UA123054C2 - Composition having antiviral effect - Google Patents

Abstract

The present invention relates to a pharmaceutical composition which comprises tyrothricine, benzalkonium chloride, and/or benzocaine for use in antiviral therapy or in the treatment of a viral infection.

Description

The field of technology to which the invention belongs

The invention relates to a pharmaceutical composition containing thyrothricin, benzalkonium chloride and/or benzocaine for use in antiviral therapy or in the treatment of viral infection.

Prior art

Viruses and bacteria are pathogens of widespread acute pharyngitis. Medicines to treat these conditions have been available on the market for decades, such as DoritricinF SiIazvis sore throat lozenges. They are usually used with the antiseptic and antibacterial agents contained in them for clinically effective relief of the typical symptoms of pharyngitis, such as pain on swallowing and redness of the throat.

Thyrothricin is a mixture of various linear and cyclic polypeptides from the groups of gramicidins and tyrocidins effective against bacteria. They are formed as endotoxin-like molecules of the anaerobic spore-forming bacterium VasiShy5 apeigipousysy5 (synonymous Vasiy5 Bhemiv).

The spectrum of activity includes mainly gram-positive bacteria, but also some gram-negative bacteria and various types of fungi, such as Sapaeida aibisap5. Thyrothricin contains 50-70 95 tyrosidines and 25-50 90 gramicidins, which together make up at least 85 95 active ingredients. In addition, there are small amounts of other structurally related polypeptides.

Benzalkonium chloride is a mixture of alkylbenzyldimethylammonium chlorides, the alkyl fragment of which consists of C8-C18 chains. It has a general disinfecting and preservative effect and is effective against bacteria, fungi, yeast and algae (even against viruses, but to a lesser extent).

Benzocaine (4-aminobenzoic acid ethyl ester) is a local anesthetic and is mainly used to treat local soreness of the skin and mucous membranes, such as in the mouth and throat, as well as in cold medicines, antitussives, pain relievers remedies such as solutions or lozenges for problems with the throat, stomach and teeth, astringents.

Brief description of the invention

The invention relates to a pharmaceutical composition containing thyrothricin, benzalkonium chloride and/or benzocaine, for use in antiviral therapy or in the treatment of viral infection.

According to the best embodiment, the composition contains all three active ingredients in

From a combination.

Brief description of the drawings

Figures 1-4: inhibition of the activity of NK14, NIMI and K5M viruses expressed in percentage by solutions of doritricinaf with different degrees of dilution;

Fig. 1: synergistic antiviral effect of all active ingredients - thyrothricin, benzalkonium chloride and benzocaine in combination with increasing dilution. The synergistic effect is confirmed when comparing with fig. 2-4;

Fig. 2: weak antiviral effect of tyrothricin alone with increasing degree of dilution;

Fig. 3: very weak antiviral effect of benzalkonium chloride alone with increasing degree of dilution;

Fig. 4: very weak antiviral effect of benzocaine alone with increasing degree of dilution.

Detailed description of the invention

The dose-dependent antiviral effect of the above-mentioned active substances against human pathogenic viruses KOM, NKM14 and NIMI was studied in the IP study. The data of this study demonstrate that the solution prepared from active ingredients and brought to a high physiological concentration of active ingredients reduces viral activity by 68.4 Fo (NAM'14), 52.7 95 (NIM) and 74.3 95 (K5M) . The antiviral effect could no longer be detected only at a very high degree of dilution of the active ingredient. Thus, the pharmaceutical composition containing tyrothricin, benzalkonium chloride and benzocaine is characterized by pronounced, dose-dependent and synergistic antiviral properties against EM, HEM'14 and NIMI viruses.

This observation is new and unexpected due to the active ingredients: - tyrothricin belongs to antibiotics acting against bacteria, - benzalkonium chloride and benzocaine belong to analgesics effective against pain (1-4).

Thus, this invention belongs to a pharmaceutical composition that contains - tyrothricin, - benzalkonium chloride and/or - benzocaine, for use in antiviral therapy or in the treatment of viral infection.

The composition according to the present invention may include, consist of or essentially contain the above-mentioned active substances. The expression "consists essentially of" means that the pharmaceutical composition contains these components as active ingredients. Nevertheless, it may contain one or more solvents, auxiliary substances, etc., which are necessary for the production of the pharmaceutical composition, but do not contribute or do not contribute significantly to the pharmacological effectiveness.

The term "pharmaceutical composition" as used herein includes, in particular, oral dosage forms such as solid, semi-liquid or liquid compositions for oral administration. According to a preferred embodiment, the composition is in the form of a tablet, in particular, a lollipop or a sore throat tablet.

However, as an alternative, other dosage forms are also possible, such as solid oral dosage forms, such as powders or capsules. In this case, limited additions of magnesium stearate or calcium behenate can be used as lubricants, and starch can be used as disintegrants (disintegrant). In the case of granulation, an aqueous solution of lactose, ethanol and appropriate concentrations of starch pastes can be used.

Common auxiliaries used in the production of sore throat lozenges include sorbitol (E 420), talc, esters of fatty acids and sucrose, sodium saccharinate dihydrate, peppermint oil, povidone 25 and carmellose sodium.

In addition to oral dosage forms, the pharmaceutical composition according to this invention may include parenteral forms, that is, liquid diluted solutions for injection and liquid solutions for rubbing, as well as ointments, suppositories, eye drops and nasal drops.

In a preferred form, the composition is used to treat an infection caused by rhinovirus, influenza viruses and/or pneumoviruses.

According to an embodiment, the pharmaceutical composition is used in a method of treating a viral infection, and the viral infection is caused by NEKM14 (human rhinovirus type 14), K5M (human respiratory syncytial virus) and/or NIMI (influenza A NIMI virus).

One dose of the pharmaceutical composition preferably contains about 0.5 mg of thyrothricin, about 1.0 mg of benzalkonium chloride and/or about 1.5 mg of benzocaine and one or more

From pharmaceutically acceptable excipients. In this context, the term "about" means a range of deviations of 250 95, preferably 20 95, most preferably 10 95 with respect to the mass of the active substance given above.

The pharmaceutical composition according to this invention is administered in a daily dose of approximately 2-4 mg of thyrothricin, 4-8 mg of benzalkonium chloride and 6-12 mg of benzocaine.

The present invention will be further explained with reference to the following examples of implementation, in which the effects of the composition according to the present invention are explained.

Implementation examples 1. Introduction

Viruses and bacteria are pathogens of widespread acute pharyngitis. This disease, also known as catarrhal pharyngitis, is an inflammation of the mucous membrane of the pharynx and lower pharyngeal structures, which is accompanied by typical symptoms of pain on swallowing, scratching, burning, dryness of the throat and redness of the mucous membrane of the pharynx (1).

Acute pharyngitis requires primarily symptomatic treatment to relieve pain.

Antibiotic therapy is less common and should be used only in 10-15 95 patients with streptococcal pharyngitis (1-3) or in cases where a late complication such as hemorrhagic fever is to be prevented. Sore throat lozenges such as doritricin have been shown to be clinically successful in symptomatic treatment (4). The combination of antibacterial and analgesic agents: thyrothricin, benzalkonium chloride and benzocaine, contained in it, effectively relieves the typical symptoms of pharyngitis (4).

Interestingly, the individual components of this combination are not only effective against bacteria, but, as described by the inventors herein, they are also active against viruses. It is questionable whether the combination of active ingredients contained in doritricinief) has an antiviral effect in aggregate. With this in mind, the study below was conducted to examine the dose-dependent antiviral effect of doritricin sore throat tablets? Syiasis in relation to human-pathogenic ip miigo viruses. 2. Materials and methods

Doritricinf tablets (MEBISE, Iserlon, Germany) in solution were studied in different degrees of dilution in relation to viruses that cause respiratory tract infections in humans. One tablet contains 0.5 mg of thyrothricin, 1.0 mg of benzalkonium chloride and 1.5 mg of benzocaine. The following viruses were evaluated: human rhinovirus type 14 (NKM14, non-enveloped RNA virus), respiratory syncytial virus (K5U, paramyxovirus, non-enveloped RNA virus) and influenza A virus

NIM! (RISHA, NIM, orthomyxoviruses, enveloped RNA virus).

Doritricin tablet to obtain the test solution? first crushed, and then dissolved in 1 ml of dimethylsulfoxide (OM5O), diluted with distilled water 1: 100 and sterilized (using a filter with a cell size of 0.45 μm). A series of filtrate dilutions with a pH adjusted to 7.4 was prepared step by step with a dilution of Iod2 or Iod10 using a complete nutrient medium. The solvent prepared according to the described procedure (0OM50O) was used as a solvent control.

In order to exclude artifacts due to the effects of the test solutions on the cells for the study of antiviral activity, which reduce viability and metabolism, the test solutions were first subjected to the study of viability in cell culture. In parallel approaches, the investigated solutions at all stages of dilution, as well as the control with a solvent, were added to growing Neba virus-sensitive, MOSC, and HEr-2 cells and cultured in total for 3 days (NeHa, MOSC) or 6 days (HEr- 2) at 377" Si 595 002. As an additional control, only cells cultured with a medium for cell cultivation were included in the cell culture. Assessment of possible effects on the reduction of viability was carried out on 1, 2, 3 and 2 days after the introduction of the substance using MT T-test: Treated cells were incubated with MTT solution for 45 minutes (Hep-2, Nega) and 60 minutes, respectively.

The reduction of the yellow dye 3-(4,5-dimethylthiazol-2-yl)y-2,5-diphenyl-tetrazolium bromide (MIT) in solution to blue-violet formazan can only be carried out by intact living cells, and it is proof of cell viability. After solubilization of formazan crystals in 195 OM5O, the optical density of cell culture supernatants was determined photometrically at a wavelength of 570 nm. A higher optical density indicates a higher number of viable cells. Moreover, a possible change in the morphology of the cells was assessed using microscopy on the 3rd day (NeHa, MOSC) and 6th day (НЕр-2) after the addition of the substance.

The OO values of the test samples for each approach were normalized to the OO values of untreated controls, which was defined as 10095 viability, and displayed as percentages accordingly. Thus, it is possible to determine the average inhibitory concentration (IC50) for samples from several parallels in the form of dose-effect curves for the test samples up to a concentration that did not show any deterioration of cell viability caused by the test substance and, therefore, was suitable for application in studies of antiviral activity.

After viability studies, antiviral activity against NKM14, RItsA and

KZM Virus-sensitive cells were infected with the corresponding viruses at an average infectious dose (MI) from 0.0004 to 0.0008 (NeHa were infected with NKM14; MOSCs were infected with RiIshA; HEr-2 were infected

E5M). Virus-infected cells were treated 1 hour after infection with the tested solutions in physiological concentrations of substances in a semipermeable agarose supernatant.

On the 2nd day (RiIshA), on the 3rd day (NEM14) and on the 5th day (K5M), in accordance with the approach to the study, viral plaques (plaque-forming units/ml; RES/ml) formed in the cell layer were counted using the reading system of AIO EiYibroi (Ashoiittype Oiadpovika strN, Strasberg,

Germany) and evaluated as percent inhibition of plaque count, defined as complete virus infection in a control approach that does not involve the addition of any active ingredient.

In addition to virus control and negative control, the antiviral active ingredient virazol? ribavirin, which is an antiviral agent against RNA viruses, was used as an internal laboratory control to verify the study procedure described above. The effectiveness of the reference substances was confirmed in the test system. All controls were exposed to all culture conditions for antiviral activity assays. 3. Results

For the study of antiviral activity, the investigated solutions were used, with concentrations of active ingredients corresponding to the degree of dilution of the tablet doritricinaef), which is 1: 400 or higher (table 1). With these concentrations of active ingredients, no viability-decreasing effects were detected on the NeHa, MOSC and HErg2 cells used in the viability study.

As a result, the combination of the active ingredients doritricinaF in a dilution of 1: 100 and 1: 400 showed a comparable antiviral effect and significantly reduced the number of plaques of RNA viruses 60 HEM'14, NIMI and E5M by 97 95 and 68 9", 53 9b and 74 95, in accordance. This effect on plaque formation decreased with increasing degree of dilution. Only at high levels of dilution, the effect was no longer evident: for NEM14 from 1: 3200, for K5M from 1: 6400 and for NIMI from 1: 64000.

The relative inhibitory effect, which depends on the degree of dilution of doritricinaf, is shown in fig. 1.

The synergistic effect of C active substances is proven by the same series of experiments with individual substances, which demonstrate only very weak antiviral activity at a dilution of 1:100 (Fig. 2, C, 4).

Ribavirin (KIVA), used as a control, showed a reduction in the number of viral plaques of 53 95 in NKM14 (RKIVA 10 μg / ml), 96 95 in RISHA (RKIVA 3 μg / ml) and 97 95 in KM (KIVA 1 μg / ml).

The underlying mechanism of action was studied in additional experiments. It was evaluated whether it is based on a synergistic effect, and which of the three components and on which viruses has the main effect.

Table 1

Concentrations in μg/ml of active ingredients that are used in the study of antiviral activity and belong to the components of DoritricinaeF

Ingredients doritritsinyaye Z | 77777777 4. Conclusion

In addition to the known antibacterial and analgesic effects, the pharmaceutical composition according to the present invention is also characterized by pronounced dose-dependent and synergistic antiviral properties against CM viruses,

NEM14 and NIMI. 5. Sources of information: (1) Roviau-Wate KM. Iptesiop5 ip rediayisv: oi api pem/ disease. Zmiz May 5 UUKIu 20127142:m/13654. (2) Vivpo AG, Segreg MA, Smaypeu UM, "g., Kariap EG, Zspugagi: VN. Rgasiise dcideiipev Tog te diadposiv apa tapadetepi oi dor A zigeriosossa! rpagupaoiiv5. IpTesioiv Oizease5 Bosieiu oi

Ategiisa. Siip Iptesi biz 2002 di! 15;535(2):113-25. (3) 5pytsItap 5T, Vizpo AI, Syed9 NMU, Setbeg MA, Kariap EI, Gaye S, Yei AI. Siipisa! rgasiise diyideiype Tog ype adiadpo5i5 apa tapadetepi oi dgoir A 5igeriosossa! rpagupoiv: 2012 iraaie ru She

Intesiioiv Rizeazev 5-axis of Ategis. Siip Iptesi Biz 2012 Mom 15555(10):1279-82.

Ko) (4) Zspoiep T, Rashieg M, Kobeg S. Oogiingisip bei aksheg Rpagupadiiiv. Oetsivsne AroipeKeggeshipd

Claims (6)

2005:;145(1):93-4. FORMULA OF THE INVENTION 1. A pharmaceutical composition containing: - thyrothricin, - benzalkonium chloride and - benzocaine, for use in antiviral therapy or in the treatment of viral infection. 2. A pharmaceutical composition for use in the method of treating a viral infection according to claim 1, and the viral infection is caused by a rhinovirus, influenza viruses and/or pneumoviruses. 3. Pharmaceutical composition for use in the method of treating a viral infection according to claim 1 or claim 2, and the viral infection is caused by NEKM14 (human rhinovirus type 14), CM (human respiratory syncytial virus) and/or NITMI (influenza A NIMI virus). 4. A pharmaceutical composition for use in a method of treating a viral infection according to one or more of the previous items, containing: approximately 0.5 mg of thyrothricin, approximately 1.0 mg of benzalkonium chloride, approximately 1.5 mg of benzocaine per dose and one or more pharmaceutically acceptable excipients. 5. A pharmaceutical composition for use in the method of treating a viral infection according to one or more of the previous items, which is a tablet for sore throat. 6. A pharmaceutical composition for use in the method of treating a viral infection according to one or more of the previous items, and the specified composition is administered in a daily dose of approximately 2-4 mg of thyrothricin, 4-8 mg of benzalkonium chloride and 6-12 mg of benzocaine. ibutivyzhuszhusna zakonenisty dovitvyat VA fivsi Z zktvyni inguezients Z ! wai and se Notebook СЬ4--А- - - - F 5 F F F F F « F « « F 6 E E E 2 E E E E E E E E E E E E E E E E - 2 - « - -4e- both "fe NAU ZA sh y: s-- -. I'm from VU! 5 le what she in J and se Ser nd freon, I a day. Щ Sonya ana -- and tk ХО Yakob Z КЯДО кор о тхЗО зкю лк Eozviveda Fg.i shhe and PM Protivshusna zIinJENET terOotritsna separate p. -- than I «o same VVU 5 eh y 3 oo Sh E o den pi pi otv tk NE iha00 How 100 with Zk Zk, Breeding I.