasing its bioavailability and providing a prolonged action.

The technical result is achieved by the fact that in the preparation for the treatment and prevention of viral diseases containing remantadine fixed to the polymer, sodium alginate is used as the polymer, and the preparation additionally contains sugar syrup or sorbitol syrup 20-60%, the preparation obtained by mixing remantadine, sodium alginate and the specified syrup, taken in amounts corresponding to the following ratio of starting components, wt.%:
Remantadine - 0.05-5.0
Sodium Alginate - 0.05-5.0
Sugar syrup or sorbitol syrup 20-60% - Else
and it is preferable to take remantadine and sodium alginate in a ratio of 1: 1.

 The soluble form of remantadine in combination with sodium alginate allows the use of sugar syrup form for the preparation of the drug, which allows it to be used to treat children. Solid forms of the preparation may also be used, for example in the form of tablets or capsules.

Getting the drug
Alginate is an acidic mucopolysaccharide of algae. When interacting with the base with aminoethyladamantane (remantadine), alginate-Na forms a strong salt complex. Complexation was checked by gel chromatography on columns with Sepharose-2B. The saturation of alginate with rimantadine was judged by the amount of remantadine detected in the second peak of elution. The first peak eluted with high polymer alginate.

 Thus, the remantadine / alginate complex is a polymer form of remantadine.

 In the previously developed Polymer preparation (RU 2071323), α-1-methyl-α (1-adamantyl) methylamine (remantadine) is bound to a polymer carrier based on a copolymer of vinyl alcohol with N-vinylamino succinic acid. The polymerization of remantadine in such a system significantly improves the properties of the drug.

 When choosing alginate, the aim was to replace a synthetic polymer with weak activity in the induction of interferon by a natural carrier with pronounced immunomodulating properties, which is a natural mucopolysaccharide - alginate.

 To obtain the target product, a syrup is prepared, for example, 60% sugar syrup, and at the same time a mixture of remantadine with sodium alginate is prepared in the calculated quantities. After a three-hour incubation, the mixture is added to the syrup with slow stirring. The resulting syrup is mixed and packaged.

 Table 1 shows examples of formulations for the preparation of the drug.

 The resulting preparation was investigated for bioavailability and pharmacokinetics.

Bioavailability and pharmacokinetics of remantadine alginate
Bioavailability was investigated by determining the concentration of remantadine in the blood after intragastric administration through a probe of remantadine alginate in mice. After 1.5 hours, rimantadine appears in the blood and reaches a maximum after 5.5 hours. The concentration of remantadine remains stable for 12 hours and then decreases to trace concentrations by the end of the day. Remantadine monomer has a shorter half-life in peripheral blood. Reaching a maximum after 1.5-2 hours, rimantadine monomer is completely eliminated from the blood after 6 hours.

 Thus, remantadine alginate has a high bioavailability and a long period of elimination from the blood, which allows us to recommend taking 2 times a day.

 Also analyzed the toxicity of the obtained funds.

Toxicity of remantadine in combination with sodium alginate
To assess the toxicity of the drug, a remantadine concentration of 5.7 mg / kg body weight of the experimental mice was used, which corresponds to a ten-fold equitherapeutic dose.

 The experiments were carried out on two groups of mice of immature age, one of which received a daily dose of remantadine in syrup without sodium alginate, the other an equal dose with sodium alginate. For three months, the dynamics of changes in body weight and hematological parameters were evaluated.

 As follows from the data given in table. 1 and 2, taking remantadine at a dose of 5.7 mg / kg weight without sodium alginate significantly worsens the dynamics of the increase in body weight in experimental animals and reduces hematological parameters. Whereas in parallel experiments in mice treated with rimantadine in the same dose, but in combination with sodium alginate, the indices mentioned do not practically differ from the control, which convincingly indicates a significant decrease in the toxicity of remantadine when taken in combination with sodium alginate.

Results of a study of the effect of alginate-Na and remantadine on the frequency of isolation of rimantadine-resistant influenza viruses from patients with influenza infection
The observation was carried out on 56 patients with influenza in the epidemic period 1998/1999. , in January-February, when there was a peak in influenza in St. Petersburg. The observation group selected children aged 10 to 16 years. Taking remantadine, half of the patients were prescribed immediately 1 teaspoon 4 times a day. In patients, 2 days after the start of taking remantadine, nasopharyngeal swabs were taken in syrup and the MDCK cell culture highly sensitive to influenza viruses was infected.

 The data on the isolation of influenza viruses are presented in table 3. As can be seen from table 3, the maximum frequency of isolation is characteristic of H1N1 viruses (21.4%). H3N2 influenza viruses were released with a frequency of about 14%, and influenza B - about 18%. Matching data with an excess of the effectiveness of virus excretion by 32% was obtained in the control group of children who did not receive rimantadine in syrup, but who took antigrippin and other symptomatic agents. Viruses isolated were examined after two passages for resistance to remantadine. To isolate rimantadine-resistant viruses, MDCK cells and remantadine were used at a concentration of 10 μg / ml. As a result, it was found that in children receiving rimantadine in syrup, only one strain of influenza virus resistant to rimantadine was isolated. In the control group, one strain of the H3N2 influenza virus resistant to remantadine was also isolated. From these data it follows that remantadine with alginate does not cause an increase in the frequency of excretion of viruses resistant to remantadine with the recommended regimen.

 According to Hayden, the frequency of isolation of rimantadine-resistant influenza viruses when used in the regimen of 1 tablet (50 mg) 4 times a day in his epidemiological studies reached 26-32% (for different age groups).

 The results obtained indicate that the combination of alginate and remantadine in syrup, which favorably changes the pharmacokinetics of remantadine, reduces the incidence of remantadine-resistant viruses.

 Example 1 use of the funds received.

 To confirm the therapeutic effect of the proposed drug, two groups of sick children from 6 to 10 years old were taken in total of 20 people each with signs of influenza, confirmed by laboratory analyzes of swabs from the nasopharynx. Group 1 received 4 tsp algir syrup. per day for 10 days. Group 2 did not receive special antiviral treatment.

 During the observation, it was found that in the first group of patients on the 4-5th day after the start of taking the drug, the clinical manifestations of influenza infection significantly decreased, the temperature returned to normal, and there were no complications.

 In the second group, a significantly more severe course of the disease, prolonged hyperthermia was observed, some patients showed signs of pneumonia and neurological symptoms. In some cases, intensive therapeutic measures were required.

 The results obtained confirm the high effectiveness of the drug in the treatment of influenza.

Example 2
The results obtained in the study of the proposed drug in an epidemic situation, confirmed its high efficiency. In family foci, people in contact with patients for prophylactic purposes took 2 teaspoons of remantadine with alginate (algir) (20 mg in terms of remantadine - base). The effectiveness of influenza prophylaxis in an epidemic as a result of taking algirem was more than 2 times higher than that of remantadine hydrochloride (Table 4). The effectiveness index of the proposed drug with a high degree of reliability (p <0.01) is 2.1 times higher than the remantadine hydrochloride index.

Example 3
The effectiveness of algirema against tick-borne encephalitis virus was determined on a mouse infection model with the introduction of tick-borne encephalitis virus, strain Absettarov, isolated from the patient’s blood. To reproduce a lethal infection, the peripheral (subcutaneous) route of administration of the virus cultured in the brain tissue of mice was used. Infection was carried out with the introduction of 0.2 ml / mouse suspension of brain tissue in physiological saline (aqueous NaC1 mass fraction of 0.85%) containing 1-10 LD _{50 of the} virus.

 In the experimental groups of animals, algire was administered orally at a dose for each dose, calculated on the basis of a content of 0.5 μg / kg body weight.

 The introduction of the drug was carried out according to various schemes. Algire was administered once 24 hours before infection (Scheme 1) or 1 hour before infection (Scheme 2). Algire was administered three times 1 hour before infection and 48 and 96 hours after infection (Scheme 3). The drug was administered 24 hours before infection and 4 hours and then 24 hours after (Scheme 4).

 The effectiveness of the drug was evaluated by the level of death of animals compared with the control group of mice that were given a placebo (physiological saline) according to the experimental group.

 For all the algirem administration schemes used, a marked decrease in the death of infected mice was observed (Table 5). The effect was achieved already with a single injection of the drug. Higher efficiency with the introduction of treatment and prophylactic regimens (3 and 4) is due to the peculiarity of the pathogenesis of tick-borne encephalitis infection, which is characterized by a two-wave course. The observed differences in the protection of the drug with different schemes of its administration depend on some fluctuations in the death of animals in the control groups.

Claims (1)

1. The drug for the treatment and prevention of viral diseases, containing rimantadine, fixed to the polymer, characterized in that it contains sodium alginate as a polymer and additionally contains sugar syrup or sorbitol syrup 20-60%, and the drug is obtained by mixing remantadine, sodium alginate and the specified syrup in the following ratio of starting components, wt. %:
Remantadine - 0.05-5.0
Sodium Alginate - 0.05-5.0
Sugar syrup or sorbitol syrup 20-60% - Else
2. The drug according to p. 1, characterized in that it is obtained by mixing these components with a ratio of remantadine and sodium alginate equal to 1: 1.

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