Using compounds of antiviral class for preparing agent for treatment and prophylaxis of respiratory tract viral infection
FIELD: medicine, virology, pharmacy.
SUBSTANCE: invention relates to using derivatives of dithiocarbamate of the formula: R1R2NCS2H and oxidized forms of these compounds, in particular, their dimmers and their pharmaceutically compatible salts for preparing an agent used in treatment or prophylaxis of infection caused by RNA-containing viruses that damage respiratory tract and inducing disease. Also, invention relates to a disinfecting agent containing dithiocarbamate compound and a method for disinfection of surfaces, media and cell cultures.
EFFECT: valuable medicinal properties of compounds.
19 cl, 14 dwg, 14 ex
The technical field to which the invention relates.
The invention relates to the use of compounds of dithiocarbamate, the sanitizer air and the method of disinfection of surfaces, environments and cultures of the cells.
The level of technology
There are many viruses that cause diseases of the respiratory tract of humans and mammals. Although these respiratory pathogenic viruses and may vary structurally and belong to different virus families, but common to them is that they can penetrate the body through the respiratory tract and affect, for example, specific cells of this tract, such as the epithelial cell layer of the respiratory tract, alveolar cells, lung cells, etc. are Common and classic symptoms of influenza infection, characterized by local inflammation and symptoms in the respiratory tract (such as runny nose, hoarseness, cough, vesicles, sore throat).
Viral infections, primarily in the respiratory tract, causing pathological changes in the respective cells, particularly in cells of epithelia due to oxidative stress. Reactive intermediate forms of oxygen (reactive oxygen intermediates; ROIs), produced, for example, white blood cells, pulmonary epithelial cells or xanthine-oxidase, act as intermediaries for such cleto is different, caused by viruses violations. During this oxidative stress can occur activation of oxidant - specific transcription factor NFkB (nuclear factor-kB). This factor NFkB has been detected in various cell types and was installed its causal relationship with gene activation in inflammatory and immune responses.
Antioxidants can block the activation of NFkB, catching reactive intermediate forms of oxygen, causing the activation. Therefore, it was proposed to apply antioxidants primarily in the treatment of infections with latent viruses; however, it was found that effective treatment of such latent infections only one antioxidant is impossible, and what is required, if any, conduct, combination therapy using a mixture of different (i.e. it has a different action) antioxidants and other virusology funds (US 5, 686, 436). However, inhibition of viral replication or even virus infection via antioxidants to date has not been reached, in particular, inhibition of infections caused by influenza viruses and picornaviruses (Knobil et al., Am. J.Physiol. 274 (1) (1998) (134-142).
On the other hand, different proposed for the treatment of viral infections antioxidants are very different among themselves in relation to antioxidant activity. So, for example, L-ascorbin the th acid and vitamin E protect glutathione; vitamins K, a and E act as antagonists of peroxynitrite and other strong oxidants in the body. Anti-inflammatory steroids, neoglycoconjugate lazaridi, dithiocarbamate and N-acetyl-L-cysteine described as inhibitors of NFkB activation.
Depending on the carrier of the genetic information of viruses can be divided into DNA viruses and RNA viruses, and nucleic acid present in a single or double strand and surrounded by a protein shell.
This single-stranded RNA such RNA viruses is present either in the form of a positive circuit (mRNA), or as negative. In addition, the genetic information of the virus can also be represented in the form of several fragments, as is the case, for example, in influenza virus.
The human rhinoviruses (HRV), related to the picornaviruses, are the primary cause occurring in the world of cold. Frequent manifestation of HRV, the risk of severe secondary infections, and economic consequences, as expressed in medical costs, doctor's visits, the level of disease workers, doing HRV important and dangerous pathogen diseases. Despite frequent manifestation, at the present time is not effective against this viral disease, not including treatment of symptoms. On the other hand, the consequences of, for example, from infection caused by renover the catfish, are not as severe or even life threatening, so had to put up with medicines that have a high risk of side effects. So the funds that you want to apply against such viruses must have only minor side effects or does not have. To the group of picornaviruses animals include virus And equine rhinitis (ERAV), which, like the virus belongs to the genus of aphthovirus.
Other important families of viruses responsible for diseases of the respiratory tract, are Orthomyxo - and Paramyxoviridae virus human influenza as the most important representative.
The emergence of new pandemic strains of influenza is usually associated with new subtypes containing the new hemagglutinin or neuraminidase gene. These new viruses differ immunologically from pre-existing influenza viruses. In order to become infectious, the virus and influenza requires 8 segments of RNA viruses With influenza - only 7. Viruses a, b and C influenza can form in vivo homotypic varietate, but not between types. Theoretically, from the corresponding 8 segments of the two viruses And the flu can be formed 256 varieties. However, such random segregation does not occur because the protein levels of some proteins need a partner, due to his ..... the IOM. It has been specifically shown with respect to the virus Avian Influenza-Virus A/chicken/Germany/34 (H7N1) FPV Rostock, which is encoded by the segment 4, capable of forming a functional virus only specific with your specific strain of the M2 protein encoded by segment 7 (Grambas, S., Hay A.J.Maturation of Influenza A virus hemagglutinin-estimates of the pH encountered during transport and its regulation by the M2 protein. Virology 1992; 190: 11-18) (Grambas, S., Bennet H.S., Hay A.J. Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses. Virology 1992; 191: 541-549). One of the main strategic measures in the annual combating viral infection of influenza in a population is vaccinated against influenza. However, despite extensive program of vaccination influenza remains a cause of morbidity and mortality worldwide and a major cause of disease and death in patients with immunocompromised and elderly persons. Antiviral activity of amantadine and rimantadine reduces the duration of clinical symptoms of influenza, however, described the important side effects and the emergence of resistant mutants (FIELDS et al., Virology, 3. Edition (1995) Lippincott-Raven Publ., Philadelphia, Vol.1, p.434-436). Currently on the market there is a new group of antiviral products, which inhibit the neuraminidase of influenza virus. Zanamivir and oseltamivir are, for example, neuraminidase inhibitors viruses and influenza; however, these drugs only reduce the duration of a symptom of the century
In the US 5686436 described a method of suppressing the reproduction of retroviruses and latent viruses in humans and animals, such as, for example, non-human influenza virus (HIV), when this medicine is injected, containing, among other things, antioxidants and induction inhibitors of factor NFkB. In contrast to the effective suppression of infections caused by respiratory viruses, it is necessary to effectively fight the virus is already in the acute phase of infection; treatment, which can only be performed on the stage of latent viruses, is unsuitable for the prevention or treatment of acute viral infections of the respiratory tract.
Thus, there remains a need in high-performance active substance against viral infections of the respiratory tract, in particular, of the person, which would not at all or only insignificantly adverse effects would be cheap and could be produced in large quantities.
The present invention is solved through the use of compounds of dithiocarbamate with the structural formula R1R2NCS2H, where R1and R2denote, independently of one another, an unbranched or branched alkyl with 1-4 carbon atoms or form together with the nitrogen atom an aliphatic ring with 4-6 carbon atoms, and R1, R2or aliphatic number is CH optionally substituted by one or more substituents, selected from HE, NO2, NH2, COOH, SH, F, Cl, Br, I, methyl or ethyl, and oxidized forms of these compounds, in particular, their dimers, as well as their pharmaceutically compatible salts to obtain funds for the treatment or prevention of infection caused by RNA viruses affecting the respiratory tract and causing it to disease. Under "respiratory tract" means in the context of the invention all the organs and parts, starting with the holes in the body (nose, mouth, eyes (including lacrimal canadian) and ears) to the pulmonary vesicles. While pharmaceutically acceptable salts are, in particular, Na, K, Ca, Mg, NH4, Zn. For the first time it has been unexpectedly discovered that compounds of dithiocarbamate according to the invention may be effective against infections caused by RNA viruses affecting the respiratory tract and causing his disease named in the framework of this proposal, "respiratory RNA viruses." Contrary to observations Knobel et al. (Am. J.Physiol. (1998), str-142), in the framework of the present invention clearly shows the antiviral effect of the compounds of dithiocarbamate according to the invention against infections caused by respiratory RNA viruses, for example, HRV (human rhinoviruses and influenza viruses. This is all the more surprising that other antioxidants such antiviral effect against respiratory RNA viruses poblador and that changes in redox potential is not solely responsible for the antiviral effect of the compounds of dithiocarbamate, according to the invention. For example, according to the invention can clearly show that the antioxidants: vitamins C and E, 2-mercaptoethanol and N-acetyl-L-cysteine absolutely have no effect against respiratory RNA viruses. Additionally, the effectiveness of the compounds of dithiocarbamate according to the invention in respect of infections caused by respiratory RNA viruses, and reproduction of these viruses is not explained solely by inhibition of NFkB activation, since it was found that by using the compounds of dithiocarbamate according to the invention, in the framework of this proposal under them must also understand the oxidized form, in particular, dimers, is purposefully stops reproduction respiratory RNA viruses.
In DE 1963223 AS disclosed agent for the treatment of viral infections of the brain, and this tool should include an inhibitor of the biosynthesis of monoamines norepinephrine, dopamine and 5-hydroxy-tryptamine. Provided in this publication, the example indicates complex α-methylthiophenethylamine ether in mice with a simplex infection herpes. In the manner described in this publication, the mechanism of inhibition of the biosynthesis of special monoamines applies only to treat infections caused by DNA viruses in the brain. Treatment for respiratory RNA viruses according to the invention is carried out on a different principle and it is not applicable for viral infections of the brain, as evidenced by the negative examples in relation FSME (example 13) and EMC (example 14). Therefore, DE 1963223 relates to other fields of application, cannot be compared with the intended use according to the invention and thus is not a close analogue.
In J.G. Calvert, Interferon Research 1990 (10), p.13-23, described the effect of diethyldithiocarbamate (DDTC) at mengovirus, it was found that the DDTC inactivate virions mengovirus. However, mengovirus are the causative agents of severe encephalomyocarditis not affect the respiratory tract and does not provoke in him of the disease.
In WO 95/03792 A1 reports the use of compounds of the thiol to obtain a pharmaceutical composition for the treatment of viral diseases, and the thiol compound destroys the disulfide bridges in proteins of viruses. In this document, among the many viruses are also referred RNA viruses, in particular, Picornaviridae. It also provides many examples on tirinya connection, in particular, is mentioned in quite General terms dithiocarbamate. From many different combination possibilities are only given examples of the following: as tylnej compounds: N-acetylcysteine (NAC), cysteine, systeemiteoria and complex N,S-deacetylation-ethyl ester (DACEE), as viral diseases are only hepatitis b virus and smallpox virus rabbit.
Consequently the nutrient not only were the examples for the most part, compounds and viral diseases, but it was discovered that disclosed in this publication, the treatment does not really meet in full its description: some picornaviruses (those not strike the respiratory tract and nerve cells) by PDTC not oppressed and their reproduction is not reduced. Therefore, not all combinations are triggered by viruses, diseases and tylnej compounds achieve the goal, and the selected application according to the invention compounds of dithiocarbamate that WO 95/03792 A1 is not disclosed for the treatment or prevention of infections caused by respiratory RNA viruses, is not obvious.
In GB 861043 And reported compositions used among other things to protect against viruses. These compositions contain, for example, dithiocarbamate, however, specific viruses are not here disclosed.
Knobil et al., Am. J.Physiol. (1998), str-142, report on the study of oxidants and their impact on gene expression, caused by a virus. It was clearly established that neither NAC nor PDTC does not inhibit the infection caused by the influenza virus, or replication.
In DE 2555730 AND described an antimicrobial agent containing the compound of dimethyldithiocarbamate, and this compound is a complex of 8--metal-N,N-dimethyldithiocarbamate. But in this publication reported only about antifungal and antibacter the real action.
In WO 99/66918 A1 deals with the application of a disulfide derivative dithiocarbamato for reduction of NOx in a patient and for inhibition of NFkB. However, this volume provides an extensive list of illnesses without the detailed descriptions of viral diseases.
Flory et al., J. Biol. Chem., 24.03.2000, 275 (12), str-8314, report on the study of the influence of different proteins of the virus And influenza activation-dependent NFkB expression.
Tai D.I. et al., Hepatology, March 2000, 31 (3), str-787, report on the study of the inhibition of NFkB activation using PDTC, and it was assumed that the infection is caused by HCV virus could cause antiapoptosis processes due to the activation of NFkB.
Schwarz et al., 1998 (J.Virol; Vol.72 (7), str-5660), investigated the effect of NFkB on the multiplication of the virus encephalomyocarditis (EMCV), related rhinovirus person. In cells without NFkB (shock separation (knockout) p50-/- or P65-/-), which were infected with EMCV virus, the replication of the virus, although it has declined, but there was increased cell death from apoptosis. This is in sharp contradiction with the data presented here: these examples clearly show that the compounds of dithiocarbamate according to the invention not only inhibit replication related rhinovirus, but also prevent caused by a virus cell death. Therefore, inhibition of NFkB is not the decisive factor for the effectiveness of compounds ditioner the Amata according to the invention.
According to the invention antiviral effect of the compounds of dithiocarbamate also does not depend on combinations of certain substances. Connection dithiocarbamate according to the invention can be used completely independently from other additives, in particular, from the antioxidants that is absolutely necessary for US 5686436 to achieve antiretroviral action of inhibitors of NFkB activation, as it was unexpectedly found that the antiviral action not only helped to overcome oxidative stress, but also due to the connections dithiocarbamate according to the invention it has become possible to suppress the infection/replication.
It was shown that the compounds of dithiocarbamate according to the invention induce genes that act as transcription factors, inducing antioxidants (Meyer et al., EMBO J. 12, 2005-2015, 1993). You can induce a heterodimeric transcription factor API using N-utilzation (NAC) and compounds of dithiocarbamate according to the invention, which leads to DNA binding and TRANS-activation. Activation of AP1 connections dithiocarbamate according to the invention depends on protein synthesis and involves the transcription of genes c-jun and c-fos. However, only the activation of AP1 does not provide a reliable inhibition of the virus according to the invention.
Pyrrolidin-dithiocarbamate (PDTC) is already known in ka is este Pro - and antioxidant, an inhibitor of the activation of the transcription factor NFkB, zinc ionophore and metal halinowego agent. Sherman et al., Biochem. Biophys. Res. Comm., 191 (3): 1301-1308, 1993, described PDTC as an inhibitor of activated factor NFkB and NO synthesis. In WO 01/00193 A2 reported compositions containing diethyldithiocarbamate in the Pico - and nanomolar range, which is characterized by selenopyran action against apoptosis.
It was shown that the compounds of dithiocarbamate according to the invention have selenopyran action against RNA viruses - respiratory RNA viruses that infect the respiratory tract and provoking in him the disease, both in vitro and in vivo.
Thus, according to the invention, the infection warned already at a very early stage, before significant cellular damage or even death.
In the framework of the present invention under respiratory RNA viruses include any viruses of humans and mammals, affecting the body through the respiratory tract, i.e. the respiratory tract and lungs, and penetrating into it, while they cause respiratory tract disease. Obviously the biological processes occurring during such infections are so similar that the effect of the compounds of dithiocarbamate according to the invention is equally effective despite the biological heterogeneity of this group of viruses. what, however, was marked and return the result according to which for other viruses penetrated the body other infectious tracks and past other biological cycles (for example, able to integrate into the host genome in the form of latent virus) or causing disease in other organs, e.g. the brain, the action of only one connection dithiocarbamate according to the invention is insufficient for the treatment of viral infections.
Meanwhile, in this regard, it was shown that in the treatment of patients suffering from AIDS, dithiocarbamate the improvement of patients or their cure is not achieved (Multicenter, randomized, placebo-controlled study of dithiocarb (Imuthiol) in human immunodeficienty virus-infected asymptomatic and minimally syptomatic patients. The HIV87 Study Group. AIDS Res. Hum. Retroviruses 1993 Jan.; 9(1): 83-9). On the basis of these results, further clinical studies of latent adventitious virus assays by CPE was not conducted (see US 5686436).
Its special action connection dithiocarbamate according to the invention are primarily at the early stage of viral infection or, if the reception is held prior to the infection. Thus, the compounds of dithiocarbamate can prevent the beginning of a viral infection, if they are prophylactic administration of, for example, in areas and at times when there is a risk or even an increased risk of respiratory viral infections, for example, in areas where there are epidemics or deducted PR the term cold. It is preferable to apply at this link dithiocarbamate according to the invention for the prevention of viral infection.
However, it is most preferable to carry out the inhibition of the virus according to the invention primarily on early stage already begun infections caused by respiratory RNA virus. In this connection dithiocarbamate according to the invention is purposefully used for inhibiting viral replication, i.e. at the moment when the infected individual has not been significant damage. The result can be prevented not only the consequences of progressive viral infection in the individual, but also the subsequent spread of infectious viruses among other individuals; the risk of further contamination is minimized, which is of great absosmurfly effect and a great value especially in respect of the virus of human influenza.
In particular, respiratory RNA viruses in the framework of the present invention are: rhinoviruses, Coxsackie virus, Echovirus, coronaviruses, enteroviruses, orthomyxovirus person (for example, influenza virus (a, b and C), paramyxoviruses (e.g., parainfluenza virus and pneumovirus), respiratory virus sentice (RSV) and other RNA viruses, while they are able to cause disease (at least) in the respiratory tract. Viruses Ilyich strains, cause disease in the respiratory tract, and in another organ such as the brain, such as viruses meningitis, encephalomyocarditis, poliovirus, cardiovirus and others, the subject of this application are not. Respiratory RNA viruses according to the invention are inherent in the infection of epithelial cells of the upper and lower parts of the respiratory tract. In addition, can also affect other organs. The resulting local inflammation of the respiratory tract is the main reason for the appearance of typical symptoms of flu such as runny nose, sore throat, hiplot, cough, vesicles and often fever. Also frequent manifestation of secondary infections in immune-weakened individual contributes to the infection caused by these respiratory viruses.
In the framework of the present invention under the picornaviruses include any "real" picornaviruses in accordance with currently accepted classification of the Picornaviridae based on King et al. ("Picornaviridae" in "Vims Taxonomy, Seventh Report of the International Committee for the Taxonomy of Viruses" (2000), Eds. Van Regenmortel et al., Academic Press 657-673), if they affect the respiratory tract and cause in it the disease, i.e. genus: enterovirus, rhinovirus, aphtovirus, parechovirus, arbovirus, comovirus and Echovirus. These viruses of the family Picornaviridae features: similar genetic structure, protein composition, especially cultiver the cation and temperature resistance and anti-virus tools.
It was found that the present invention is particularly applicable for destruction of pathogenic for humans and animals representatives genuine respiratory Picornaviridae, in particular, from the family of antivirus (antivirus 70, 71, Coxsackievirus), rhinovirus (for example, rhinovirus person) and aphthovirus (e.g., virus), in contrast to other viruses, including picornaviruses, causing latent infection, such as HAV, the inherent advantages of the invention were not confirmed. This is probably due to the fact that the group of "real" respiratory viruses is itself such a homogeneous and pathophysiological processes infections so similar that connection dithiocarbamate appear to act the same way.
Under "viral infection" in the context of this application refers to any attack respiratory virus on cells containing, for example, one of the following: consolidation of viral particles in the cell, the introduction at a later stage of the genetic information of the virus into the cell, the production of new viral particles and expression of infectious virus particles.
Additionally, you may also use oxidized forms of such compounds, in particular, their dimers, since the latter, as is known, quickly converted at the exchange of substances in the body with the formation of reduced forms. Within n the standing applications such compounds should be understood as "oxidized form", the balance of S are oxidized. A preferred example of such oxidized dimeric form is disulfiram-tetraethyleneglycol (C10H20N2S4), also known as "Antabuse" or "Abstinent". Tetradecyltrimethyl itself known, and it is used particularly in the treatment of alcoholism: it mediates ocalenia-restorations and deactivates the aldehyde-dehydrogenase. When receiving ethanol is the concentration of acetaldehyde in the body, which has an extremely negative impact on overall health: while taking disulfiram and alcohol appear fears, nausea, loss of vision, pain in the chest, head, etc. and these symptoms are kept for 3-4 days and even weeks. So disulfiram assigned alcoholics as a therapeutic agent, as any subsequent binge drinking should be prevented such strong negative effects.
Other known action of disulfiram are the inhibition of enzymes such as, for example, fructose, 1,6-diphosphate-dehydrogenase, xanthine oxidase, glucokinase, aldehyde oxidase and dopamine-β-hydroxylase.
In addition disulfiram is used for the treatment of pediculosis, scabies and Nickel dermatitis.
IU is abolism disulfiram already analyzed in detail, see, for example, Dollery C., 1999, Therapeutic Drugs, Second Edition, Vol.1, Churchill Livingstone, Edinburgh. Described that the diethyldithiocarbamate is a major metabolite of tetraethylthiuramdisulphide in the body, and this transformation happens very quickly.
The oxidized form of the compounds of dithiocarbamate according to the invention are soluble in oil and can be used, for example, as a vehicle for oral administration, and in the stomach compounds are absorbed. This connection can be used, in particular, in the form of powder aerosols. Additionally, the oxidized form of the compounds according to the invention can be provided with hydroxyl groups to enhance water solubility and retrieve aerosol form. If connection dithiocarbamate according to the invention prepared in the form of oxidized, in particular, dimeric, drug, this tool will have a depot effect, i.e. compounds of dithiocarbamate according to the invention will be gradually deposited in the body for a certain period of time and then it will be absorbed. To obtain such a depot oxidized connection dithiocarbamate can be implanted in a known manner in the body to be treated.
A preferred compound according to the invention is characterized by the fact that R1and R2represent, independently from each other, alkyl with 1-3 tomanipulate or form together with the nitrogen atom an aliphatic ring with 4-6 carbon atoms. It has proved to be particularly positive in the treatment and prevention of infections caused by respiratory RNA virus.
It is preferable to choose the connection dithiocarbamate of pyrrolidin-dithiocarbamate (PDTC) and N,N-diethyldithiocarbamate (DDTC). These compounds are characterized by a pronounced activity against infections caused by respiratory RNA viruses.
Especially preferred for this infection, caused by picornaviruses, orthomyxoviruses or paramyxoviruses. Connection dithiocarbamate according to the invention are particularly active against infections caused by these viruses.
Preferably, orthomyxovirus was a non-human influenza virus, originating in particular from the group consisting of viruses And influenza, paramyxovirus, parainfluenza virus or pneumovirus. Preferably, orthomyxovirus was a virus And influenza mammals, and picornavirus - rhinovirus, in particular, rhinovirus human or a horse, enterovirus, in particular, enterovirus 70, 71 or Coxsackie virus or aphtovirus, in particular, the virus or virus And equine rhinitis. In this case, as already explained above, exclude such viruses and their strains that cause no disease in the respiratory tract, as for example in the brain or nerve cells, for example, cardiovirus and the and the polioviruses, related picornaviruses. Such viral infections particularly effective in the cure or prevention of a connection according to the invention.
The family Picornaviridae covers a number of small RNA viruses, including rhinoviruses (e.g., human rhinovirus), enteroviruses (e.g., enterovirus 70, 71, Coxsackievirus, which, for example, the causative agent of hand, foot and mouth), aphthovirus (for example, the FMD virus, and in this case the disease is caused in the mouth, as well as virus And equine rhinitis). In relation to, for example, FMD is particularly interesting to note that by using the compounds according to the invention it is necessary to bridge the gap of time between vaccination and the occurrence of vaccine efficacy (about 10-12 days), because during this time the vaccinated animals remain benzamidine and probably after infection emit large amounts of virus, they would not while becoming ill. Therefore, the connection according to the invention could be introduced animals simultaneously with vaccination and may last for about 2 weeks after that.
The names of orthomyxo - and paramyxoviruses assigned after the division of the former collective designation of influenza viruses and other similar viruses. Human paramyxoviruses cause measles, mumps, respiratory and neurological disease is. For paramyxoviruses include, among other things, parainfluenza virus, mumps virus, the virus pseudocode birds, respiratory virus syncytium (RSV), measles virus and cowpox plague. To orthomyxoviruses include, among other things, the viruses a, b and C, which cause human influenza.
Influenza viruses of type a is responsible for more outbreaks of the flu and all of a pandemic. Although influenza a viruses and are found among horses and pigs, as well as among birds, for example, as the causative agent of classical avian plague, but only influenza viruses of humans and mammals, for example, horses can be thought of as respiratory viruses in the truest sense of the word, because the biology of the virus of bird flu (avian influenza (AI)) is completely different from the biology of the virus of human influenza. So the AI virus cannot be considered as a respiratory virus. The ducks AI virus initially replicates in the intestinal tract, which in humans is not happening. As a result, the AI viruses can be isolated from faeces of birds (Hinshaw et al., 1980, Canad. J.Microbiol. 26, 622-9). In addition, the rate of change of the nucleotides of AI viruses below the same speed of the viruses that can be isolated from mammals. Evolution of viral proteins in organisms other than the body of the bird shows, as a rule, the rapid accumulation of mutations that AI viruses found (Gorman et al., 991, J.Virol. 65:3704-14; Ludwig et al., 1995, Virology 212: 555-61). Receptor specificity varies between different influenza viruses. Most AI viruses is principally associated with receptor A2-3-galactose-sulinowo acid. In contrast, the human influenza virus primarily associated with the receptor of A2-6-galactose-sulinowo acid (Rogers + Paulson, 1983, Virology 127, 361-73; Baum + Paulson, 1990, Acta Histochem. Suppl. 40: 35-8).
Because the viruses according to the invention cause in humans and mammals a number of common diseases, anti-virus effect connection dithiocarbamate according to the invention is of particular importance in the light of these viruses. Given that connection dithiocarbamate according to the invention have a pronounced activity against these viruses, these compounds are particularly suitable for the production of a variety of tools for the treatment and prevention of such viral infections. Connection dithiocarbamate according to the invention can be manufactured in large quantities easily and cheaply, and even in elevated concentrations almost do not have a toxic effect on being treated cells.
According to a preferred variant of the connection of dithiocarbamate according to the invention are contained in the medium at a concentration of 0.01-5000 mm, preferably 1-300 mm, particularly preferably 10-100 mm. Under such con is intratech connection dithiocarbamate according to the invention are particularly effective against infections, caused by respiratory RNA viruses, and almost or completely do not have side effects. The applied concentration is selected depending on the exposed treatment of viral infection, its intensity being treated organism, e.g. a human or an animal, and age.
Especially optimally to the concentration of the compounds of dithiocarbamate according to the invention in the medium was 10 mm-1 M In this case, the connection dithiocarbamate according to the invention are present in a highly concentrated form, and the tool may be diluted before treatment to the desired concentration.
Preferably, the medium contained in addition pharmaceutically compatible carrier. This can be used any known specialist in the field of pharmacy media, such as a saline phosphate buffer (PBS) or otherwise bateriafina solutions of salt or composition with the content of the liposomes, and in this case, the carrier may be chosen based on treatment, viral infection and are subject to the treatment of the body.
Preferably, the tool could be administered orally, through the nose, intravenous, parenteral, rectal or had the appearance of eye drops or ear drops, mouthwash or spray. This route of administration depends in particular on the subject of treatment is viral infection. Infection in the respiratory tract can be treated, for example, by insertion through the nose, for example, in the form of aerosols containing compounds of dithiocarbamate as the treatment of viral infections is the place to virus attack. Depending on the application selects a specific concentration of the compounds of dithiocarbamates or tool contains additional substances that are effective in this form of application. It is also possible to apply the tool in a dry form, and before treatment it is diluted with an appropriate solvent.
Particularly effective use is achieved in the case when the tool provides additional antiviral agents. Thus it becomes possible to inhibit a viral infection in the respiratory tract with multiple parties and at the same time can reduce or completely destroy a whole set of different viruses. Such additional antiviral agents are, for example, substances that suppress the replication, immune-boosting substances, neutralizing antibodies, etc. and if necessary, substances that can boost overall immune system.
Preferably, the tool contains a combination of at least two different dithiocarbamates according to the invention, in particular, a mixture of pyrrolidin-dithiocarbamate and N,N-diethyl-dithiocarbamate. Link is dithiocarbamate according to the invention is performed in the cells the function of Pro - and antioxidants. Their antioxidant action involves the removal of hydrogen peroxide, superoxide radicals, peroxynitrite, hydroxyl radicals and lipid products perechisleniya. Due to this removal of the oxidation of dithiocarbamates in turondale. Turangalila responsible for procyclically actions dithiocarbamato, and in some cases the formation of Turnov depends on the presence of metals. Described that the anti-apoptotic activity of dithiocarbamato likely is determined by the inactivation of caspase caused by oxidation of the thiol.
Particularly preferably, the tool according to the invention contain additional substances selected from antibiotics, vaccines, immunosuppressants, stabilizers, immunostimulatory agents, blood products, or mixtures thereof. If, in addition, apply antibiotics, along with respiratory viruses can be eliminated and other infectious bacteria. If the tool further comprises a vaccine, which imply both passive and active vaccines simultaneously with the treatment or prevention of viral infections according to the invention are warned and some other additional viral infection that can easily infect weakened body. To increase the stability during storage or for renewal note the indicators can be added stabilizers. Blood products are, for example, plasma, blood cells, clotting factors, and so on, depending on the treatment of the patient.
It is advisable to use a means for inhibiting the propagation of viruses. This ensures the non-proliferation of past infection or, otherwise, its a very fast treatment.
In accordance with another aspect of the invention, the latter relates to the sanitizer air, containing at least one compound of dithiocarbamate according to the invention, as described above. Under "disinfectant" in the framework of the present invention is defined as any means used outside the human or animal to fight viruses, for example, on the surface, inside of, among others, in the media, or cell cultures. These disinfectants can be used, in particular, in the case where the processed material has sensitivity towards other aggressive antiviral substances. For example, a disinfectant containing compounds of dithiocarbamate according to the invention, can be used as an additive to the environments or for processing cells or cultures of cells that are sensitive to the effects of other, more aggressive disinfectants or anti-viral compounds. Connection dithiocarbamate according to the SNO invention has proved particularly effective in the treatment and prevention of infections of respiratory cell and cell cultures, caused by respiratory RNA viruses.
Particularly effective is the disinfectant in the case when it contains compounds of dithiocarbamate according to the invention at a concentration of 10 μm-5 M, in particular 30 μm to 1 M. the concentration of effective, on the one hand, against infections caused by respiratory RNA viruses, and, on the other hand, this sanitizer is extremely gentle, for example, in the case when it is used as an antiviral substances for cell cultures. However, this concentration is determined by the type and stage of development of viral infection used in the treatment of substances, for example, on the type and sensitivity of the cells. Needless to say, it is also possible to prepare dezinfitsiruyuschee means in the form of a concentrate, and before application to bring the appropriate solvent to the desired concentration of the compound of dithiocarbamate according to the invention.
Preferably, the disinfectant contained additional disinfectants, in particular, antiviral substances. Such substances known to any expert in the field of Microbiology, added in the case when it is necessary to destroy other viruses, for example, DNA viruses. Needless to say that may be added besides antibacterial what's the matter, in particular, antibiotics.
Another aspect of the present invention is a method for the disinfection of surfaces, environments and cultures of cells, and the disinfectant according to the invention, as described above, is applied to the surface or in the cell culture or introduced into the environment. For disinfection of surfaces is sufficient, for example, treatment with a disinfectant. In the case of the environments and cultures of cells disinfectant can be affected for a longer time, and, as described above, the concentration of the disinfectant may vary depending on the destination.
According to another aspect of the invention, the latter relates to the treatment and prevention of infections caused by respiratory RNA viruses, with the use of compounds of dithiocarbamate according to the invention. With this tool, containing compounds of dithiocarbamate according to the invention, as described above, is administered to a patient or animal in an appropriate form and concentration.
Read more the present invention is illustrated below using examples and drawings that it is not limited.
Brief description of drawings
Figure 1 graphically represents the dampening effect of pyrrolidin-dithiocarbamate (PDTC) on the replication of rhinovirus man in the cell culture;
Fig. represents the inhibition pyrrolidin-dithiocarbamato cytopathic action induced rhinovirus;
Figure 3 shows the increase in cell viability using pyrrolidin-dithiocarbamate and N,N-diethyl-dithiocarbamate (DDTC);
Figure 4 shows the effect of pyrrolidin-dithiocarbamate in the treatment depending on the duration of use;
Figure 5 shows the cleavage of eIF4GI;
6 explains the analysis Western Blot to determine the expression of kapitbahayan of rhinovirus;
Fig.7 shows the effect of other antioxidants infection is caused by rhinoviruses person (HRV);
Figures 8 and 9 show the effect of pyrrolidin-dithiocarbamate on the replication of influenza virus;
Figure 10 illustrates the dependence of the action pyrrolidin-dithiocarbamate on Vero cells infected with the influenza virus, his concentration;
Figa and 11B illustrate the effect of pyrrolidin-dithiocarbamate on mice infected with influenza virus;
Fig shows the effectiveness of pyrrolidin-dithiocarbamate against virus And equine rhinitis (ERAV);
Figa and 13B illustrate the effectiveness of pyrrolidin-dithiocarbamate against MKS;
Fig shows lack of action pyrrolidin-dithiocarbamate virus encephalomyocarditis (EMCV).
Example 1. Reduced production of infectious particles rhinovirus using pyrrolidin-dithiocarbamate (PDTC)
To determine the effectiveness of infections caused Rin the virus man (HRV), after infection of cells with different HRV serotypes were injected additive PDTC.
HeLa cells were infected with serotypes 1A, 2, 14 and 16 of the virus HRV when TCID50(dose 50% infection of tissue culture) on one cell. Simultaneously to the medium was added PDTC at a concentration of 125 μm. After 4 h of infection were removed excess virus and again added PDTC in a fresh environment. After 24 hours (figure 1, top) and 48 hours (figure 1, bottom) after infection (R) has collected the supernatant and determine the amount of viral progeny with tests TCID50. Treatment of cells with PDTC reduced after 24 h the titer of virus in 103(figure 1, top). The supernatant treated with PDTC cells, which was collected within 48 h after infection, also showed a significant reduction in virus titre (figure 1, bottom). These tests suggest that PDTC has a strong antiviral effect on different HRV serotypes.
Example 2. Inhibition of the cytopathic action induced by virus HRV, and increase the viability of infected cells with PDTC
In the later stages of rhinoviral infection manifested morphological changes of cells, known as "the cytopathic effect (CPE). These induced using HRV "cytopathic effect" characterized by rounding of cells, shrinkage, deformation of the nucleus and condensation chromatherapy infection of HeLa cells, caused by a virus HRV2, HRV14, HRV1A and HRV16, at 100 TCID50one cell was observed unambiguous cytopathic effect after 8 h after infection. Additive PDTC during infection inhibited the appearance of these cytopathic action. Figure 2 shows that the morphology of the cells after 8 h after infection in the presence of 125 μm PDTC (see lower right Fig.) did not differ from the morphology of uninfected cells (see the top two figures).
Thus, the use of PDTC leads to cure your viral infection at a very early stage, resulting in a prevention of significant destruction of cells.
Example 3. Improving the viability of infected cells using pyrrolidin-dithiocarbamate (PDTC) and N,N-diethyl-dithiocarbamate (DDTC)
Were experience on the proliferation of cells to verify the validity of PDTC and DDTC on the viability of cells during viral infection.
This determined the titer cell device 96®Aqueousfor non-radioactive tests for the rapid multiplication of cells (Promega; Madison, Wisconsin, USA) according to the manufacturer's instructions. The day before infection, cells were placed on plates with 96 corrugations. These cells were infected with different serotypes of HRV at 20 TCID50/cell. Cell viability was determined by adding tetrazole, incubation was performed for 2 h at 37°and measurement is whether the absorbance at 492 nm.
At 24 h after infection, caused by HRV2, cells ZeLa found compared with uninfected HeLa cells (mock infection medium = MIM-false infectious environment) complete loss of their metabolic activity. Figure 3 (top: DDTC; bottom: PDTC), you can see that the viability of the cells increased significantly after supplementation PDTC or DDTC in low concentrations. Only PDTC or only DDTC effect on uninfected cells only slightly. However, the concentration used for the inhibition of viral infections, toxicity is not caused ("-" means "without").
Example 4. The effectiveness of PDTC depending on time
To determine the stage in the life cycle of the virus, which begins to act PDTC, the latter was added at different time points after virus infection at 20 TCID50/cell and have tested on the proliferation. Managed to establish that the addition of PDTC (125 microns) within the first six hours after infection ("-" means "without") provides the best protection against induced by viruses loss of proliferation (figa). This effect is not typical for a single serotype, as well as applied serotypes 1A, 2, 14 and 16 of the virus HRV. Only treatment of cells with PDTC (25 μm) more than 8 hours after infection reduced the protective effect.
Similar results were obtained in the case when the definition is Yali titres of virus in the supernatant of infected and treated with PDTC cells (see figv). Additive PDTC for up to four hours after infection reduced the titers produced HRV2 103. Even if treatment with PDTC was started 6 hours after infection produced titers of virus were significantly understated. These results suggest that the addition of PDTC has a antiviral action and when it is too late stages of infection and increases cell viability and reduces the number of infectious viruses.
This suggests that the antiviral effect is manifested not only in the early stages of the virus life cycle, for example, during the coupling of the receptor or its introduction into the cell.
Example 5. Study of the process flow of an infection by HRV, in the presence of PDTC
To study the process flow rhinoviral infection in the presence and absence of PDTC were analyzed by different proteolytic activity. Typical proteolytic activity, which is manifested at the time of infections caused by Rhino - and enteroviruses, is the enzymatic cleavage of the cellular translation initiation factors 4GI (eIF4GI) and 4GII viral 2A-proteinase, resulting terminates protein synthesis of the host cell. Depending on the serotype HRV proteins eIF4G split at an early stage of infection. the optional, briefly described splitting activity in the rhinoviral infection is splitting intermediator filament protein cytokeratin 8. Also, and this splitting depends on the 2A protease, but occurs at the late stage of infection. To determine the effect of treatment with PDTC on the course of viral infection have been analyzing the "Western Blot", which analyzed the substrates 2A protease.
In the analysis of "Western Blot" at the appropriate times removed Wednesday. Cells were literally by adding 100 ál of protein buffer (8% sodium dodecyl sulfate, 20% β-mercaptoethanol, 20% glycerol, and 0.04% bromophenol blue). 20 µl of extract protein share per one track using SDS-PAGE and spend blotting on PVDF membrane. Incubation with antibodies was performed at 0.1% of Tween 20 and 5% skim milk powder in TBS. When determining immunity used polyclonal rabbit antibodies against eIF4GI. As secondary antibodies used antiimmunoglobulin rabbit conjugated with alkaline phosphatase. Staining for alkaline phosphatase, the size of the molecules were determined using pre-stained marker SDS-7B (Sigma).
In HeLa cells infected by HRV2 (100 TCID50for a cell), the cleavage of eIF4GI was detected 4 hours after infection ("cf" means the product of cleavage, "cleavige product"), full Rasse the separation occurred 8 hours after infection (figure 5). By this point in the infected cells in the presence of PDTC eIF4GI cleavage were observed. In later periods, approximately 24 hours after infection, could be seen in cells treated with PDTC, a slight splitting of eIF4GI.
This suggests that either the function of the protease was blocked, or the number of viral protein decreased dramatically.
Example 6. Identification of epithelial proteins of viruses
To determine the effect of PDTC on the expression of proteins of the viruses was determined by the capsid proteins HRV2 in protein extracts of HeLa cells infected with HRV2 (100 TCID50), through the analysis of "Western Blot" (see Fig.6). Analysis of the "Western Blot" was performed, as described above in example 5, were used polyclonal anticigarette rabbit against HRV2. In untreated cells 6 hours after infection were found significant amounts of protein rhinoviruses VP1, VP2 and VP3. Treatment with PDTC prevents the expression of these capsid protein within the first 8 hours after infection. Weak expression of VP1, VP2 and VP3 was discovered only later, after approximately 24 hours after infection.
In the result, it was shown that PDTC induces delayed the production of proteins of the virus, and this explains the anti-rhinoviral effect of PDTC and protection of cells.
Example 7. The definition according to antivi osnago actions redox potential
It was investigated the inhibitory effects of other antioxidants during infection of HeLa cells by the virus HRV2 (Fig.7). HeLa cells were infected for 6-hole tablet virus HVR2 (100 TCID50/cell) (n. i. means "not infected"). After 1 h after infection, the medium is removed and add fresh medium or medium with PDTC or NAC in different concentrations. After 24 hours, the cell layer is washed with saline phosphate buffer (PBS) and stained crystal violet. On figa you can see that layer of cells have been destroyed by the infection caused by the virus HRV2, this effect can be prevented by PDTC, but not NAC. The mechanism of action of vitamin C, Trolox and β-mercaptoethanol (2-ME) is determined as described in example 3 (figure 7B, C, D). Unexpectedly, it was found that all these antioxidant substances had no protective effect in the reproduction of viruses. In order to control were tested for toxic effects of substances in the absence of virus. A large dose of vitamin C (100 μg/ml) strongly inhibited the growth of cells.
Thus, it was shown that the antiviral effect of PDTC not explained by its antioxidant action, and is obviously associated with other properties.
Example 8. The effect of PDTC on the replication of influenza virus
Influenza virus A/PR8/34 or Vienna/47/96 at the 0.01 multiplicity of infection (m.o.i.=multipliciy of infection) infect 5× 105the Vero cells and incubated for 1 h at room temperature. Then, the inoculum is removed and infectious medium was added 5 μg/ml of trypsin and 600 μm PDTC. After 48 h and 72 h remove the supernatant and measure it the titer of the virus using standard research on the tablet. As shown in Fig, the titer of the virus A/PR8/34 decreased in the presence of PDTC more than 2 logarithmic steps in comparison with the control sample (s).
To determine the effectiveness of PDTC the tests TCID50(50%dose infection of tissue culture) using PDTC and without it. TCID50calculated according to the method Cerbera (Kaerber) for any concentration: 96-well microplates infect the indicated virus at twofold dilution, 1 hour after infection remove the supernatant and add the environment with the content of PDTC in the indicated concentrations. The number of infected wells determined after 4 days. Figure 9 shows how at a concentration of 300 μm PDTC TCID50decreased to 76.4% and 82.2% for A/PR8/34 and A/Vienna/47/96.
Inhibition of in excess of 99.9% for both viruses was achieved at a concentration of 1200 microns.
Example 9. Determination of effective concentration of PDTC
The effective concentration of PDTC determined by analysis of the reduction of cytopathic effect (CPE Reduktion-Assay) (CPE = cytopathic effect): Vero cells cultured on 96-well m is croplasma and infect at 5 TCID 50and 50 TCID50on one hole influenza virus A/PR8/34 and under 5 TCID50on one hole influenza a/Vienna/47/96. After 1 h after infection remove the supernatant and add medium with 5 μg/ml of trypsin and twice divorced PDTC, an initial concentration which was 1200 μm. In the next four days the tablets visually examined for cytopathic effect. The manifestation of the cytopathic effect calculated in relation to the control sample for each concentration. Positive 100% means complete lysis of all wells. Figure 10 one can see that the decrease of the positive holes of 50% was obtained at a concentration of from 50 to 100 μm PDTC, the complete cessation of the cytopathic effect for all viruses occurred at a concentration of 600 μm PDTC.
Example 10. Defining actions PDTC in vivo
10 mice C57/BL6 inoculant through the nose with a lethal dose (50 μl of 107pfU) of virus A/PR8/34. After 1 h after they enter through the nose 25 μl of 600 mm PDTC and 25 µl of physiological solution with phosphate buffer. Mice are examined and treated within the first 48 hours, every 12 hours, then every 24 hours the weight of the mice was measured and expressed as % of initial weight (% w, figa). On FIGU you can see that all are treated with PDTC mouse (square) was sick with a viral infection and 7 days after infection gained weight. All mouse OperatingSystem solution with phosphate buffer (diamonds), died within 12 days after infection (%s = % survivors).
Thus, it was shown that already one PDTC has a strong effect against infections caused by influenza virus in vitro and in vivo.
Example 11. Antiviral activity PDTC against virus And equine rhinitis (ERAV)
The reduction of the reproduction of the virus And equine rhinitis addition of PDTC examined as follows: Vero cells infect 10 TCID50a cell with a virus ERAV. At the same time add PDTC in different concentrations (1 mm-50 μm). 4 hours after infection remove the inoculum and add fresh medium with the content of PDTC. 24 hours after infection remove the supernatant and determine the titer of the virus using standard research on the tablet.
On Fig shows how the titer of virus (vt) in the supernatant liquid is reduced compared with untreated cells.
Example 12. Effect of PDTC on the replication of the virus of foot and mouth disease (MKS) in the cell culture
By analyzing the reduction of cytopathic effect (CPE Reduktions-Assay) to determine the effective concentration of PDTC when exposed to cells destroyed by the virus.
Cells IB-RS-2 cultivated microplate with 96 wells, infect with the virus of FMD O-Manisa at 0.1 TCID50per cell and incubated for 1 h at 37°C. Then, the inoculum is removed and add infectious environment with the specified concentration of PDTC (10 µm to 1200 µm). After 4 h to determine the number of positive holes by observation through a microscope for cytopathic effect.
On figa shown that the number of infected wells (% pos.) after 24 hours depended on the concentration of PDTC. 600 μm PDTC protects cells on 100% of the cytopathic effect caused by viruses. 50%reduction of the positive holes is achieved at a concentration in the range of 75-150 microns.
To determine the effects of PDTC on the replication of FMD virus cell IB-RS-2 infect in flasks KZT25 cm2the FMD virus is O-Manisa when 0,001 TCID50per cell and incubated for 1 h at 37°C. Then remove the inoculum and add infectious environment with the content of PDTC in the indicated concentrations (0 μm-200 μm). 24 hours after infection remove the supernatant and determine the titer of virus (TCID) in the standard analysis on the tablet.
As shown in figv, the titer of the virus 0-Manisa FMD decreased in the presence of PDTC more than 2 logarithmic steps in comparison with the control sample (0). In the control sample PDTC was not added.
Example 13. The effect of PDTC on the multiplication of the FSME virus in cell culture
The FSME virus does not belong to the picornaviruses and diseases in the respiratory tract is not called.
Continuous monolayers of cells KSS-21 (ATSS) infecting virus FSME at 10 pfu/cell (Neudörfel) in the presence of PDTC in the following concentrations: 1000 μm, 500 μm, 250 μm, 125 μm, 62.5 μm, of 31.25 ám and 15.6 μm, and 7.8 μm to 3.9 μm, of 1.95 μm, 0,975 microns.
The cells are then incubated in the tech is of 4 days at 37° And the monolayer of cells analyzed under a microscope. The multiplication of viruses is determined using immunofermentnogo analysis. Microscopic analysis showed no signs of toxic action PDTC. Quantifying the propagation of viruses suggests that PDTC in any of the investigated concentration is able to reduce the multiplication of viruses.
A similar study was conducted to determine the effect of PDTC on the replication of the virus in cells in suspension: tested the same concentration as above. And in this experience, there is no confirmation of the reduction of the reproduction of the virus under the effect of PDTC.
Example 14. The effect of PDTC on mice infected with EMC virus
Mouse SV were infected intraperitoneally with EMC virus at 10 TCID50. The control group injected intraperitoneally once a day, 50 μl of physiological solution with phosphate buffer. The other two groups injected with 50 μl of 50 mm PDTC in the day, and in one group the treatment is started simultaneously with infection (PDTC), in the other group 24 hours after infection (PDTC, 24 h after infection).
On Fig shows the weight change of mice after infection. It is obvious that the treated and untreated mice are not different. Among infected mice of all groups the death occurred after an average of 5.5 days.
So would what about the shows that PDTC no effect on the infection caused by a virus EMC, which, although it relates to the picornaviruses, but the disease is not in the respiratory tract, and in nerve cells.
1. The use of compounds of dithiocarbamate structural formulas R1R2NCS2H, in which R1and R2denote, independently of one another, an unbranched or branched (C1-C4) alkyl or form together with the nitrogen atom an aliphatic ring with 4-6 carbon atoms, and R1, R2or aliphatic ring optionally substituted by one or more substituents selected from HE, NO2, NH2, COOH, SH, F, Cl, Br, I, methyl or ethyl, and oxidized forms of these compounds, in particular, their dimers, as well as their pharmaceutically compatible salts to obtain funds for the treatment or prevention of infection caused by RNA viruses affecting the respiratory tract of humans and animals and calling it a disease.
2. The use according to claim 1, wherein R1and R2signify, independently from each other, (C1-C3) alkyl or form together with the nitrogen atom an aliphatic ring with 4-6 carbon atoms.
3. The use according to claim 1 or 2, characterized in that the connection dithiocarbamate is selected from pyrrolidinedithiocarbamate and N,N-diethyldithiocarbamate.
4. The use according to claim 1, ex is different, however, what viral infection is an infection caused by picornaviruses, orthomyxoviruses or paramyxoviruses.
5. The use according to claim 4, characterized in that orthomyxovirus is a non-human influenza virus, in particular originating from a group of viruses, and influenza and paramyxovirus is a parainfluenza virus or pneumovirus.
6. The use according to claim 4, characterized in that orthomyxovirus is a virus And influenza mammals.
7. The use according to claim 4, wherein the picornavirus is a rhinovirus, in particular, rhinovirus human or a horse, enterovirus, in particular, enterovirus 70, 71, or Coxsackie virus or aphtovirus, in particular the FMD virus, or the virus And equine rhinitis.
8. The use according to claim 1, characterized in that the preparation contains the compound of dithiocarbamate at a concentration of 0.01-5000 mm, preferably 1-300 mm, particularly preferably 10-100 mm.
9. The use according to claim 1, characterized in that the medium contains compounds of dithiocarbamate at a concentration of 10 mm-1 M
10. The use according to claim 1, characterized in that the tool includes the additional pharmaceutically compatible carrier.
11. The use according to claim 1, characterized in that the tool is intended for use orally, through the nose, intravenously, rectally, parenterally or in the form of drops for eyes or ears or the form of a gargle for the throat or aerosols.
12. The use according to claim 1, characterized in that the tool includes the additional antiviral agents.
13. The use according to claim 1, characterized in that the tool contains a combination of at least two different compounds of dithiocarbamate.
14. The use according to claim 1, characterized in that the tool contains additional substances selected from antibiotics, vaccines, immune suppressors, stabilizers, immunostimulatory agents, blood products, or mixtures thereof.
15. The use according to claim 1, characterized in that the tool is used for inhibiting the propagation of viruses.
16. Disinfectant to eliminate RNA viruses that infect the respiratory tract of humans and animals, containing at least one compound of dithiocarbamate described in any one of claims 1 to 15.
17. The disinfectant according to item 16, characterized in that it comprises connection dithiocarbamate at a concentration of 10 μm-5 M, in particular 30 μm-1 M
18. The disinfectant according to item 16 or 17, characterized in that it further comprises a disinfectant, in particular, antiviral substances.
19. The method of disinfection of surfaces, environments and cultures of the cells, characterized in that the disinfectant on 17 or 18 is applied to the surface or the cell culture or introduced into the environment.
Priority points and features:
In terms of the application of dithiocarbamato except dimers (oxidized form) for the treatment or prophylaxis of viral infections in the respiratory tract of all items installed priority date 16.07.2001 according to the application AT 1103/01.
In terms of the application of dithiocarbamates and including oxidized forms, in particular dimers for the treatment and prevention of viral infection in the respiratory tract for all claims priority set by date 17.12.2001 according to AT 1973/01.
In terms of the application of dithiocarbamates, as well as their oxidized forms, in particular dimers for the treatment or prevention picornaviruses infection on all claims priority set by date 17.12.2001 according to the application AT 1972/01.
FIELD: medicine, pharmacy.
SUBSTANCE: invention relates to a cycloferon-containing medicinal agent used in prophylaxis and treatment of influenza and acute respiratory diseases, herpetic infection, chronic viral hepatitis B and C and prophylaxis of oncological diseases. Agent comprises a physiological solution in the following ratio of components: 10-29 - 10-2 mg of cycloferon in 1 ml of physiological solution. Also, invention relates to a method for preparing this medicinal agent. Proposed medicinal agent enhances antiviral and antitumor activity of natural killers by 1.8-fold, not less.
EFFECT: improved preparing and using method, valuable medicinal properties of agent.
7 cl, 1 dwg, 19 ex
SUBSTANCE: claimed strain is obtained by hybridization of epidemic virus with cold adapted and temperature sensitive virus, which represents attenuation donor and is harmless for adults, and infants of 3-14 years old. Said virus makes it possible to obtain reassortant vaccine strains from new epidemic viruses. Strain is effectively cultivated in germinative hen embryos at 32°C, and has temperature sensitivity and cold adaptation. Reassortant has two genes derived from epidemic virus encoding surface proteins (hemagglutinin and neuroamidinase) and six genes derived from attenuation donor encoding non-glycosilated proteins. Strain has no reactogenicity in relation to adults and infants of 3-14 years old at intranasal application.
EFFECT: strain for living influenza intranasal vaccine with good biological properties and reactogenicity.
3 tbl, 2 ex
SUBSTANCE: one should perform the following stages: a) removal of contaminants out of plant; b) plant's reducing; c) treatment of reduced plant with laser radiation; d) suspending the mixture obtained at stage c) in water; e) maceration of suspension obtained at stage d) and f) separation of liquid developed. Composition should be obtained due to this technique. It should be applied at treating hepatitis C as an aqueous extract. It should be applied as aqueous extract as immunostimulant. Pharmaceutical preparation includes aqueous extract as an active constituent.
EFFECT: increased biological activity of the product.
43 cl, 16 ex, 1 tbl
FIELD: organic synthesis.
SUBSTANCE: invention provides group of novel antiviral nitrogen-containing compounds, in particular adamantane derivatives having general formula:
, wherein R represents chlorine or ethyl.
EFFECT: increased choice of biologically active compounds suitable for use in medicine as antiherpetic agents.
2 cl, 6 tbl, 3 ex
SUBSTANCE: biologically active additive has propolis and pot marigold tincture, ascorbic acid, calcium gluconate, benadryl, rutin and auxiliary substances like starch, calcium stearate, talc taken in known proportion. The biologically active additive is produced as tablets of mass 0.55 g.
EFFECT: enhanced effectiveness of prophylaxis.
SUBSTANCE: method involves making patient contact with effective quantity of one or several compounds. Pharmaceutical composition comprises one or several compounds applicable in absence of exogenous antigen for treating or alleviating the cases of infectious, autoimmune diseases and allergy.
EFFECT: enhanced effectiveness of treatment or prophylaxis.
FIELD: virology, biotechnology, medicine, pharmacy.
SUBSTANCE: invention proposes the envelope HCV protein (hepatitis C virus). Protein comprises 80% of glycosylated sites. Such protein is more preferable for diagnostic, prophylactic and therapeutic using. Also, invention proposes a method for preparing such protein, and a drug, vaccine and different pharmaceutical compositions also comprising such protein. Proposed group of inventions can be used in medicine for diagnosis, treatment and prophylaxis of HCV infection and to prediction of clinical effectiveness of treatment.
EFFECT: improved preparing method, valuable medicinal properties of protein.
37 cl, 77 dwg, 16 tbl, 29 ex
FIELD: genetic engineering, virology, pharmacy.
SUBSTANCE: invention proposes the recombinant modified virus OF VACCINE Ankara able to express structural antigens of hepatitis C virus. Virus comprises DNA sequences encoding structural antigens of hepatitis C virus or their functional regions or epitopes of hepatitis C virus structural antigens. Also, invention proposes a pharmaceutical composition comprising such virus, eucaryotic cell infected with such virus, a method for preparing such virus and a method for preparing hepatitis C virus structural polypeptides. Invention can be used in virology and medicine for preparing hepatitis C virus antigen.
EFFECT: valuable properties of virus.
20 cl, 14 dwg, 1 tbl
FIELD: medicine, obstetrics, gynecology.
SUBSTANCE: uterine cavity should be drained in the course of operation, moreover, irrigator's distal end should be withdrawn through operation wound at anterior abdominal wall, and 2 h after the end of operation uterine cavity should be washed through irrigator with 400 ml of cooled 0.06%-sodium hypochlorite solution at perfusion rate being 200 ml/h, 6 times every 12 h up to 3-4 d; after each perfusion one should introduce 1 g kanamycin directly into uterine cavity, moreover, in case of availability of bacterioid and/or anaerobic flora in uterine cavity according to the results obtained due to pre-operational antibioticogram one should add 100 ml 3%-hydrogen peroxide solution into perfusion solution. The present innovation enables to efficiently sanitize uterine cavity due to intrauterine injection of antibiotics by taking into account antibioticogram performed at all stages of operative treatment.
EFFECT: higher efficiency of prophylaxis.
1 cl, 2 ex
SUBSTANCE: method involves administering 1-methyl-2-phenylthiomethyl-3-carbetoxi-4-dimethyl-aminomethyl-5-oxibromoindol or its salts selected from a group containing hydrochloride monohydrate (arbidol) earlier known as immunomodulator, besilate, fumarate, maleate, citrate or their hydrates.
EFFECT: enhanced effectiveness in healing organism from rotavirus.
4 cl, 8 tbl
FIELD: organic chemistry, biochemistry, medicine, virology.
SUBSTANCE: invention relates to derivatives of 2'=amino-2'-deoxynucleosides of the formula:
wherein R means hydrogen atom (H), alkyl, aminoalkyl; R1 means -(R2NR3) wherein R2 and/or R3 means H, -OH, -NH2, alkyl, benzyl under condition that R doesn't represent H or methyl when R2 and R3 mean H. Compounds elicit an antiviral activity with respect to measles and Marburg viruses exceeding that of ribavirin.
EFFECT: valuable properties of compounds.
4 tbl, 2 dwg, 18 ex
FIELD: medicine, virology, pharmacy.
SUBSTANCE: invention proposes an agent for treatment and prophylaxis of infection caused by coronaviruses, in particular, for treatment of atypical pneumonia (SARS), and pharmaceutical composition of indicated designation based on thereof. Agent represents 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxybromoindole or 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxybromoindole monohydrate hydrochloride (arbidol) known early as an immunomodulator and preparation used against influenza viruses. Invention provides reducing accumulation of coronaviruses (on example with TOPS virus) in lung.
EFFECT: valuable medicinal properties of agent.
2 cl, 7 tbl, 9 ex
FIELD: organic chemistry, biochemistry, medicine, pharmacy.
SUBSTANCE: invention relates to macrocyclic peptides of the general formula (I): wherein W means nitrogen atom (N); R21 means hydrogen atom (H), (C1-C6)-alkoxy-, hydroxy-group or N-(C1-C6-alkyl)2; R22 means hydrogen atom (H), (C1-C6)-alkyl, CF3, (C1-C6)-alkoxy-group, (C2-C7)-alkoxyalkyl, C6-aryl or Het wherein het means five- or six-membered saturated or unsaturated heterocycle comprising two heteroatoms taken among nitrogen, oxygen or sulfur atom and wherein indicated Het is substituted with radical R24 wherein R23 means hydrogen atom (H), -NH-C(O)-R26, OR26, -NHC(O)-NH-R26, -NHC(O)-OR26 wherein R26 means hydrogen atom, (C1-C6)-alkyl; R3 means hydroxy-group or group of the formula -NH-R31 wherein R31 means -C(O)-R32, -C(O)-NHR32 or -C(O)-OR32 wherein R32 means (C1-C6)-alkyl or (C3-C6)-cycloalkyl; D means a saturated or unsaturated alkylene chain comprising of 5-10 carbon atoms and comprising optionally one-three heteroatoms taken independently of one another among oxygen (O), sulfur (S) atom, or N-R41 wherein R41 means hydrogen atom (H), -C(O)-R42 wherein R42 means (C1-C6)-alkyl, C6-aryl; R4 means hydrogen atom (H) or one-three substitutes at any carbon atom in chain D wherein substitutes are taken independently of one another from group comprising (C1-C6)-alkyl, hydroxyl; A means carboxylic acid or its alkyl esters or their derivatives. Invention relates to pharmaceutical compositions containing indicated compounds and eliciting activity with respect to hepatitis C virus and these peptides inhibit activity of NS3-protease specifically but don't elicit significant inhibitory activity with respect to other serine proteases.
EFFECT: valuable biochemical and medicinal properties of peptides.
106 cl, 9 tbl, 61 ex
FIELD: medicine, medicinal technology.
SUBSTANCE: invention relates to physics of aerodispersed systems, namely to methods for preparing hygroscopic submicrone aerosols of alkaline metal iodides. Method for preparing hygroscopic submicrone aerosol of alkaline metal iodides in atmosphere air involves feeding atmosphere air into heated quartz cylindrical reactor with evaporating alkaline metal halide, saturation the air flow with its vapors, mixing vapor-air mixture with cold air in outlet from quartz cylindrical reactor and its cooling up to the supersaturation degree 102 and their condensation to form submicrone aerosol of alkaline metal halide. As alkaline metal halide method involves using potassium iodide, sodium iodide or their mixture. Atmosphere air is fed into heated quartz reactor with its relative moisture above 30% at temperature 10-40°C and saturated its with vapors of potassium iodide and/or sodium iodide at temperature 550-750°C with simultaneous formation of bipolar gaseous ions and saturated molecular iodine vapor. Vapor-air flow in outlet from quartz cylindrical reactor is cooled initially to temperature 150-250°C with additional coaxial flow of atmosphere air for formation of bipolar charged submicrone aerosol of potassium iodide and/or sodium iodide with addition of molecular iodine followed by cooling aerosol flow to temperature 30-50°C with simultaneous its dilution of its concentration by turbulent stirring with additional coaxial atmosphere air flow. Invention provides formation of hygroscopic bipolar charged submicrone aerosol alkaline metals iodide (KJ and/or NaJ) with addition of molecular iodine (J2) that is stable by size and the mass concentration in atmosphere used in system for conditioning atmosphere air and creating the curative microclimate into compartments, and for providing the possibility for its using for medicinal aims for inhalation. Invention can be used in systems for conditioning air and creating the curative microclimate into compartments, and in medicine in treatment of diseases with using atmosphere air comprising hygroscopic submicrone aerosol of alkaline metals iodide.
EFFECT: improved preparing method, valuable medicinal properties of aerosol.
6 cl, 1 dwg, 2 ex