Synergistic inhibition of viral replication with long-chain hydrocarbons and nucleoside analogs

FIELD: medicine.

SUBSTANCE: the present innovation deals with antiviral preparations that contain aliphatic alcohol C21-C28 in combination with either nucleoside or nucleotide analog or phosphoformic acid in pharmaceutically acceptable carrier. It is necessary to mention that n-docosanol is considered to be a preferable aliphatic alcohol. Concentration of aliphatic alcohol C21-C28 corresponds to 0.05% to 40% by weight. Concentration of either nucleoside or nucleotide analog or phosphoformic acid corresponds to 0.1% to 10% by weight. The innovation, also, deals with the ways to treat viral infections due to applying such compositions. Aliphatic alcohols C21-C28 synergistically intensify antiviral activity of nucleoside analogs directed against replication of several herpetic viruses and that of cow's pox.

EFFECT: higher efficiency of inhibition.

28 cl, 13 dwg, 21 ex, 6 tbl

 

The existing level of technology

This invention relates to the treatment of viral infections with long-chain hydrocarbons in combination with nucleoside analogues and, in particular, to the local application therapeutic compositions containing n-docosanol in combination with nucleoside analogue or phosphorus-formic acid (PMK).

Viral infections pose a serious threat to public health. Such viruses as herpes simplex viruses (HSV-1 and HSV-2), cytomegalo-virus (CMV), a virus of Epstein-Barr (EBV), varicella zoster virus (OIE), the influenza viruses, lipotropnye human viruses (e.g., virus T-cell lymphoma person, WTLC-1) and the human immunodeficiency viruses (e.g. HIV-1), lead to significant morbidity and mortality. Viruses HSV-1 and HSV-2 are associated with inflammation and lesions of the skin and mucous membranes, including those with lesions of herpes fever, lesions of simple herpes of the lips and lesions of genital herpes. BBO causes shingles, and EBV associated with mononucleosis. Influenza viruses cause symptoms of epidemic influenza and can lead to death. The HIV virus causes acquired immune deficiency that weakens and kills the infected people. Although these viruses can remain latent in some cells over different periods of time, usually in the originate replication leads to irreversible destruction of infected cells, creating a variety of clinical manifestations caused by these viruses diseases.

Most modern anti-virus therapies using nucleoside analogues, such as purine nucleoside analogue acyclovir (ACV) and pyrimidine nucleoside analog, azidothymidine (AZT), which interfere with viral replication in infected host cells. These nucleoside analogues are converted to their triphosphorylated (nucleotide) derived by viral and/or cellular kinases, while they block the elongation of viral DNA. Gianniny analogue 9-(2-hydroxy)-ethoxymethylene called ACV that have potent antiviral activity. Examples of therapeutic nucleoside analogues associated with ACV, and methods for their preparation are disclosed in U.S. patent No. 4199574, 4294831 and 4360522 issued Schaeffer, in U.S. patent No. 5580571 issued Hostetler, in U.S. patent No. 5756737 issued Turchetta et al., and in U.S. patent No. 5567816 issued Schloemer et al.; descriptions of these inventions included here by reference. One of the major problems associated with the use of these nucleoside analogues are their limited phosphorylation in some cells and cytotoxic side effects triphosphates of nucleoside analogues. In addition, these antiviral drugs can potentially act as mutagens and/is any teratogens in host cells. Thus, despite a strong antiviral effect of nucleoside analogues, a search for less toxic effective therapeutic agents.

Among the alternatives nucleoside analogues for the treatment of viral infections are a variety of long-chain alcohols, fatty acids, alkanes and similar compounds. First work with these compounds was focused on their direct antiviral action. For example, it was reported that unsaturated alcohols having from 14 to 20 carbon atoms and from one to four double bonds, possess antiviral activity. The most effective of these unsaturated alcohols was γ-linalilovy alcohol, With 18-alcohol with double bonds at positions b, 9 and 12 (Sands et'al., Antimicrob. Agents &Chemother. 15:67-73, 1979). It was also shown that formulations containing oleic acid (18, one double bond), exhibit antiviral activity against herpes virus (patent application PCT WO 9602244A1).

It has been shown that aliphatic alcohols having from 20 to 32 carbon atoms, possess antiviral and anti-inflammatory activity. Therapeutic compositions containing such long-chain aliphatic alcohols and similar compounds described in U.S. patent No. 4874794, U.S. patent No. 5071879, U.S. patent No. 5166219, U.S. patent No. 5194451 and U.S. patent No. 5534554 described here p is a means of links.

It was reported that some compounds structurally related to long-chain aliphatic alcohols, possess antiviral activity. For example, U.S. patent No. 4513008 discloses antiviral activity linear polyunsaturated acids, aldehydes or alcohols containing from 20 to 24 carbon atoms, having from five to seven double bonds. Compounds having long-chain fatty Alloway group containing at least three bonds attached to the nucleoside or nucleoside analogue, is also disclosed as an antiviral drug in U.S. patent No. 5216142. Related U.S. patent No. 5276020 discloses antiviral compounds having long-chain fatty Alloway group with 16, 18 or 20 carbon atoms, attached to a nucleoside analogue, and a method of treating viral infections using these compounds. Indeed, Hostetler et al. recently reported improved oral absorption and antiviral activity of 18-carbon derived ACV, 1-D-octadecyl-sn-glycero-3-phospho-ACV (Hostetler et al., Biochem. Pharmacol. 53:1815-1822, 1997).

It was also reported local therapeutic compositions containing various alcohols, fatty acids and amines. For example, it was reported antiviral activity of liposomal AL721, a mixture of neutral glycerides, phosphatidylcholine, and phosphatidylethanolamine (Antonian et al., Neursci. Biobehav. Rev, 11:399-413, 1987). Antimicrobial compounds for topical treatment containing complex literally monoether lauric acid with 15 carbon atoms or an ester of a polyhydric alcohol lauric acid with a mixture of fatty acids (capric acid with 10 carbon atoms and Caprylic acid with 8 carbon atoms), were disclosed in U.S. patent No. 5208257. Treatment of herpetic lesions using applied topically compositions containing anesthetic, surfactant and local media, has been disclosed in U.S. patent No. 5380754. A method of treating inflammation by topical application of ethyl-CIS,CIS(9,12)octadecadienoate (ethyl linoleate) was disclosed in U.S. patent No. 4025645 as treatment of herpetic fever.

Katz et al. {Proc. Natl. Acad. Sci. USA 88:10825-10829, 1991; U.S. patent No. 5534554) showed that one of the long-chain aliphatic alcohols, n-docosanol (22 carbon atoms), has a strong systemic and local antiviral activity against several viruses, including herpes simplex virus (in vitro and in vivo, HIV-1 (in vitro), respiratory syncytial virus (in vitro) and Friend virus (in vitro and in vivo). Unlike unsaturated alcohols having from 10 to 18 carbon atoms, which are detergent-like antiviral activity, n-docosanol not deactivate the virus directly (Katz et al., Proc. Natl. Acad. Sci. USA 88:10825-10829, 1991; Snipes et al., Antimirob. Agents Chemother, 11:98-104, 1977). Progressive linking and perception n-docosanol cells can be attributed to its antiviral activity, since pre-incubation of cells with alcohol gives optimal antiviral activity. Moreover, 70% associated with cell n-docosanol found in components of the cell membrane, and the rest is associated with soluble parts of cells (Pope et aL, J. Lipid. Res. 37:2167-2178, 1996). The introduction of n-docosanol in the plasma membrane did not inhibit binding of the virus with the cell surface. Early synthesis of viral protein inhibited more than 80% and no virus was localizability in nuclei (Katz et aL, Proc. Natl. Acad. Sci. USA 88:10825-10829, 1991). The fusion of the virus with the plasma membrane of cells is inhibited (Pope et al., Antiviral. Res. 40:85-94, 1998).

It seems that for the inhibition of synthesis of viral proteins and antiviral activity of n-docosanol required cellular metabolism of alcohol (Pope et al., J. Lipid Res. 37:2167-2178, 1996; Katz et al., Ann. N.Y. Acad. Sci. 724:470488, 1994). In addition, although the intracellular metabolic conversion of n-docosanol can be attributed to its antiviral activity (Katz et al., Annals. N.Y. Acad. Sci. 724:470488, 1994), n-docosanol is not cytotoxic at concentrations up to 300 mmol.

Such compounds as n-docosanol, pharmacological actions are mediated by cellular metabolism, can change the way meth is balibali and expression of the second medication. In addition, it is known that viruses strongly modulate the metabolism of the host cell. These interactions medication may cause unwanted effects in patients treated with multiple medications. However, there may occur also beneficial drug interaction. Of course, there are many reports about the interactions between nucleoside analogues, such as ACV, and compounds for modulation of cellular metabolism (Spector et al., Proc. Natl. Acad. Sci. USA 86:1051-1055, 1989; O'brien et al., Antimicrob. Agents Chemother. 34:1178-1182, 1990; Hirsch et al., 1996 Antiviral Agents. In Fields Virology Third Edition, B. N. Fields, D. M. Knipe, P. M. Howley, eds. Lippincott-Raven Publishers, Philadelphia, pp. 431-466). Usually this mechanism includes one or more steps in the absorption or metabolism nucleoside, resulting in more efficient expression of antiviral activity.

Because patients with recurrent herpes is a viral disease can competition be treated with 10% cream n-docosanol and acyclovir (ZOVIRAX™), we investigated the possibility of harmful and useful drug interactions. The present invention is based on the discovery that n-docosanol synergistically activates antiviral activity of nucleoside analogues against replication of several herpes virus and cowpox virus.

The invention

This is General the invention relates to an antiviral composition, containing long-chain aliphatic compound and nucleoside or nucleotide analog or phosphorus-formic acid (PMK) in a pharmaceutically acceptable carrier. In particular, long-chain aliphatic compound is selected from the group consisting of primary alcohols containing 18-28 carbon atoms, erollover alcohol, brassicicola alcohol, n-docosane, n-docosanoic acid, erucamide and stearic acid, or mixtures thereof.

Aliphatic compound represented at a concentration in the range from approximately 0.05% to approximately 40%. Nucleoside or nucleotide analogue in the antiviral composition is selected from the group consisting of ACV, adefovir, azidothymidine, brivudine, cidofovir, ddC, ddI, famciclovir, ganciclovir, idoxuridine, lamivudine, lobucavir, penciclovir, ribavirin, sorivudine, trifluridine, trimethoprim, valaciclovir and Agha A. Nucleoside or nucleotide analog FMF presents at concentrations in the range from approximately 0.1% to approximately 10%.

In the preferred implementation of this anti-virus composition also contains a non-ionic surfactant. This surfactant may contain bifunctional block-polymer, which is polyoxyalkylene derivative of propylene glycol with a molecular weight of from about 1000 to is roughly 25000, block copolymer of ethylene oxide and of propylene oxide with a molecular weight of from about 6000 to about 12000, or non-ionic surface-active agent is octoxynol-9 or octoxynol-10.

Preferably, the antiviral composition of the present invention contains n-docosanol and nucleoside analogue from the group consisting of ACV, or pyrophosphatase analog PMK, ribavirin, trifluridine and yeah, in a pharmaceutically acceptable carrier, and n is docosanol presents at a concentration in the range from approximately 0.05% to approximately 40%, and the nucleoside analogue is presented in a concentration in the range from approximately 0.1% to approximately 10%.

Pharmaceutically acceptable carrier in accordance with the present invention contains sucrose stearate in a concentration of from about 1% to about 25%, mineral oil at a concentration of from about 1% to about 25%, propylene glycol USP at a concentration of from about 1% to about 25%, benzyl alcohol at a concentration of from about 0.1% to about 10% water.

Disclosed antiviral compounds can be used in the treatment of viral infections, and long-chain aliphatic compound is introduced in combination with nucleoside or nucleotide analogue. Long-chain aliphatic compound and nucleate the hydrated or nucleotide analog can be independently adapted for insertion from one to five times a day in any way from the group, consisting of local, oral, permucosal, resembling penetration and intravenously.

To use long-chain aliphatic compounds and nucleoside or nucleotide analog according to the present invention also refers in the preparation of drugs for the treatment of viral infections. This medicine may be injected with a dosage of from about 0.01 to about 10 g with a frequency of approximately one to five times per day for about one to fourteen days. Medication may be administered by any means from a group consisting of local, oral, permucosal, resembling penetration and intravenously.

Also disclosed is a method of treatment of viral infections. This method includes introducing a composition containing aliphatic compound and a nucleoside analog or PMK in a pharmaceutically acceptable carrier, the composition may be injected local image from three to five times a day, or parenterally, or by resemblance penetration through the gastrointestinal tract, through the respiratory system or through the urinary system.

In the preferred implementation of the method for the treatment of viral infection includes the introduction of a composition containing n-docosanol and either ACV, PMK, ribavirin, trifluralin or Agha And pharmaceutical is acceptable carrier.

It should be understood that both the foregoing General description and the subsequent detailed description are for example only and explanation and do not limit the invention described in the claims. The accompanying drawings illustrate various implementation of the invention and together with the description serve to explain the principles of the invention.

Brief description of drawings

Figure 1 is a graph showing the inhibition of the formation of plaque, HSV-2 in Vero cells in vitro suspensions of n-docosanol (22 carbon atoms,), n-tetracosanoate (lignocellulose) alcohol (24 carbon atoms, ⋄), n-hexacosanol (26 carbon atoms,) and n-octacosanol (28 carbon atoms Δ), at the concentrations shown on the X-axis (the data represent the percentage of observed plaques compared to control cultures, which were affected by the suspension of surface-active substances without long-chain alcohol).

Figa is a diagram showing that the increase of the ratio of surfactant to n-docosanol reduces the formation of viral plaques at incubation of Vero cells with suspension for 12 hours before adding the virus HSV-2; the ratio of surfactant: n-docosanol were as follows: 1:1 ( ), 3:1 (Δ), 5:1 (and 10:1 (On).

Figb shows control samples corresponding to the samples on figa using the same concentration of surfactant in the suspension, as for each the ratio of surfactant: alcohol, shown in figa, but without n-docosanol (using the same symbols as on figa).

Figure 3 is a chart showing that suspension of n-docosanol with surface-active octoxynol () inhibit the formation of plaque HSV-2 in Vero cells, incubated with the suspension and HSV-2 within 48 hours, increase in the inhibition correlating with increasing concentration of n-docosanol, while the control culture, incubated with HSV-2 and surface-active octoxynol (O), showed no inhibition (i.e. were equivalent to untreated control cultures, with approximately 50 plaques on deepening); rectangles above and below the data points show the standard deviation for duplicate samples.

Figure 4 is a chart showing that the suspension of surfactant/n-docosanol () and surfactant/n-docosane () inhibit the formation of viral plaques HSV - cultural Vero cells, incubated with these substances during the 12 hours before adding HSV-2.

Figure 5 is a chart showing that suspension stearyl alcohol (18 carbon atoms,and archipelogo alcohol (20 carbon atoms, Δ) toxic for cultural neoplastic b-cells, incubated for 48 hours with suspensions at concentrations shown on the X-axis, compared with the control cultures incubated with suspensions of surface-active substances without alcohol (Oh), as was determined by the introduction of 3H-thymidine into DNA (data are percentage of control cultures incubated only with the environment).

Figa and 6B schematically show protivovpalitelnoe cellular action of suspensions of surfactant/n-docosanol () foreskin fibroblasts compared with cells incubated with suspensions of surfactant/n-dokusan (Δ) or with control cultures incubated with a suspension of surfactant without active ingredient (A), at the concentrations shown on the X (averages of duplicate tests, calculated after 96 hours incubation of cells infected with 1000 cells/recess (figa) or 30,000 cells/dredging (figb) in plates with 96 cavities).

Fig is a chart showing time dependence protivovrashchatelnogo cellular effects of suspension surfactant/n-docosanol after 72 hours () and 96 hours (About) incubation by means of the methods described for figa.

Fig shows the inhibition of skin diseases HSV-2 in hairless Guinea pigs using the combination formula of the cream consisting of n-docosanol and ACV. Data are average results of two independently conducted experiments and were analyzed using t-student test with two "tails".

Fig.9 shows synergistic against HSV activity of n-docosanol and ACV in cultures of Vero cells. Data are expressed as median and RMS errors for plaques observed in triplication recesses/analysis.

Figure 10 shows a synergistic inhibition of n-docosanol and ACV playback of HSV-1 in cultures of Vero cells. Data Express the mean value EOB observed when triplicate primordial cell cultures; the RMS error does not exceed 15% of the respective average values (not shown).

11 shows a synergistic inhibition of replication of HSV-1 in vitro n-docosanol and non-AZW nucleoside analogues. Data expressed as EU90for inhibition of vos is proizvedeniya HSV-1, derived from the average value EOB observed when triplicate original cell culture/analysis.

Fig shows additional antiviral activity of n-docosanol and PMK in relation to the replication of HSV-1 and the lack of interaction with cowpox virus. Data expressed as mean values EOB when quadruplicate cultures.

Fig shows the improvement using n-docosanol antiviral activity of nucleoside analogues in relation to the replication of vaccinia virus. Data expressed as EU50(panel A) and the EU90(panel B) for inhibition of reproduction of HSV-1 derived from the average EOB observed when triplicate the original cell culture.

A detailed description of the preferred execution

In its broadest implementation of the present invention is a composition useful as a treatment for viral infections. This composition contains long-chain aliphatic compound in combination with nucleoside or nucleotide analog or PMK in a pharmaceutically acceptable carrier. Also disclosed is a method of treatment of viral infections, introducing long-chain aliphatic compounds in combination with nucleoside or nucleotide analog or PMK.

Aliphatic compounds suitable for use in the present invention, selected from the group consisting of saturated aliphatic alcohols, mono-unsaturated aliphatic alcohols, aliphatic alkanes, mono-unsaturated aliphatic amides and aliphatic acids having a length of carbon chain of 18 to 28 carbon atoms (C18-s). The preferred composition contains stearyl alcohol, aracelly alcohol, passively alcohol, arachidonoyl alcohol, n-docosanol, n-dokusan, n-docosanol acid, erucamide and stearic acid, or a mixture thereof. Most preferably aliphatic compound is n-docosanol. Aliphatic compound may be used in accordance with a preferred variant of the present invention in concentrations ranging from approximately 0.05% to approximately 40%. Most preferably n-docosanol is used at a concentration in the range from approximately 1% to approximately 20%.

Methods of synthesis of n-docosanol and Aracinovo alcohol (CIS-13-dokusen-1-ol) known in the art (see for example U.S. patent No. 4186211). Stearyl alcohol can be synthesized in accordance with the method described by Brown et al. (J. Am. Chem. Soc. 78:2582, 1956). Methods of synthesis of alkanes, aliphatic alcohols, amides and aliphatic acids are well known from the existing state of the art (for example, see A. Streitwieser, Jr. & C. N. Heathcock, Introduction to Organic Chemistry, 2nd ed., Macmillan Publishing Co., New York, NY, 1981, article is anizah 160, 243-247, 303-307, 311-312, 315-317, 401-406, 447-453, 515-516, 544, 548-555, 604-605, 670, 753-754 and 950).

Nucleoside or nucleotide analogue in the antiviral composition of the present invention can be selected from the group consisting of ACV, adefovir, azidothymidine, brivudine, cidofovir, ddC, ddI, famciclovir, ganciclovir, idoxuridine, lamivudine, lobucavir, penciclovir, ribavirin, rifampin, sorivudine, trifluridine, valaciclovir and Agha A. Nucleoside analog or PMK is present in concentrations ranging from about 0.1% to about 10%. Most preferably used ACV, PMK, ribavirin, trifluralin or Agha And in concentrations in the range from approximately 0.1% to approximately 10%.

Methods of synthesis of nucleoside or nucleotide analogues in accordance with the present invention are well known from the existing art. Synthesis of acyclovir disclosed in U.S. patent No. 4199574 issued Schaeffer, U.S. patent No. 5567816 issued Schloemer, and in U.S. patent No. 5756737 issued Turchetta, and is well known to specialists.

Phosphorus-formic acid can be synthesized by alkaline hydrolysis of triethylphosphite, as described by Nylen, P. (Chemische Berichte 57:1023-1038, 1924).

The antiviral composition in accordance with one of the executions may contain a surfactant which is a nonionic detergent, as duhf the purpose ground receiving stations block polymer, which polyoxyalkylene derivative of propylene glycol having a molecular weight from about 1000 to about 25,000 or above. Preferably surface-active agent is a block copolymer of propylene oxide and ethylene oxide (poloxamer 188)having a molecular weight between 6000 and 12000, more preferably approximately 8400 (for example, PLURONIC F-68®). Other preferred surfactants are octoxynol-9 and/or octoxynol-10 (for example, TRITON X-100®), deoxycholate or a mixture of nonionic detergents. Active ingredients (long-chain aliphatic compound and a nucleoside analog or PMK) ranges from approximately 0.1% to approximately 50% of the weight of the final composition, preferably from 1% to 10% by weight. Optimal antiviral activity of the active ingredient depends on the ratio of surface-active substances and active ingredients, which can vary from 1:1 (by weight) to 10:1 (by weight), and preferably is 5:1 (by weight).

Active agents and optionally surfactants are combined with a carrier that is physiologically compatible with the skin and membrane tissue of a human or animal to which it is entered. That is, the media is almost inactive, except for the surface-active properties that are used in prigot is no suspensions of the active ingredients. The compositions may contain other physiologically active components that do not interfere with the effectiveness of saturated aliphatic alcohols, mono-unsaturated aliphatic alcohols, aliphatic alkanes and aliphatic acid or nucleoside analogues. An example of the composition disclosed in U.S. patent No. 3592930.

Suitable carriers include water and oil media, such as, for example, white petrolatum, isopropyl myristate, lanolin or lanolin alcohols, mineral oil, monooleate sorbitan, propylene glycol, cetylstearyl alcohol (together or in various combinations)with detergent (e.g., polyoxyl a stearate or sulfate sodium-lauryl), mixed with water to produce a lotion, gel, cream, or semi-solid composition. Other suitable carrier materials include mixtures of emulsifiers and emollients such solvents, such as sucrose stearate, cocoat sucrose, distearate sucrose, mineral oil, propylene glycol, 2-ethyl-1,3-hexanediol, polyoxypropylene-15-stearyl ether and water. The media can also be added such preservatives like methylparaben, propylparaben, benzyl alcohol and tetraacetate salt of ethylene diamine. Soluble suspension without thickeners are most suitable for application to the skin surface in the form of aerosol sprays, with the help of a well is izvestnyh methods of application. The composition may also contain a plasticizer, such as glycerol or polyethylene glycol (molecular weight from 800 to 20000), and these improve the penetration of substances, as azone. The composition of the medium may vary within such limits as he does not interfere with the pharmacological activity of the active ingredients.

The compositions may also contain antimicrobial agents, other antiviral agents, antifungal agents, antioxidants, buffering agents, sunscreen and cosmetic agents such as coloring agents, fragrances, lubricants and wetting or drying agents. Antimicrobial agents that are useful to introduce into the composition, are polymyxin b and tetracycline. Other anti-viral agents included in the formula may be cytokines. Antifungal agents that may be introduced into the composition, are micatin or tolnaftate. Can be entered antioxidants such as vitamin E. Can be entered such sunscreen substances as para-aminobenzoic acid. The drying agents that may be introduced into the composition, are well known, such, for example, phenol and benzyl alcohol. May also be entered such lubricants as synthetic or natural beeswax. Thickeners added to the formulations may include pullulan, xanthan gum, polyvinyl pyrrolidone or carbox is methylcellulose.

Optimally, these compositions are effective in reducing viral titer of the entire curable individual, in particular for systemic treatment, and in places of destruction, in particular, for the local treatment of the affected areas of the skin or mucous membranes. Disclosed methods of treatment can also reduce the symptoms of viral infection (such as pain associated with caused by a virus lesions) and help more rapid recovery than without treatment.

The method according to the present invention provides an introduction to the composition containing the active ingredients and, optionally, a surfactant, a person or an animal to treat or prevent a viral infection. The introduction is preferably carried on the skin or mucous membrane with the help of a cream, lotion, gel, ointment, suspension, aerosol spray, or semi-solid formulas (e.g., candles), which are all formulated using well-known from the existing prior art methods. However, parenteral and resemblance penetration also means, in accordance with some executions of the present invention. If the route of introduction of the local route or resemblance penetration, the composition may contain an optional improving the penetration of a substance well known to the current level of technology, such to the to the azone and dimethylsulfoxide. The application consist of from one to ten of the coating composition in an amount from 10 mg to 10 g per one application within one to fourteen days. Usually applying to happen once in twelve hours, and more frequently, up to once every four hours. It is most preferable to produce from two to five of coating composition per day, from 0.1 to 5 g per application, within one to seven days, and this is sufficient to prevent or treat viral infections. When the local application, the compositions are preferably applied daily to the lesions as soon as symptoms (eg, pain, or inflammation).

These compositions and methods useful for the prevention or treatment of various viral infections such as caused by herpes viruses, including HSV-1, HSV-2 and HSV-6, CMV, EBV or BBO, influenza viruses, limfotropnami the human viruses (e.g., WTLC-1), the human immunodeficiency viruses (e.g. HIV-1), human papilloma virus and respiratory syncytial virus. Due to the cytostatic activity of some compounds and potential interactions with anticancer drugs with nucleoside analogs, compositions and methods can also be useful for the inhibition of malignant cell growth and/or metastasis. This cell inhibition and combined chemioterapia can be combined with horoscopist ways of treating cancer (e.g., radiation and/or surgical interventions), resulting in complete or partial remission of a tumor or other cancerous cell growth.

If not stated otherwise, all technical and scientific terms used herein have the meaning commonly understood specialists in the relevant field. Unless otherwise noted, the methods used and implied herein are standard methodologies well known to the specialist. Examples of executions are for illustrative purposes only.

Working examples

The source of the chemicals and reagents - n-docosanol (>98% purity, molecular weight 326) was purchased from company M. Michel and Company, Inc., New York, NY. Benzyl alcohol, mineral oil, polypropylenglycol, stearic acid and stearate sucrose were obtained from the company Croda Inc., New York, NY. Powder acyclovir was obtained from the company Burroughs Wellcome Co., Research Triangle Park, NC. Adenine 9-b-D-arabinofuranoside, phosphorus-formic acid, ribavirin, rifampicin and trifluralin deoxyribose were purchased from Sigma Chemical Co., St. Louis, MO. PLURONIC F-68® (poloxamer 188) was purchased from BASF, Parisappany, NY.

Source animals, viruses, cell lines - strain makin-Taira virus HSV-1 (#VR-539), strain MS virus HSV-2 (#VR-540), the strain Ellen varicella zoster virus (OIE, #VR-1367), the strain of Town cyto-megalovirus (CMV, #VR-977) and strain WR cowpox virus (#VR-19) were obtained from American Type Culture Collection (ATCC), Rockville, MD. Viral material HSV and cowpox virus were prepared in cultures of Vero cells (kidney African green monkey, ATCC #CCL-81), at that time, as materials input and CMV were produced in the cell line MRC-5 lung of a human embryo (ATCC #CCL-171). Levels of units the formation of plaque (EOB) for these viruses was determined in the original cell line, and the materials were stored at -85°C.

The methods of preparation of the anti-virus part - If not noted otherwise, the local creamy emulsion of n-docosanol used in the working examples was prepared with the help of (in weight %) 10% n-docosanol, 5% sucrose stearate, 8% mineral oil, 5% propylene glycol USP, 2.7% of benzyl alcohol, the rest is water (Katz et al., in Slow Infections of the Central Nervous System. Ann. N.Y. Acad. Sci. 724:472-488, 1994). Control filler cream contains 10% stearic acid, which was attended by a total of 10% of aliphatic hydrocarbon. Components was heated up to 80°and mixed while cooling to room temperature. Typically, the mixture solidifies when the temperature drops to 30°C. Each day during treatment with 0.3 ml of newly restored (water) ACV was mixed with 2.7 ml containing n-docosanol cream, giving the drug with 5% ACV and 9% of n-docosanol; the corresponding control creams mixed with 0.3 ml of water. The mixture was mixed for 5 minutes in a vessel SPEX (SPEX Industries, Inc. Metuchen, NJ) with SPEC Mixer.

n-Docosanol was suspenderbelt in PLURONIC F-68® (poloxamer 188; molecular weight of 8400), as described (Katz et al., Proc. Natl. Acad. Sci. USA 88:10825-10829, 1991; Pope et al., J. Lipid Res. 37:2167-2178, 1996). PLURONIC F-68® was diluted to 12 mmol in sterile saline at 37°and then was heated to 50°C. n-Docosanol was added to a solution of PLURONIC F-68® 90 mmol), the mixture was subjected to sonication using an ultrasonic device Branson 450 (Branson Ultrasonics, Danbury, CT) for 21 minutes at an output power of 65 watts, so the mixture was heated to 86°C. the resulting suspension consists of a very small round particles of an average size of 0.1-0.5 micron that was measured by transmission electron microscopy (Katz et al., Proc. Natl. Acad. Sci. USA 88:10825-10829, 1991). Control filler for this suspension contained only PLURONIC F-B8® in saline solution.

Ways to optimize antiviral activity the Antiviral activity of pharmaceutical compositions containing long-chain aliphatic compound and a nucleoside analogue, has been optimized using four different tests, including (1) infection of the skin of the Guinea pig virus HSV, (2) the formation of viral plaques HSV, (3) many infected with the herpes virus cells, and (4) inhibition of reproduction of the virus.

System in vivo studies used Bessa is osyh Guinea pigs (250-400 g), which were obtained from Charles River Laboratories, Wilmington, MA. Their backs were purified with ethanol and sterile saline and was inoculable virus HSV-2 under General anesthesia using Ketamine (Parke-Davis, Morris Plains, NJ) and Nembutal (Abbott Laboratories, North Chicago, IL). Saline solution (75 μl)containing 5×105JOB HSV-2, was applied to plots 4×4 cm on the backs of Guinea pigs with subsequent inoculation with tattoo tool. This is a common experimental method for the evaluation of local therapy in the treatment of mediated HSV skin diseases (Spruance et al., Antiviral Res. 9:295-313, 1988). Each animal had 6 sites of inoculation. Treatment with 200 μl of cream was made with a glass rod with gentle circular Pomerania 2 times a day. Plots were evaluated from the point of view of the number of vesicular lesions at the marked points in time.

Study of the formation of plaques in vitro for HSV was performed using Vero cells caused 1.5×105/ml in 16-millimeter deeper (1 ml) or 35-mm deeper (2 ml) in DMEM with 5% fetal calf serum, 1 mmol of pyruvate sodium, 4 mmol L-glutamine, 50 IU/ml penicillin, 50 mg/ml streptomycin and 10 mmol of HEPES buffer. Varying the concentration of a suspension of n-docosanol or corresponding control n is politely (without n-docosanol) was added at the initial stage of cultural development. After 24-hour incubation was added to test antiviral medication (for example, ASV), and then all cultures insulinomas with the required value EOB HSV. Cultures were incubated (10% CO2in the air, high humidity) for an additional 44 hours, stained (abrasively/binder consists of 1.25 mg/ml carbol-Magenta plus 2.5 mg/ml methylene blue in methanol), and counts the number of HSV plaques with the preparation of the microscope (magnification 10 times).

Research playback viruses in vitro for HSV and cowpox virus were initiated and described for the formation of plaques HSV in Vero cells in 16-millimeter cavities, but the plates were incubated in the amount of 3 days after inoculation with 500 EOB/recess, as shown. At this point, cultural surfactant fluid was collected and dissolved in fresh culture of Vero cells (1×105/ml, 0.1 ml/recess in the plate with 96 holes) to study content EOB. These secondary plates were incubated for 72 hours before fixation, staining and counting of cytopathology for HSV.

Studies of CMV infection and BBO was initiated with cells MRC-5 as described above to obtain JOB HSV in 16-millimeter cavities. Two days after infection, culture medium was ameena fresh medium without inhibitor. After an additional 2 days of incubation, the cells were collected by trypsinization and studied on the subject of infected cells in the test infectious center using cells MRC-5. In short, trypsinization cells were diluted in cell cultures of MRC-5 at the plate with 24 holes. After 6 days incubation of the secondary culture was stained, and counted cytopathology for BBO and CMV.

EXAMPLE 1

Antiviral activity of aliphatic alcohols C21-s

Aliphatic alcohols was suspendibility in surface-active substance PLURONIC F-68® using the following procedure described for alcohol n-docosanol. Surfactant was diluted to 10 mg/ml in the medium Needle, modified the highest glucose Dulbecco at 37° (DMEM; Whittaker Bioroducts, Walkersville, MD); and the solution was heated to 50°C. n-Docosanol was added to a final concentration of 30 mmol of sodium, surface-active substances, and the mixture was treated with ultrasound for 21 minutes at initial power of 65 watts using an ultrasonic device (Branson 450), which caused heating of the suspension to 88°C. the Resulting suspension contained all particles average size of about 0.3 microns, as determined using transmission electron microscopy. Control solutions containing PLURONIC F-68® without add the military aliphatic alcohol and suspension contains great concentrations of surfactants and/or n-docosanol, were prepared using almost the same procedure.

Suspension stearyl alcohol (C18), archipelogo alcohol (C20), heneicosane (C21), lignocellulose alcohol (C24) and n-hexacosanol (s) were prepared using almost the same Protocol as described for the suspensions of n-docosanol. For aliphatic alcohols, longer than C22, the mixture was heated before sonication up to 80°for lignocellulose alcohol (C24) and up to 90°for n-hexacosanol (s) and 1-octacosanol (s). n-Hexadecanol was obtained from Aldrich Chemicals (Milwaukee, WI); stearyl alcohol and arachidonoyl alcohol were obtained from M. Michel (New York, NY), and other compounds from Sigma Chemical Co. (St. Louis, MO).

Strain MS of herpes simplex virus 2 (HSV-2; obtained from the American Type Culture Collection, Rockville, MD; ATCC No. VR-540) was used for infection of kidney cells of the African green monkey (Vero cells; ATCC No. CCL 81), to determine the impact of suspensions of aliphatic alcohol in the efficiency of formation of plaques. The Vero cells were cultured in 6×105cells in 1.8 ml of medium in 35-mm deepening or 3×105cells in 0.8 ml of medium on a 16-mm hole in DMEM, supplemented with 5% serum, fetal calf, sodium pyruvate, L-glutamine, penicillin/St is atomizing and 1 mmol of Hepes buffer at 37° C in humidified incubator containing 10% CO2. Control suspension surfactants or suspension containing aliphatic alcohols, are added at the beginning of the cultivation. After 24 hours the cultures were added to the virus HSV-2 with 175 EOB/35-mm deepening and/or 50 EOB/16-mm deepening.

After about 42 hours after the addition of HSV-2 culture were washed once with physiological saline. Cells were fixed and stained with methanol containing carbol-fuchsin (1.25 mg/ml) and methylene blue (2.5 mg/ml), and was counting plaques. Data presented are averages of duplicate cultures, which generally vary less than 10%, and statistical comparisons were made using student's criterion.

The suspension containing aliphatic alcohols C21, C24, s or s, inhibited the formation of plaques of virus HSV-2 in Vero cells around curves dose response similar to the curves for n-docosanol (C22). Typical results are shown in Table 1. Effective concentration (µm)required for 50% inhibition (EC50) formation of plaques in table 1.

No obvious effect of chain length on the inhibition of the formation of plaques HSV-2. All alcohols from C21 to s inhibited formation is Laski virus HSV-2, and none of the compounds showed significantly greater activity than C22. Connection with odd length chains, heneicosane (C21), also inhibited the formation of plaques by the virus HSV-2, showing that there is no obvious influence of the chain length (i.e. molecules with odd length chains operate in the same way as molecules with an even length).

Suspension stearyl alcohol (18) and archipelogo alcohol (C20) were toxic to Vero cells when added in such quantities, at which the observed inhibiting virus activity of n-docosanol. At concentrations that were not cytotoxic (0.2 µmol for stearyl alcohol and 2 µmol for archipelogo alcohol) is equivalent to the concentration of aliphatic alcohols With 18 and 20 did not show inhibition of the formation of viral plaques. Control suspensions of surface-active substances, which were absent aliphatic alcohol, were not cytotoxic and did not show antiviral activity.

EXAMPLE 2

Action increase the ratio of surfactant to aliphatic alcohol

Antiviral effect of increasing the ratio (weight) of surfactant to the aliphatic alcohol has been demonstrated by increasing the ratio of PLURONIC F-68® and n-docosanol (compared with Example 1, which used the demonstrated ratio of 1:1 (by weight) of surfactant to alcohol). Suspension 1:1 had a molecular ratio of 26:1 for molecules of n-docosanol (molecular weight 326,57) and surfactants (molecular weight of 8400). In General, increasing the amount of surfactant reduces the size of particles in suspension and causes the formation of smaller single-disk, and not megaplatinum bubbles (Sandra et al., J. Biol. Chem. 254:2244-2249, 1979). This leads to the fact that more alcohol is on the surface of the particles, where possible, to interact with the cells.

Suspension of n-docosanol were obtained almost in the same way as described in Example 1 using a constant amount of alcohol, but with increasing amounts of surfactants to achieve ratios of 3:1, 5:1 and 10:1 (by weight) between PLURONIC F-68® and n-docosanol in the final suspension. The increase in the ratio between the surface-active substance and alcohol increases the antiviral effectiveness of the suspension in the culture of Vero cells (Figure 2). That is, the suspension with a ratio of 3:1 between the surface-active substance and alcohol showed greater antiviral activity than suspension with a ratio of 1:1 (at a concentration of n-docosanol ≥8 mmol); suspension with 5:1 ratio showed increased antiviral activity compared with a suspension ratio of 1:1 (at a concentration of n-docosanol ≥4 mmol); and suspension with appropriate what achenium 10:1 showed greater antiviral activity compared with a suspension ratio of 1:1 (at a concentration of n-docosanol ≥ 1 mmol). Antiviral activity was dependent on n-docosanol in suspension because of the control culture, incubated with the same concentrations of surfactants in the suspension, as for each tested above ratio, showed practically no antiviral activity (figb).

Increased ratio of surface-active substance and alcohol correlated with an increase in the number associated with the cells n-docosanol, which was determined using Vero cells, incubated for 24 hours with suspensions of surfactant-n-[1-14With] docosanol. Cells incubated with suspensions, containing a ratio of 4:1 between the surface-active substance and n-docosanol connected 7,8×10-6μg/cell, while the equivalent culture, inkubirovaniya in suspension with 1:1 ratio, was associated 3,1×10-6μg/cell. Optimal antiviral activity of n-docosanol was achieved when the ratio between the surface-active substance and alcohol from about 4:1 to 5:1 (by weight).

Antiviral activity of aliphatic compounds was not a property unique combination of aliphatic compounds and certain non-ionic surfactants in the suspension. Then there are other detergents gave effective is antivirusni suspension aliphatic alcohol. Suspension of n-docosanol with nonionic octoxinol a detergent (TRITON X-100®, Rohm & Haas) were prepared by: (a) fusion 2.5 g of n-docosanol with 1.5 g of detergent at 90°b) mixing the molten solution with 500 ml of saline solution at 90°and 1.15 g of polyvinylpyrrolidone (PVP), b) processing the hot mixture microdigital at 1300 pounds per square inch for 5 cycles, and d) ultrafiltration treated mixture through the cartridge with hollow fiber to eliminate excess detergent and PVP. A control suspension of the detergent was prepared in a similar manner except for the absence of n-docosanol. Deoxycholate suspension/n-docosanol (weight ratio 1:1 between the surface-active substance and alcohol) were prepared almost the same way as described above.

And octoxynol, and deoxycholate suspension of n-docosanol inhibited the formation of plaques of virus HSV-2 test on Vero cells. Typical results are shown in Table 3. Suspension of octoxynol/n-docosanol inhibited the formation of plaques compared to octoxinol control suspension at concentrations of n-docosanol greater than or equal to 2 mmol EU50approximately 4.5 mmol. The nonionic surfactant used to prepare suspensions of aliphatic alcohol, had no effect on the antiviral activity is here suspensions.

The increase in the ratio between the surface-active substance and n-docosanol significantly increased antiviral activity suspension. That is, the number of n-docosanol in suspension required for 50% inhibition of the formation of plaques, decreased (for example, 15 mmol to 3 mmol).

EXAMPLE 3

Antiviral activity of aliphatic alkane, n-docosane

Suspension of surfactant/n-docosane (Sigma Chemical Co.) was prepared almost the same way as described in Example 1. Antiviral activity of suspensions of surfactant/n-docosane was compared with the activity of such suspension surfactant/n-docosanol using the test on Vero cells for measuring the inhibition of the formation of plaques HSV-2 is almost the same as described in Example 1.

As shown in figure 4, the suspension of surfactant/n-docosane inhibited the formation of plaques by the virus HSV-2 in cultures of Vero cells with the curve of the dose response curve similar to the curve of the dose response of suspensions of surfactant/n-docosanol. Suspension of n-docosanol () and n-docosane (in PLURONIC F-68® inhibited the formation of viral plaques HSV-2 in cultured Vero cells, incubated with suspensions for 12 hours prior to adding the Oia HSV-2. Control suspensions of surfactant did not show antiviral activity (data not shown). Consequently, an aliphatic alcohol, with 22 carbon atoms, and alkane showed comparable antiviral activity, indicating that hidroxilasa share is not required for activity, as measured by the inhibition of the formation of viral plaques.

EXAMPLE 4

Oxidation of 1-gidroksamovoi share n-docosanol causes cytotoxicity

Suspension nonionic detergent surfactant/n-docosanoic acid (Sigma Chemical Co.) was prepared and tested for antiviral activity using Vero cells and HSV-2 is almost the same as described in Example 1. Fatty acid with 22 carbon atoms was toxic to Vero cells when used at concentrations equivalent to the concentrations at which it appeared inhibition of virus n-docosanol (see Table 2). When the cultures was added to a suspension of n-docosanoic acid at the concentration of 4 mmol 15 mmol, cells were rounded and depart from the plate. When transferred concentrations of n-docosanoic acid (≤ 2 mmol) antiviral activity was approximately similar to the activity observed with suspensions of n-docosanol at the same concentrations, but significantly less than was observed with suspensions of n-docosanol when the concentration is the Nations from 4 to 15 mmol. Thus, fatty acid C22 have shown some antiviral activity, being diluted to portable cell concentrations, but had increased compared with the corresponding aliphatic alcohol cytotoxicity.

EXAMPLE 5

Antiviral activity monounsaturated aliphatic alcohols with 22 carbon atoms

Suspension of surfactant/erollover alcohol (CIS-13-dokusen-1-ol; Sigma Chemical Co.) were prepared and tested for antiviral activity using Vero cells and HSV-2 is almost the same as described in example 1 to determine the effect of unsaturation of the hydrocarbon chain. Suspension of surfactant/erollover alcohol was toxic to Vero cells, when added to the cultures at the concentrations at which it was effective n docosanol (2-15 mmol). However, as shown in Table 2, portable cell concentration (≤ 1 mmol) showed a significant inhibition of the formation of plaques of virus HSV-2 (up to 93%). In addition, when the concentration erollover alcohol 1 mmol was not observed no toxicity to the cells. The effective concentration required for 50% inhibition of the formation of plaques erollover alcohol (EC50=0.15 mmol)was 60-fold lower than the concentration required for n-docosanol (EC50

Likewise determined the antiviral activity of the TRANS-isomer monounsaturated alcohol to 22 carbon atoms, brassicicola alcohol (TRANS-13-dokusen-1-ol). Suspensions were prepared with other non-ionic surface-active agent, TETRONIC-908® (BASF), and tests on inhibition of virus were carried out using the HSV-1 instead of HSV-2 by essentially the same procedures described in Example 1. As shown in Table 2, passively alcohol showed antiviral activity similar to the activity of n-docosanol. Cellular toxicity brassicicola alcohol was significantly lower than the toxicity erollover alcohol.

On the basis of these results, the addition of one double CIS-connection (but not TRANS) at position 13 of aliphatic alcohol with 22 carbon atoms greatly increases antiviral activity. Alcohol with double TRANS-communication was less toxic than alcohol with dual CIS-relationship. Increased cytotoxicity may be caused by bending of the molecule due to the double Cys-connection.

Suspension of surfactant/erollover alcohol had no direct virusbouncerules actions. That is, the incubation of the virus HSV-2 with a suspension of surfactant/erollover alcohol within 2 hours't have disabled the virus, which was identified by subsequent formation of plaques on Vero cells.

EXAMPLE 6

Testing erucamide in cultures of mammalian cells

Erucamide (CIS-13-docosenoic; molecular weight=337,59) is a long-amidon to 22 carbon atoms with one double bond, such structure erasilova alcohol. Suspension of non-ionic surfactants/erucamide (Aldrich Chemical Co.) was prepared using TETRONIC-908®, and tested for antiviral activity using Vero cells and HSV-2 is almost the same as described in Example 1. Amide 22 carbon atoms was toxic to Vero cells when used at concentrations of 3 mmol or higher, like toxicity observed in Aracinovo alcohol and n-docosanoic acid (see Table 2). When the cultures were added to the suspension of erucamide with concentrations of 3 mmol 15 mmol, cells were rounded and separated from the plate. At lower concentrations of erucamide in suspension showed a significant antiviral activity. When hyphenated concentrations erucamide (≤ 1.7 mmol) antiviral activity suspension erucamide was less than that of suspensions erollover alcohol with almost equivalent concentrations, but more than that suspensions of n-docosanol, n-docosane, n-docosanoic acid or brassicicola alcohol. That is, the percentage of inhibition of the formation of plaques for suspensions of erucamide was 78%, 7 mmol, 68% at 1.5 mmol, 58% at 1.2 mmol, 44% at 0.89 mmol, 42% at 0.59 mmol and 34% at 0.03 mmol. Thus, the amide 22 carbon atoms showed significant antiviral activity in portable cell concentrations, but increased cytotoxicity compared with a saturated aliphatic alcohol with 22 carbon atoms (n-docosanol) and similar toxicity of mono-unsaturated erollover alcohol having 22 carbon atoms.

EXAMPLE 7

Cytotoxicity in cultures of mammalian cells

n-docosanol shows minimal cytotoxicity toward cultured cells even during prolonged incubation. Three tests were used to calculate the influence of aliphatic alcohols on the survival of cells and proliferation: 1) counting cells using hemocytometer and determine the number of cells without Trypanosoma blue; 2) measurement of implementation3H-thymidine into cellular DNA by adding3H-thymidine (obtained from New England Nuclear) to a culture medium, lizirovania cells with water and collect the DNA on the filter paper; and 3) measurement of total cellular protein using sulforhodamine test, adapted for use plates microtitre with 96 cavities (Skehan et al., J. Natl. Cancer Inst. 82:1107-1112, 1990). All of these methods are well-known tests on cell survival and cytotoxicity.

The tested cells were Vero cells (see Example 1), WI-38 diploid cell line human embryo lung (ATSS No. CCL 75), HFL1, diploid cell line human lung of the fetus (ATSS No. CCL 153) and the cells of the foreskin of the human fetus (ATSS No. CCL 1635). Hybridoma line b-cells mouse (indicated by the MBI-9) was designed and cultured as described previously (Marcelletti et al., J. Immunol. 148:3857-3863, 1992), however, other tumor lines and hybridoma, such as any of the cell lines was ATSS TIB or HB, equivalent can be used to determine the effects of aliphatic compounds in suspensions on cell proliferation. All cells were cultured in DMEM, supplemented with 10% serum, fetal calf, sodium pyruvate, L-glutamine and penicillin/streptomycin using procedures well known to the current level of technology. Suspension of aliphatic alcohols were prepared almost the same way as described in Example 1.

In the first test of Vero cells were cultured for up to 72 hours in the presence of 9 mmol of n-docosanol contained in suspension with a surface-active agent without significant destructive effects of the inoculation of cultures in the amount of 6×105cells in 1.8 ml of medium in 35-mm deepening or 3×105cells in 0.8 ml of medium on a 16-mm recess. Typical data are presented in Table 3,they show what is the total number of viable Vero cells and foreskin fibroblasts remained unchanged after incubation with suspension aliphatic alcohol within 24 hours to 72 hours. Other tested cell lines, including fibroblasts of normal skin (ATSS CRL 1900), lung cells, WI-38, human lung cells of the fetus and hybridoma b cells showed similar cell viability in the presence of a suspension of n-docosanol when the cells were inoculated with high enough densities. Control suspensions of surface-active substances without aliphatic alcohol also showed no cytotoxicity for Vero cells, but showed a time-dependent cytotoxicity for cells of the foreskin of the fetus, which was not observed for containing alcohol suspension. For cell lines of the foreskin of the fetus adding aliphatic alcohol has obviously reduced the cytotoxic effect of surface-active substances.

Despite the fact that cell lines remained immune to Trypanosoma blue even after 72 hours of incubation with n-docosanol, normal skin fibroblasts, foreskin fibroblasts, cells WI-38 human lung cells of a fetus was found significant morphological changes in the analysis using light microscopy. P is the following 72 hours incubation with suspensions of alcohol in the cytoplasm of the cells appeared numerous light transmitting sections and cells became aquaitance. Cells treated with control suspensions of surfactant, showed no vacuolization after 72-hour incubation.

In contrast to the absence of cytotoxicity, usually observed with suspensions of n-docosanol, suspension stearyl alcohol (18) and archipelogo alcohol (C20) were highly cytotoxic to all these cell lines. In the presence of these aliphatic alcohols 18 and 20 carbon atoms cells growing in monolayer were detached from the plates and were visualized. Suspended cells were also visualized in the processing of suspensions and stearyl archipelogo spirits.

Viability was calculated for different cell lines or by measuring penetration3H-thymidine into DNA, or by measuring total cellular protein by staining sulforhodamine. Typical results are illustrated in figure 5 and show inhibition penetration3H-thymidine into DNA of hybridoma b cells at various concentrations of aliphatic alcohols with 18, 20 and 22 carbon atoms. IC50for stearyl alcohol (18) for the line b-cells and other cell lines was equal to less than 35 μmol; for archipelogo alcohol (C20) IC50approximately 1.7 mmol. On the contrary, IC50for n-docosanol estimated through extrapolation, equally the elk approximately 20 mmol and was more than observed using only surface-active substances. Thus, we observed an approximately 50-fold reduction IC50when shortening aliphatic alcohol with 20 carbon atoms at the 2 carbon atom.

The data shown in figure 5, were obtained after 48 hours of incubation with suspensions; however, the apparent toxicity was noticeable within 24 hours of incubation. Suspension heneicosane (C21) and suspension of alcohols with longer carbon chain, lignocellulose alcohol (C24), n-hexacosanol (s), and n-octacosanol (s) showed the same minimum level of cytotoxicity observed for suspensions of n-docosanol.

The impact of suspensions of n-docosanol and n-docosane on cell proliferation (citistat) was calculated using the test staining sulforhodamine performed on cultures of fibroblasts foreskin man, incubated in plates with 96 cavities. The results shown in figa and 6B show that the inhibiting effect of suspension of n-docosanol depended on the initial density of the cells in vivo cultures, while suspension of n-docosane did not show noticeable antiproliferative effect compared with the control suspension surfactant at any density cells. The results, shown in Fig.7, to show the shape, the cells associated with the suspension of n-docosanol, showed greater inhibition of proliferation depending on the total time of incubation. That is, a longer incubation resulted in greater inhibition of cell proliferation.

The foreskin fibroblasts deposited on the plate with suspensions aliphatic alcohol or without it, or with the control suspensions of surface-active substances with a density of 1000 cells/recess (figa and 7) or 30,000 cells/dredging (figb) on the plate with 96 holes. After incubation for 72 hours or 96 hours at 37°the cells were precipitated with trichloroacetic acid, stained with sulforhodamine and were calculated by measuring OD540the reader plate microtitre. 6 shows the results obtained for cells incubated for 96 hours, and Fig.7 shows the results for cells incubated for 72 hours, compared to 96 hours (1,000 cells/recess).

Suspension with a concentration of n-docosanol more than 3 mmol inhibited cell proliferation, put on a plate with a density of 1000 cells/cavity, tested after 96 hours of incubation (figa). On the contrary, the suspension alkane with 22 carbon atoms, n-docosane, showed minimal antiproliferative effect compared to the control suspensionoverdosing-active substances (figa). At higher initial densities of cells (figb) or a shorter time of incubation (Fig.7) or at concentrations less than 3 mmol n-docosanol not inhibited cell proliferation compared with the control suspensions (only surfactant in the suspension). Similar results were observed when n-docosanol were incubated with cells WI-38, human lung cells of the fetus and normal skin fibroblasts, using the same test on the proliferation, as described for 6, and 7.

The suspension containing aliphatic alcohols with more than 20 carbon atoms, showed a small cell toxicity. Obvious cytotoxic effect was observed only when cells were placed on the plate with low densities and were incubated with concentrations of n-docosanol more than 3 mmol within 72 hours and longer. Control suspension, which was absent aliphatic alcohol, did not show cytotoxic activity.

The length of the carbon chain aliphatic alcohol affects its toxicity to cells, in contrast to the results presented in Example 1, showing the lack of apparent effect of chain length on antiviral activity. IC50decreased from more than 15 mmol for alcohols with 22 or 21 carbon atoms up to 1.5 mmol of alcohol with 20 carbon atoms and up to less than the than 35 µmol for alcohol to 18 carbon atoms. A significant increase in toxicity of aliphatic alcohol containing just four carbon atoms less than that of the alcohol to 22 carbon atoms that were not expected.

EXAMPLE 8

Antiviral activity of compounds with stearic acid

Was measured antiviral activity and cytotoxicity of stearic acid (molecular weight 284,5), dissolved in ethanol or suspended in TETRONIC 908®, practically the same as described in Example 1. Antiviral activity was measured as the percentage of inhibition of the formation of plaques of HSV-2 in the culture of Vero cells, almost the same way as described in Example 1. Cytotoxicity was investigated by examination under a microscope of cells for cell growth and integrity of cell cultivation plates in comparison with untreated control cultures. Was not determined noticeable toxicity, if the monolayers treated cells were indistinguishable from untreated cells. Moderate toxicity was determined, if the monolayer cells were purified as compared to the control cultures. Toxicity was determined in such concentrations that destroyed the monolayer treated cells, as evidenced by the separation of cells from the cultivation plate. There was little toxicity in suspensions of stearic acid in TETRONIC 908® concentration walls is inovas acid 11 µm and 22 µm and for a solution of stearic acid in ethanol with a concentration of stearic acid 3.5 µmol. All these treatments showed no more than 10% inhibition of the formation of plaques HSV-2 compared with infected control cultures. Moderate toxicity was observed after treatment of a suspension of stearic acid in TETRONIC 908® with the concentration of stearic acid 11 44 µmol µmol and a solution of stearic acid in ethanol with a concentration of stearic acid 35 µmol; antiviral activity could not be determined because of the state of the cells. All suspensions and solutions with a concentration of stearic acid 88 µm and 350 µm were toxic, and antiviral activity could not be determined because the monolayer of cells was destroyed.

EXAMPLE 9

Antiviral activity applied topical compositions containing n-docosanol or stearic acid, for animal model

Antiviral activity of compounds containing stearic acid, was confirmed in vivo using a model of infection of Guinea pigs with the virus HSV-2. Hairless Guinea pigs (six males in the test, each weighing 200-300 g; obtained from Charles Rivers Laboratories, Wilmington, MA) were subjected to anesthesia and inoculated with virus HSV-2 (strain ATS VR-540 grown in Vero cells and purified using standard methods). On day 0, each animal was inoculated in six places of inoculation within the site area of 4 cm2STIs is e 75 μl of physiological saline, containing 9,75×106EOB/ml 24 hours after inoculation (day 1) animals were treated topically three to five times a day creams, described below, or water as a negative control, and processing is continued under the same conditions during days 2, 3 and 4. The inoculation was evaluated for skin irritation and the formation of bubbles daily on days 2, 3 and 4. Irritation was scored on a scale from 0 to 4: 0 for normal skin with no erythema; 1 for mild erythema; 2, moderate erythema; 3 severe erythema and 4 for severe erythema, accompanied by bleeding. Bubbles was defined as white, fluid-filled pustules.

Compositions for topical application were: cream containing n-docosanol; cream containing stearic acid; and placebo. n-Docosanol cream contains 10% by weight of n-docosanol (Michel and Co., New York, NY), 5% by weight of sucrose stearate (Croda, Inc., New York, NY), 8% by weight of mineral oil NF (Witco Corp., Newark, NJ), 5% by weight of propylene glycol USP, 2.7 percent by weight benzyl alcohol NF (Ruger Chemical Co., Irvington, NJ) and 69.3% of purified water USP. Cream with stearic acid contained 10% by weight of stearic acid (Henkel, Cincinnati, OH), 5% by weight of sucrose stearate (Croda, Inc., New York, NY), 8% by weight of mineral oil NF (Witco Corp., Newark, NJ), 5% by weight of propylene glycol USP, 2.7 percent by weight benzyl alcohol NF (Ruger Chemical Co., Irvington, NJ) and 69.3% of purified water USP. Both cu is mA were prepared by combining all ingredients, with the exception of water, heated to 80°and mixing the ingredients at a speed of 400±5 rpm (with the help of stirrer Heidolph RZR), which at 85°water was added with increasing the stirring speed to 1900±5 rpm After 3 minutes at a temperature of 80°the mixture was left to cool with constant stirring until 30°With (approximately 8 minutes). Placebo was prepared by heating to 70% of polyethylene glycol (PEG) 400 NF and 30% PEG 3350 NF to 65°while PEG 3350 didn't melt completely, then continuous stirring of the mixture at 400 rpm until then, while the mixture was cooled to 30°C.

The results of these tests are presented as average values in Table 4. Readings on day 2 was filmed at 48 hours after inoculation; day 3 - 72 hours after inoculation; and day 4 - 96 hours after inoculation (total of six seats each reading). As can be seen from table 4, on the second day none of the creams did not influence significantly on the amount of bubbles compared to the processed water control instances, and neither it from places showed no irritation. On the third day the place treated with n-docosanol cream showed significant inhibition of the number of bubbles with respect to the treated water control. It seems that three of the application of a cream containing n-docosanol, the day would be the about enough because five applications a day gave almost the same level of inhibition. On the third day designated, processed cream stearic acid three times a day, showed moderate inhibition of bubbles compared with the treated water control seats, while the areas which were treated five times a day, showed a statistically significant inhibition of bubbles. Application polietilenglikoli placebo five times a day did not reduce significantly the number of bubbles compared to the treated water control places at any time.

On the third day some irritation was observed for cream n-docosanol, and cream with stearic acid. On the fourth day of treatment, three times a day cream containing n-docosanol, significantly reduced the number of bubbles in comparison with the control seats, although there was slight irritation. On the fourth day of treatment five times a day cream containing n-docosanol, or cream containing stearic acid, significantly reduced the number of bubbles compared to control and placebo, although mild erythema was observed in both treatments.

These are obtained in vivo results show that topical treatment of infections of HSV-2 creams containing n-docosanol is as active ingredient, or stearic acid as the active ingredient, can significantly reduce the amount of bubbles caused by infection. Cream containing n-docosanol as the active ingredient, is more effective in the treatment of viral infections as a significant reduction in the number of bubbles was observed at only three applications per day, while to notice a reduction in the number of bubbles when you use a cream containing only stearic acid as the active ingredient, required five applications a day.

EXAMPLE 10

Antiviral activity of topically applied n-docosanol and stearic acid in a clinical study in humans

Antiviral activity of compounds containing stearic acid, was confirmed in vivo in clinical studies for the treatment of oral herpes in 648 immunocompetent patients who started treatment within 12 hours after localized episode of oral herpes (that is, when the initial feeling of prodrome, erythema or papule, but not when the bubbles). These patients had a history of acute cases of lip herpes with an average fixed-length episodes of 8.9 days (from the beginning of sensations and/or erythema until full recovery). This duration is consistent with the usual will continue what inetu from 8 to 10 days for episodes of oral herpes in the published reports of this disease (R. J. Whitley, in Fields Virology, p. 2316).

In these studies, patients randomly received either creams containing 10% n-docosanol, or 10% stearic acid, prepared almost the same way as described in Example 9. Patients applied the cream topically on a localized area exposed to herpes, five times a day for at least five days (25 application schedule with re-application after active exercise, showering, or washing, re-application was not considered an application for schedule). If an episode of herpes continued after five days, the patient continued to apply the cream for up to ten days (50 application schedule). Patients kept a diary of the time of application, focal pain and scabby symptoms and examined twice daily during the treatment period to examine the effectiveness of treatment.

The criteria used to assess treatment contained during recovery, which contains the interrupt episode (defined as complete elimination associated with an episode of symptoms before reaching the bubble phase) or complete recovery (defined as the absence of a peel without traces of active lesions, regardless of whether there were any residual after the defeat of the skin changes such as erythema, peeling or asymmetry); time of termination of the reproduction of the virus (only investigated the I number 1); time pain relief; pain; time to stop itching and time of the cortical stage.

For comparison, we used data of the patients ' histories and published the results (Spruance et al., New Engl. J. Med. 297:69-75, 1977) is not affected by the lesions.

Table 5 shows the results of two independent studies (indicated by numbers in parentheses in the table). These data show that the duration of herpes fever is significantly reduced to an average of 5.5 days after treatment with either a cream containing n-docosanol, or by using a cream containing stearic acid, in comparison with the marked for patients still 8,9 days duration of untreated herpes fevers. Thus, the duration was significantly reduced by more than 35% (P≤0.0001)and if patients were treated from the very beginning of the episode using any cream containing n-docosanol or cream containing stearic acid. In addition, treatment at an early stage with the help of any such cream has shortened the duration of pain symptoms associated with recurrent episodes of herpes with approximately 6 days, when the disease is not treated, to less than 3 days for the treated areas.

EXAMPLE 11

Improved healing of lesions of the virus In The G-1 after local treatment using the formula, containing n-dakotan

Ten patients with previous histories of occurrence of lesions of the virus HSV-1 on the face (herpes fever) was given a cream formula with 5.0 mg/ml n-docosane, suspended in 20 mg/ml poloxamer block copolymer surfactant; formula cream contains 5-8% by weight of mineral oil NF as a softener, 5% by weight of propylene glycol USP as a humectant and preservative, 1-3% by weight benzyl alcohol, NF as a secondary preservative and balanced purified water as the aqueous medium.

People were given the instructions for applying the cream on the affected areas or early inflammation around the mouth, when people discovered herpes fever. These people had herpes fever, untreated, for an average of ten days, while all untreated herpes fever developed in the bubbles, which turned into scabs and passed. People were instructed to apply the cream to the affected skin areas at least twice a day and up to four times a day. People were also instructed to record the stage of infection (erythema to papules and until bubbles to swelling and to scab), which they observe, and record subjective observations regarding pain associated with lesions in the Rus HSV-1.

Each person was treated at least one herpes fever during the study. All patients noted a decrease in pain in the treatment of herpes fevers cream containing n-dokusan, compared with the previous lesions, which were not subjected to treatment. For each person, the duration of infection of HSV-1 compared with previous infections decreased from 20% to 60% (i.e., the duration ranged from 4 to 8 days depending on the person). At least half of the people who participated in the study, treating herpes fever four times a day by using a cream containing n-dokusan, herpes fever has not progressed to the bubble stage. Instead, for local treatment of lesions of the virus HSV-2 at the stage of erythema or papule lesions usually not progressed beyond the stage of papules and recovered without further development of the lesion. These results show that formulas containing n-dokusan are effective in the prevention and treatment of viral infections in the local application.

EXAMPLE 12

Treatment of gruppovoj infection with formulas containing aracelly alcohol and erucamide

Aqueous suspension of 0.15 mmol erollover alcohol 1.4 mmol nonionic poloxamer 188 as surfactants containing propylene glycol USP (0.5% of the rake) and benzyl alcohol NF (2% by weight) as preservatives, were prepared in standard flexible bottles of nasal spray is capable of compression to produce the suspension in the form of an aerosol. Similarly, the preparation containing 1.5 mmol of erucamide, were prepared in containers for nasal spray to obtain a suspension in the form of an aerosol. The drugs were administered during gruppovogo season two groups (one for testing erollover alcohol and one for testing erucamide) twenty healthy people in each that have not been vaccinated against influenza in the previous 12 months.

People were provided with instructions for using the obtained suspension as a nasal spray from one to five times per day (one to two pumps in each nostril at intervals of 2-4 hours) upon detection of flu-like symptoms (congestion in the respiratory tract, pain in the body, increased sensitivity of the eyes, fever, nausea, or any combination thereof). People were instructed to record their subjective and objective observations about the severity of flu symptoms (duration of symptoms, body temperature during fever, the duration and severity of pain in the body) in the period during which they discovered the symptoms. People also instructed to record data about the use of suspension in the form of a nasal spray (number of injections and the time of reception) in order during this period. People were asked to summarize their subjective observations about the severity of symptoms when using aerosol surfactants/erollover alcohol or aerosol of surfactant/erucamide compared with the previous experience of taking gruppovoj infection.

Approximately half of participating in a study of people who used the aerosol surfactants/erollover of alcohol, as prescribed, noted a reduction in flu-like symptoms compared with previous episodes of influenza. Those who used the spray in an average of five times a day (one or two injection in each nostril), noted greatly reduced the congestion in the respiratory tract associated with influenza infection, compared with men, not treated. Those who used the spray in an average of five times a day, noted a significant decrease in the frequency of fever (from one to three times per episode of influenza), compared with men, not treated (from two to five times per episode of influenza), and a significant reduction highest recorded body temperature (average of 37.8° (C) in comparison with men, not treated (average of 38.9°). The average duration of flu symptoms in approximately half of people treated is erusalem of surfactant/erollover alcohol, 1.7 days at a time, like people, are not subjected to treatment, the average duration of flu symptoms was 3 days. These results show that the suspension of surfactant/erollover alcohol has therapeutic antiviral effect when applied to mucous membranes.

Similar results were obtained for patients treated with nazalnam spray suspension erucamide. Approximately half of the patients noted a decrease in flu-like symptoms compared with previous episodes of influenza using spray surfactant/erucamide immediately upon discovery of the symptoms. Most people have experienced greatly reduced the congestion in the respiratory tract when using aerosol on average three times a day (one or two injection in each nostril) compared to previously experienced flu-like conditions. Most of those who used the spray on average three times a day, noted one episode of fever during the period of the onset of symptoms of influenza with an average marked the highest body temperature of approximately 37°C. the Average duration of flu symptoms for people who used the spray at least three times a day, was two days, compared with the duration of symptoms of influenza in people who, not treated, constituting three days. These results show that the aerosol suspension of surfactant/erucamide has therapeutic antiviral effect when applied to the mucous membranes of the respiratory system.

EXAMPLE 13

Treatment of infection of HSV-2 through the mucous membrane with brassicicola alcohol

Candles containing 8 mmol brassicicola alcohol in suspension nonionic detergent is prepared almost the same way as described in Examples 1 and 5 were formulated by adding anhydrous dextrose (300-400 mg/suppository), vegetable starch (300-400 mg/suppository) and stearate (5-10 mg/suppository) to obtain a mixture, which was pressed in candles (1-10 g one candle) for vaginal use.

Fifteen infected with HSV-2 in women with a previous history of vaginal and/or perivaginal herpes lesions was supplied with candles and instructionals about the use of from one to four candles on the day upon detection of herpes lesions or associated with herpes lesions discomfort. Women were given instructions to record your observations about the duration of active disease, severity of lesions (phase erythema, papule, vesicle, swelling or scab), the relative amounts recorded lesions compared with the preceding paragraph is yavleniya active infection and subjective evaluation of pain or discomfort, associated with an episode of active infection. Women were instructed to use candles immediately after detection of active infection or symptoms of active infection. These women have had in the past 12-day average duration of lesions that developed in the bubbles in the absence of treatment.

In all cases, each woman was treated at least one episode of active herpes infection during the study. All patients noted a decrease in pain and discomfort in the treatment of infection by these candles compared with previous episodes of infection without treatment. In all cases, the average duration of active infection of HSV-2 to 7-8 days in the treatment of candles, and five women reported an average duration of 3-4 days. In most cases, when using candles four times a day and the beginning of treatment at the stage of erythema or papules infection has not progressed to the bubble stage and was cured after reaching the stage of papules.

Alternative suspension surfactant/brassicicola alcohol were formulated in the form of ointments containing approximately 50-80% white soft paraffin, which melted at 60°for adding and dispersing the suspension of surfactant/brassicicola alcohol before cooling. The ointment was heard in compressible tubes with prescribed what to eat to use two to five times a day as needed for external treatment of active genital herpes infections. People were instructed to use a quantity sufficient for covering defeats the virus HSV-2 genital or perivaginal areas one to four times a day immediately after the discovery of the symptoms. At least half of the patients using the ointment, reported less pain and discomfort, reduced time of treatment, the lesions not progressed to the bubble stage before cure.

These results show that suspension of surfactant/brassicicola alcohol have therapeutic antiviral effect of the local application to mucous membranes.

EXAMPLE 14

Infection treatment EBV (infectious mononucleosis) using suspensions of long chain aliphatic alcohol

Ten young men (aged 14-19 years)who were diagnosed with infectious mononucleosis (acute sore throat, fever, malaise, General lymphadenopathy, atypical mononucleosis T-lymphocytes in the peripheral blood, the total number of white blood cells in the blood 12000-18000) systematically treated by means of a sterile aqueous suspension nonionic detergent surfactants containing 10 mmol of n-hexacosanol prepared almost the same way as described in Example 1. The suspension was inyecciones (intramuscularly or intravenously) in dosiro the Ah from 0.1 mg/kg to 0.2 mg/kg aliphatic alcohol, entered by the physician in the clinical setting. Symptoms patients were monitored daily during the week and weekly for three months on the symptoms of infectious mononucleosis. All patients were EBV positive result of the test conducted at the end of the study period, which was determined by the detection of antibodies to the virus EBV in their serum using standard immune research.

All patients were observed cure symptoms of acute pain in the throat and fever within one week of submission of the suspension surfactant/n-hexacosanol and reducing symptoms of febrile illness in General within two weeks of introduction. In eight of the treated patients showed a decrease in total lymphadenopathy within two to three weeks of the introduction of the suspension of aliphatic alcohol with the return of vitality. All treated patients were less atypical mononucleonic T-lymphocytes in the peripheral blood within four weeks of the introduction under normal number of white blood cells in the blood two months after treatment.

Similar results were obtained for infected EBV patients who showed symptoms of infectious mononucleosis, which systemically treated with suspensions of n-docosanol, leagues who ucarilbuga alcohol and n-octacosanol in effective concentrations.

These results show that systemic injection of selected long chain aliphatic alcohols in aqueous suspensions has therapeutic antiviral effect.

EXAMPLE 15

n-docosanol and ACV exhibit antiviral activity against HSV in hairless Guinea pigs

The potential of anti-virus interaction of n-docosanol and ACV was investigated in vivo using a model of cutaneous infection of HSV-2 in hairless Guinea pigs. The areas of skin on the backs of hairless Guinea pigs were insulinomas by HSV-2 using tattoo tool. Infected areas of the skin were treated twice a day as prescribed, starting 2 hours after inoculation. Treatment twice a day instead of the normal mode, containing from three to five treatments were chosen because they were better able to see the differences between the combined cream and odnotrahniki creams, and a control filler containing stearic acid and did not give a positive response when applying less than 5 times a day.

Caused by the virus HSV-2 bubbles were evident at 72 hours after inoculation of HSV-2 (panel And Fig). Untreated plots showed on average 54 of the bubble at this point in time. The number of bubbles decreased by 31% when using either n-docosanol cream, lipocream ACV, but this inhibition was not statistically significant compared to the untreated group. Greater inhibition (65%) was observed for cream containing and n-docosanol, and ACV, and the average value of 19 bubbles were statistically different from the untreated group and the group treated filler. Neither n-docosanol or ACV did not cause inflammation or toxicity, even when both drugs were applied alternately.

Through 96 hours after inoculation of virus HSV-2 raw space showed on average 27 bubbles. A significant inhibition of the number of bubbles was observed in all treated plots except processed by the control filler. Plots treated with the test formula containing only n-docosanol or only ACV, reduced the average number of bubbles by 63% and 50%, respectively. An even greater inhibitory activity was observed on plots treated with a combination cream with n-docosanol and ACV, 89%. The inhibition observed for the combination of cream, was statistically greater than that observed for the test formula containing only n-docosanol or ACV, p=0.003 and p=0,0015, respectively. Again, there was no inflammation or toxicity caused by n-docosanol and ACV, even when used in combination.

Analysis of area under the curve (PPC), determined the mine as the average number of bubbles, multiplied by the number of hours during which bubbles are observed, suggests the synergy of the combination of n-docosanol and ACV. Average CPD for the untreated group was 698 puzyrko-hours. Processing only n-docosanol or only ACV led to the corresponding average CPD equal 464 (34% inhibition) and 496 (30% inhibition). Theoretical additional effect of the combination of n-docosanol plus ACV would CPD equal 322 (698 × remaining after treatment with n-docosanol [0,66] × the remaining part after processing AZW [0,7]). Average CPD for combined cream was $ 206 puzyrko-hours, with a 70% inhibition, p=0.01 vs. theoretical additional effect. Thus, these observations in vivo suggest a synergy of n-docosanol and ACV when the inhibition caused by the virus HSV-2 skin diseases and indicate that n-docosanol and ACV do not interact in vivo harmful way, at least when applied to the skin.

EXAMPLE 16

n-Docosanol and ACV exhibit synergistic activity against HSV in cell culture Vero

The potential of anti-virus interaction of n-docosanol and ACV was investigated more fully on infected HSV-2 cultures of Vero cells. The Vero cells were cultivated in medium alone or in medium containing 3 mmol of n-docosanol or 0.4 mmol PLURONIC F-68® (amounts in to the lture with 3 mmol of n-docosanol). Cultures were incubated for 24 hours, then exposed to ACV and were infected with 50 units of the formation of plaque virus HSV-2; the formation of plaques was assessed after 44 hours after that. As shown in Fig.9, raw (only Wednesday) culture showed an average of 46 plaques, and ACV inhibited the formation of plaques to 50% effective concentration (EC50)equal to 5 mmol. Such EU50for ACV was obtained for cells cultured in a medium containing PLURONIC F-68®. Culture containing n-docosanol, showed 40% less plaque than untreated or treated PLURONIC F-68® control culture that reflects the antiviral activity of this drug. In particular, note that EU50for ACW containing the n-docosanol cultures was reduced to 0.2 µm. Comparison of theoretical curve for incremental effect ACV plus n-docosanol has confirmed that this is a 25-fold improvement in activity ACV was more than expected for added effect. Cellular toxicity, such as cytoplasmic vacuoles were observed in containing ACV cultures, regardless of the presence or absence of n-docosanol.

EXAMPLE 17

Synergistic inhibition of reproduction of HSV-1 n-docosanol and AZW

We also analyzed the influence ACV on EU50n-docosanol for inhibiting the formation of fucking the key HSV-2. Despite the lack of graphical representations for n-docosanol was observed EU50equal to 2-3 mmol for inhibiting the formation of plaque HSV-2 when used separately, but when used in combination with ACV in the range of 0.2-10 µm were observed EU50for n-docosanol equal to 2-3 mmol. As outlined on isobologram (not shown), these results suggest that ACV has little effect on the antiviral activity of n-docosanol, even though the last medication significantly improves the activity of the first.

A significant decrease in EU50for ACV using n-docosanol was also observed when inhibiting the formation of plaque virus HSV-1 (not shown). This was associated with a significant decrease in EU90for ACV for inhibiting the offspring of HSV-1 (panel And figure 10). The Vero cells were treated as above indicated concentrations of n-docosanol, PLURONIC F-68® and ACV and infected with HSV-1 (500 EOB culture, 0,002 EOB/cell). Emerges at the surface of the culture was collected after 72 hours and were tested for HSV-1. In cultures containing only medium or medium plus PLURONIC F-68®, was observed EU90for ACV equal to 10 µm for inhibition of reproduction of HSV-1. n-Docosanol at a concentration of 3.3 mmol inhibited reproduction JOB by 55% and reduced EU90for ACV 17 times. Even b is greater synergy was observed when the concentration of the n-docosanol, equal to 10 µm (figure 10), when EU90was reduced to 40 times. Synergy was expressed even stronger when drug concentrations 30 mmol (EC90for ACV <0.1 µmol not shown).

To further confirm that the effects of combining n-docosanol and ACV was synergistic, the data were transformed into isobologram (panel B, figure 10). Broken line passing diagonally, shows a theoretical diagram for independent inhibitors, the curve shifts to the left displays a synergistic interaction, while the right shift would reflect antagonism (Spector et al., Proc. Natl. Acad. Sci. USA 86:1051-1055, 1989). It is clear that the experimental observation of reproduction EOB HSV-1 indicate synergy combinations of medicines n-docosanol and AZW.

EXAMPLE 18

Synergy n-docosanol and ACV when the inhibition of the replication of varicella-zoster virus (BBO person and cytomegalovirus (CMV) human cells MRC-5

The results, shown in Table 6, indicate two important points. Firstly, it was observed that the synergistic activity of n-docosanol and ACV not depended on the use of Vero cells and could also be documented for normal human cell line of fibroblasts MRC-5. Secondly, it was observed that this activity was not limited to virus and HSV could also be demonstrated PR is the inhibition of replication of human input and CMV. Diseases caused by viruses BBO and CMV, usually tend to resist treatment with ACV (Hirsch et al., in Fields Virilogy Third Edition, B. N. Fields, D. M. Knipe, P. M. Howley, eds. Lippincott-Raven Publishers, Philadelphia, pp. 431-466, 1996). Thus, the improvement of antiviral activity using n-docosanol may be clinically significant.

Cells MRC-5 were cultured (16-mm deepening, 105cells/ml, 1 ml/dredging) only in the medium or in medium containing 30, 10 or 3.3 mmol of n-docosanol or the same amount of Pluronic F-68, which was kept in cultures with a concentration of n-docosanol 30 mmol. After incubation over night acyclovir was added with different concentrations, and culture were infected with viruses BBO (groups I-V) or CMV (groups VI-X). After two days of incubation for all cultures was added to the medium without n-docosanol, Pluronic F-68 or acyclovir. After another two days, cell cultures were collected and analysed for the presence of infected cells (IR) through research in infectious center. Data about the infected cells expressed as mean values infected cells/culture, derived from four specimens (BBO) or triple copies (CMV) of the recesses in the initial group of crops (Ref. JM 1290, 460L-134/9-23-96) rvsd 4-4-97.

As shown for groups I-V in Table 6, 350000 infected Viru is Ohm input cells can be detected in cultures, containing only Wednesday, four days after infection cells MRC-5 with 500 EOB virus OIE. ACV inhibited infection BBO at EU50equal to 3 µm, and the EU90equal to 10 Microm. Replication BBO inhibited n-docosanol the EU50approximately 10 mmol. EU50EU90for ACV decreased respectively 90% and 80% of the high concentration n-docosanol equal to 30 mmol.

Similar results were obtained for CMV (groups VI-X, table 6). Four days after infection cells MRC-5 with 500 EOB virus CMV in the control cultures can be detected 200000 infected cells MRC-5, and ACV inhibited this infection if EU50EU90equal to respectively 30 and 250 µmol. n-Docosanol inhibited the replication of CMV in the EU50approximately 10 mmol. EU50EU90for ACV for inhibition of CMV replication was reduced by 90% using n-docosanol in concentrations of 10-30 mmol.

EXAMPLE 19

Nucleoside analogues, non-ACV also interact synergistically with n-docosanol when the inhibition of the replication of HSV-1 in vivo

It was interesting to determine whether to restrict anti-virus synergy with n-docosanol ATV, or other nucleoside analogues may also interact with the n-docosanol. The issue was investigated by using the reproduction JOB the PG-1 in cultures of Vero cells (11). Not processed, processed n-docosanol (15 mmol) and treated PLURONIC F-68® Vero cells were infected by 500 EOB/culture of the virus HSV-1 and were exposed to different concentrations mentioned nucleoside-analog antiviral drugs. Three days later surfaced cultural liquid was collected and analysed for posterity JOB HSV-1. In the control cultures was observed typical anti-HSV-1 EU90for ACV equal to 19 µm, which was decreased to 0.9 µmol (decrease 21 times) in the presence of n-docosanol. Nucleoside analogue of adenine arabinoside (Aga) showed EU90approximately 22 µmol when separate use, and the EU90approximately 1.4 µmol (16-fold decrease), when the culture was included n-docosanol. Trifluralin showed EU90approximately 6.8 µmol, in the absence of n-docosanol and EU90approximately 1.35 mmol (decrease 5 times)when I attended both of the medication. Similarly, ribavirin, used alone, inhibited the replication of HSV-1 with EC90approximately 24.6 mmol, which was reduced to approximately 0.33 µmol (decrease 75 times) in the presence of n-docosanol. Although these data are not shown, rifampicin is not inhibited replication In The G regardless of present or absent n-docosanol.

EXAMPLE 20

n-Docosanol and phosphate-formic acid (PMK) show additional antiviral activity against the replication of HSV-1

The potential of anti-virus interaction between n-docosanol and PMK, organic analogue of inorganic pyrophosphate presented on Fig. As shown in panel A, the raw culture of Vero cells produced approximately 107EOB virus HSV-1 three days after infection, and FMK inhibited such production EOB EU90approximately 18 µm. Similar levels of production JOB and indirect PMK inhibition was observed for cultures treated with control filler, PLURONIC F-68®. Cultures treated with only 15 mmol of n-docosanol, showed approximately 10 times less JOB HSV-1 and PMK additionally reduced production EOB EU90equal to 17 µmol. Combined exposure to n-docosanol and PMK coincide with the schedule for theoretical additive effects.

Panel B Fig illustrates two points. First, the results for cultures that did not FMK, show that n-docosanol did not inhibit the replication of vaccinia virus; 5-6×105JOB cowpox virus were reproduced regardless of the presence or absence of n-docosa the Ola or PLURONIC F-68®. Secondly, the presence or absence of n-docosanol or PLURONIC F-68® did not improve and did not suppress the antiviral activity of the company is directed against the replication of vaccinia virus, that has not happened bezmedikamentoznogo interaction for that particular virus.

EXAMPLE 21

n-Docosanol increases the inhibition of the replication of vaccinia virus nucleoside analogs

Because the vaccinia virus are not sensitive to the antiviral effects of n-docosanol, it was possible to explore the relationship between antiviral activity of n-docosanol and synergy with nucleoside analogues. As described above for panel B Fig, untreated Vero cells showed the average playback 5-6×105JOB progeny virus, vaccinia virus after 3 days after infection, regardless of the presence or absence of n-docosanol or PLURONIC F-68®. As shown in panel And Fig, replication of vaccinia virus in the control cultures (medium or PLURONIC F-68®) inhibited trifluridine, yeah-a and ribavirin EU50approximately 2, 20 and 25 Microm, respectively. EU50for each of these nucleoside analogues has been reduced at least 10-fold in cultures containing 15 mmol of n-docosanol. Vaccinia virus is usually not sensitive to the antiviral effects ACV, and treating the cells with n-docos what Nole did not change the selectivity. Panel B Fig is EU90for the same nucleoside drugs, and comparable conclusions can be derived from these results. These data show that the virus should not be sensitive to the antiviral activity of n-docosanol to n-docosanol increased antiviral activity of nucleoside analogues against this virus.

In the end, n-docosanol does not show harmful bezmedikamentoznogo interaction with ACV in any test system. Skin skin irritation Guinea-pig was not observed when these two drugs were applied separately or in combination. The skin of Guinea pigs tend to be more sensitive to irritation than human skin, it is assumed that the alternating treatment with n-docosanol and ACV also will not cause irritation. Cellular toxicity in vitro was not observed for these two drugs separately or in combination. In contrast, n-docosanol significantly improves antiviral activity ACV against HSV in vitro and in vivo. This improvement was synergistic in vitro. These results suggest that concomitant treatment of recurrent HSV disease with n-docosanol plus ACV could be very beneficial therapeutic strategy.

Antiviral synergy of n-docosanol with ACV was not limited to viruses EAP is -1 and HSV-2, and were also observed with viruses BBO and CMV. These last results are reasonable, because all of these herpes viruses are sensitive to ACV (Hirsch et al., in Fields Virology Third Edition, B. N. Fields, D. M. Knipe, P. M. Howley. eds. Lippincott-Raven Publishers, Philadelphia, pp. 431-466, 1996), although in varying degrees. This synergy was also observed for the other tested nucleoside analogues that inhibit the replication of HSV. This was expected because of various nucleoside and nucleotide analogues have a tendency to use common cellular and viral mechanisms for transport across the plasma membrane, metabolic activation and anti-virus ekspressirovali. Because viruses BBO and CMV have in common with the virus HSV steps of replication, it is likely that n-docosanol will also show synergy with nucleoside analogues, non-AZW, for the inhibition and also these viruses.

n-Docosanol shows synergy with certain tested nucleoside analogues in inhibiting replication of vaccinia virus. Replication of vaccinia virus is not inhibited by n-docosanol, which shows that the virus is not necessarily to be sensitive to n-docosanol to observe synergy with the second class of medicines. This is an important result for two reasons. First, comparison of the response of vaccinia virus with the response of the herpes virus can give details is rmatio about the molecular mechanisms of such synergy. Secondly, and more importantly, this suggests that the use of n-docosanol not necessarily be limited to the treatment of diseases caused by viruses, which are sensitive to the antiviral effects of this medication. These results indicate that n-docosanol can be used to improve the activity of nucleoside analogue regardless of viral infections, for the treatment of other diseases, such as inflammation, autoimmunity and cancer.

Antivirus selectivity tested nucleoside and nucleotide analogues appears to be unchanged in the processing of n-docosanol. The selectivity of antiviral medication type ACV depends on the characteristics of the virus, such as the expression of the viral-encoded timeinfo kinase. This explains the absence of inhibiting replication of vaccinia virus ACV regardless of the presence or absence of n-docosanol.

The degree to which n-docosanol may interact with this antiviral medication for this class of viruses, should be determined empirically. Antiviral synergy PMK and n-docosanol was not observed for HSV or cowpox virus, showing that this does not happen with all of antiviral compounds. However, there are some forecasts, such as anti-virus synergy of n-docosanol with nucleoside analogues, such is as AZT, when the inhibition of HIV replication. Influenza virus and respiratory syncytial virus are also likely candidates for the synergistic response to nucleoside analogue, n-docosanol.

Regardless of the mechanisms underlying the synergistic antiviral activity of n-docosanol and ACV, there are several advantages of the strategy of combination therapy using nucleoside or nucleotide analogue plus n-docosanol. First, it is proved by the latest advances in the treatment of HIV infections and cancers that combination therapy is generally effective. A common characteristic of such therapy is the use of two or more drugs with different mechanisms of action. Even without synergistic antiviral activity of combination therapy of recurrent HSV disease with n-docosanol and ACV would be preferred due to their non-overlapping mechanisms of action. However, n-docosanol shows synergistic antiviral activity with the nucleoside analogues, probably causing infected by the virus, the cell concentration increased levels of medication. Therefore, the second competitive advantage of using such a safe drug, as the n-docosanol, is the ability to selectively address the effect on the cell population and the increase in efficiency is Yunosti nucleoside or nucleotide analogues, shorten the time of healing, to reduce the likelihood of breeding resistant to medication mutants, and reduce the impact on the patient potentially toxic and allergenic nucleoside drugs.

A logical combination therapy from a nucleoside analogue and n-docosanol would be to use a homogeneous cream, ointment or suspension of a mixture of drugs. This application worked well in animal studies (Fig) and can be used with ACV in the treatment of patients suffering from recurrent HSV-diseases. These are associated with the herpes virus diseases as herpes zoster, CMV retinitis or sarcoma Kaposi, are also better able to respond to nucleoside therapy in the presence of n-docosanol. Virtually any diseased tissue that can be treated with cream or suspension of n-docosanol may be a target for ongoing thus improved nucleoside therapy, including the skin, gastrointestinal tract, respiratory system and certain organs of the reproductive system. A continuation of this application would be a system introduction nucleoside or nucleotide analogue and local treatment with n-docosanol. If we assume that the developed approved system formula n-docosanol, combination therapy with n-docosanol and nucleoside or nook is eating analogue could be targeted to almost any organ of the body.

The use of n-docosanol for concentration of nucleoside analogues in the diseased tissue may not be limited to diseases caused by viruses. It remains to determine whether to operate the combined n-docosanol/nucleoside therapy with intact cancer cells. However, some time ago there was interest in using the technology of gene transfer for cancer therapy using viral-encoded genes and antiviral nucleoside drugs; n-docosanol could ultimately play a role in this strategy. Of course, transfetsirovannyh or melanoma cells with the gene timedancing HSV make the cells sensitive to the toxic effects of conventional HSV-selective nucleoside drug of ganciclovir (Oliver et al., Virol. 145:84-93, 1985). It is possible that the treatment of cancer skin cells using a n-docosanol will make the response more intense. Viral diseases that are hard to cure, can also be the purpose of this application, because similar response was observed when using suicide gene therapy and nucleoside analogues in infected with the virus of Epstein-Barr In human-lympany cells (Franken et al., Nature Medicine 2:from 1379-1382, 1996) and infected with HIV-1 virus human T-cells (Caruso et al., Virol. 206:495-503, 1995).

Disclosed a method for the treatment of viral is Pecci contains the introduction of long-chain aliphatic compounds in combination with nucleoside or nucleotide analog or PMK. Preferably the active ingredients are administered together. In yet another implementation, the active ingredients are mixed and introduced into pharmaceutically acceptable carrier. In the terminology used here, the introduction of aliphatic compounds in combination with nucleoside analogue or PMK means that the compounds can be administered to the same patient at different times and in different dosages and modes of treatment, but the treatment regimens give overlapping in vivo concentrations of the two compounds, thereby simplifying the beneficial interaction between the two classes of drugs. The joint introduction of aliphatic compounds and nucleoside analogue means that these two active ingredients are introduced simultaneously, although not necessarily by the same route.

Aliphatic compound may be administered from one to five times a day orally, permucosal, intravenously or by resemblance penetration. Similarly nucleoside analog or PMK can also be given from one to five times a day orally, permucosal, intravenously or by resemblance penetration. Preferably, the aliphatic compound is applied topically on the affected tissue, and the nucleoside analogue is introduced systematically. The dosage of active aliphatic compounds in accordance with anastasimatarion range from 0.05% to approximately 40%. Most preferably aliphatic compounds are used in concentrations in the range from approximately 1% to approximately 20%.

Synergistic interaction of n-docosanol and nucleoside analogues can be used with the system of the introduction of nucleoside analogue, combined with local application of n-docosanol to diseased tissue. For example, oral administration of acyclovir in a dose of 500 mg, administered 5 times a day, leads to the maximum average concentration of acyclovir in plasma approximately 0.7 μg/ml (Tyring et al., Arch Dermatol. 134:185-191, 1998). Oral administration of acyclovir twice a day with a dosage of about 1000 mg leads to a maximum concentration of acyclovir in plasma is about 4.3 mcg/ml Injection suspension of acyclovir in a dose of 5 mg/kg by infusion of 1 every hour for 8 hours leads to a balanced concentration of acyclovir in plasma of approximately 10 µg/ml (Blum et al., Am. J. Med. 73:186-192, 1982). These schemes dosages in combination with the simultaneous introduction of n-docosanol from one to five times a day topically, orally, through the genitourinary tract (permucosal), or intravenous medicines by its absorption properties, should effectively utilize the useful interaction between these two classes of medications.

Although the present invention has been described in the context of particular examples and preferred the compulsory executions, it should be understood that the invention is not limited to such executions. On the contrary, the scope of the present invention should be determined by the subsequent claims.

1. The antiviral composition containing an aliphatic alcohol With21-C28in the concentration range of 0.05-40% by weight and nucleoside or nucleotide analog at a concentration in the range of 0.1-10% by weight in a pharmaceutically acceptable carrier.

2. The antiviral composition according to claim 1, in which the aliphatic alcohol With21-C28is selected from the group consisting of primary alcohols, erollover alcohol, brassicicola alcohol, n-docosanol and mixtures thereof.

3. The antiviral composition according to claim 1, in which the nucleoside analogue or nucleotide analogue is selected from the group consisting of acyclovir, adefovir, azidothymidine, brivudine, cidofovir, ddC, ddl, famciclovir, ganciclovir, idoxuridine, lamivudine, lobucavir, penciclovir, ribavirin, sorivudine, trifluridine, valaciclovir and Agha A.

4. The antiviral composition according to claim 1, additionally containing phosphorus-formic acid at concentrations in the range 0.1-10%.

5. The antiviral composition according to claim 1, additionally containing a non-ionic surfactant.

6. The antiviral composition according to claim 5, in which the nonionic surfactant is difunctional block polymer whom the Xia polyoxyalkylene derivative of propylene glycol, having a molecular weight of 1000-25000.

7. The antiviral composition according to claim 5, in which the nonionic surfactant is a block copolymer of ethylene oxide and propylene oxide having a molecular weight between 6000 and 12000.

8. The antiviral composition according to claim 5, in which the nonionic surfactant is selected from the group consisting of octoxynol-9 and octoxynol-10.

9. The antiviral composition according to claim 5, additionally containing a substance that improves the penetration.

10. The antiviral composition according to claim 1, additionally containing agents selected from the group consisting of antimicrobial agents, other antiviral agents, antifungal agents, antioxidants, buffering agents, sunscreen substances, cosmic agents, aromatic substances, lubricants, humidifiers, dehumidifiers, and thickeners.

11. The antiviral composition according to any one of claims 1 to 10 for use in the treatment of viral infections, in which the aliphatic alcohol With21-C28introduced in combination with nucleoside analogue or nucleotide analogue.

12. The antiviral composition according to claim 11, in which the aliphatic alcohol With21-C28and nucleoside or nucleotide analogue independently adapted for insertion from one to five times a day on a route selected from the group consisting of local, oral, permucosal,intravenous and resemblance penetration.

13. The antiviral composition containing n-docosanol at a concentration in the range of 0.05-40% by weight and nucleoside or nucleotide analog at a concentration in the range of 0.1-10% by weight in a pharmaceutically acceptable carrier.

14. The antiviral composition according to item 13, in which the nucleoside analogue or nucleotide analogue selected from the group consisting of acyclovir, adefovir, azidothymidine, brivudine, cidofovir, ddC, ddI, famciclovir, ganciclovir, idoxuridine, lamivudine, lobucavir, penciclovir, ribavirin, sorivudine, trifluridine, valaciclovir and Agha A.

15. The antiviral composition containing n-docosanol at a concentration in the range of 0.05-40% by weight and a phosphorus-formic acid at a concentration in the range of 0.1-10% by weight in a pharmaceutically acceptable carrier.

16. The antiviral composition according to item 13, in which the mixture contains 5-20% (by weight) of n-docosanol.

17. The antiviral composition according to item 13, in which the mixture contains 10-12% (by weight) of n-docosanol.

18. The antiviral composition according to item 13, further containing a nonionic surfactant.

19. The antiviral composition according to p, in which the nonionic surfactant is difunctional block polymer, which polyoxyalkylene derivative of propylene glycol having a molecular weight of 1000-25000.

20. The antiviral composition according to p, in which the nonionic surface-active the second substance is a block copolymer of ethylene oxide and propylene oxide, having a molecular weight between 6000 and 12000.

21. The antiviral composition according to p, in which the nonionic surfactant is selected from the group consisting of octoxynol-9 and octoxynol-10.

22. The antiviral composition according to item 13, which additionally contains a substance that improves the penetration.

23. The antiviral composition according to item 13, additionally containing agents selected from the group consisting of antimicrobial agents, other antiviral agents, antifungal agents, antioxidants, buffering agents, sunscreen substances, cosmic agents, aromatic substances, lubricants, humidifiers, dehumidifiers, and thickeners.

24. The antiviral composition according to item 13, additionally containing stearate based on sugar.

25. A method of treating a viral infection a composition prepared in accordance with PP-24, which is referred to in the n-docosanol is introduced in combination with nucleoside analogue or nucleotide analogue, or phosphorus-formic acid.

26. The method according A.25, where n is docosanol and nucleoside analog, or a nucleotide analog, or phosphorus-formic acid independently adapted for insertion from one to five times a day on a route selected from the group consisting of local, oral, permucosal, intravenous and resemblance penetration.

27. The method according A.25, to the m mentioned viral infection caused by herpes virus, cytomegalovirus, virus Epstein-Barr, varicella zoster virus, influenza virus, lymphotropism virus human and human immunodeficiency virus.

28. The method according A.25, in which the prepared composition for the treatment of viral infection, is injected at a dosage of 0.01-10 g with a frequency of from one to five times per day, for from one to fourteen days, while the above-mentioned composition is introduced by a route selected from the group consisting of local, oral, permucosal, intravenous and resemblance infiltration.



 

Same patents:

FIELD: biotechnology, veterinary science.

SUBSTANCE: he present innovation deals with manufacturing biopreparations for oral immunization in animals. One should grow vaccinia rabic virus, TC-80 strain in a certain passage cell culture to mix it with protective components (peptone, lactose, pectin). Mixing should be carried out at 14 : 5 : 1 ratio (viral suspension : stabilizing medium, consisting of 40% peptone solution with 8% lactose solution : 4% pectin solution, correspondingly), a semifinished product obtained after mixing should be applied onto a bait (a porous briquette) to be frozen and freeze dried for 24-30 h. The method includes decreased number of stages for manufacturing necessary vaccine being more efficient and economical. The vaccine keeps immunogenic properties for 5 d, not less at environmental temperature being up to 20 C and during storage period for 6 mo at 6-8 C.

EFFECT: higher efficiency of manufacturing.

2 ex, 1 tbl

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new imidazoquinolines of the formula (1): wherein R, R1, R2 and n have values given in the description. Compounds elicit effect of immunomodulating agents inducing biosynthesis of cytokines in animals in treatment of different pathologies, among them viral and neoplastic diseases. Also, invention relates to a pharmaceutical preparation used for inducing interferon-α or tumor necrosis α-factor, to a method for inducing biosynthesis of cytokines in animals and to methods for treatment of viral diseases and neoplasm pathologies in animals. Invention provides preparing new biologically active compounds.

EFFECT: improved inducing method, valuable properties of compounds and pharmaceutical preparation.

23 cl, 10 tbl, 231 ex

FIELD: medicine, pharmaceutical industry and technology, pharmacy.

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EFFECT: valuable medicinal properties of composition.

5 ex

FIELD: biotechnology.

SUBSTANCE: the present innovation deals with biotechnology of viral preparations. One should apply RB-71 strain of rabies virus which should be introduced into culture reservoir (0.1-0.01 MLD50/cell) simultaneously with VNK-21 at initial cell concentration being 0.5-0.5 mln cells/ml to be grown in suspension. Cultivation should be performed in suspension medium at 37 C for 5-6 d at constant mixing and maintaining pH value of 7.2-7.4. The obtained viral raw material at infectious 6.5-6.8 lg MLD50/ml and antigenic activity being 1:90-270 should be inactivated with 1,8,36-diendomethylene-1,3,6,8-tetriasecyclodecane 0.01% at 37 C for 3 d. Ready-to-use vaccine preparation meets the requirements of the standard for veterinary preparations being of 1.5-2.0 IU activity.

EFFECT: higher efficiency.

1 ex, 1 tbl

FIELD: medicine, virology, pharmaceutical industry, pharmacy.

SUBSTANCE: invention proposes the preparation used for treatment of viral hepatitis C that comprises birch bark extract with the content of betulin above 70% and a pharmaceutically acceptable carrier. The preparation is administrated to patient by oral route. The preparation promotes to the effective treatment of viral hepatitis C. Invention can be used in treatment of viral hepatitis C.

EFFECT: valuable medicinal properties of agent.

4 cl, 2 tbl

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to biologically active compounds. Agent represents 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium. The new agent elicits antioxidant properties and therefore it can be used in food industry, in pharmaceutical compositions and cosmetic products. Also, the new agent elicits antiviral activity owing to it can be used as both the independent medicinal agent and in compositions with other preparations used for treatment of viral infections.

EFFECT: expanded assortment of medicinal agents and antioxidants, realization of indicated prescription.

1 tbl, 8 dwg

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

The invention relates to the field of biotechnology

The invention relates to an LNA-modified oligonucleotide comprising at least one nucleoside analogue (LNA) of General formula I where X Is-O -; - nucleotide base; P - the place of connection magnolioideae “bridge” or 5’-terminal group selected from hydroxyl, monophosphate, diphosphate and triphosphate; R3or R3* - magnolioideae bridge 3’-terminal group; and R2* and R4* biradical selected from -(CR*R*)r-O-(CR*R*)s-, -(CR*R*)r-S-(CR*R*)s-, -(CR*R*)r-N(R*)-(CR*R*)s-, where each of R1*, R2, R3*, R3, R5* and R5not participating in the education of biradical or magnolioideae “bridge”, denotes hydrogen, halogen, hydroxy, mercapto, amino, azido; or R2and R3- biradical -(CR*R*)r-O-(CR*R*)S- while R2* is selected from hydrogen, hydroxy, and optionally substituted C1-6alkoxy group, a R1*, R4*, R5and R5* is hydrogen; where each of r and s is 0 to 4, provided that the sum r+s is 1 to 4, and each R* is a hydrogen or C1-6alkyl; or a basic salt or an acid additive salt

The invention relates to veterinary Virology and biotechnology

FIELD: medicine.

SUBSTANCE: the suggested transdermal therapeutic system (TTS) is indicated for percutaneous injection of tolterodin for several days. It is, also, described the method for its manufacturing. The suggested TTS is being a self-gluing lamellar matrix structure that contains methacrylate copolymer including ammonium groups, at least, one plastifier and up to 25 weight% tolterodin. TTS is of good tolerance by skin and is of good physical and chemical stability at prolonged storage and application, it, also, has got good adhesive properties and can provide the penetration of maximal quantity of active substance through skin.

EFFECT: higher efficiency of application.

8 cl, 2 dwg, 3 ex, 3 tbl

FIELD: cosmetology, medicine.

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EFFECT: higher efficiency of therapy.

1 tbl

FIELD: chemico-pharmaceutical industry.

SUBSTANCE: the present innovation deals with new stabilized pharmaceutical composition in its lyophilized form including the compound of formula I

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EFFECT: higher efficiency.

10 cl, 15 ex, 6 tbl

FIELD: chemico-pharmaceutical industry.

SUBSTANCE: the present innovation deals with new stabilized pharmaceutical composition in its lyophilized form including the compound of formula I

as an active ingredient and lactose disaccharide as a stabilizing agent. The present pharmaceutical compositions are of high stability at storage. As for active ingredient it is not destroyed in the course of time.

EFFECT: higher efficiency.

10 cl, 15 ex, 6 tbl

FIELD: pharmaceutics.

SUBSTANCE: the suggested composition has got viscosity being below of about 15000 cP and pH being approximately 3.0-9.0 for treating human skin diseases. He suggested composition consists of (a) therapeutically efficient quantity of, at least, one compound being useful in treating the above-mentioned disease; (b) pharmaceutically acceptable, partially bound polymer of polyacrylic acid being compatible with the compound; (c) not obligatory, a solvent being mixed with water, (d) not obligatory, a conserving agent, (e) not obligatory, a component of butyric phase and acceptable surface-active substance, and (f) water. The suggested composition is useful to treat inflammatory skin disease, acne or acne erythematosa. The composition of low viscosity has got its advantage in the fact that it is applied more accurately when in contact with a container that doses the composition in the form of drops.

EFFECT: higher efficiency of application.

23 cl, 15 ex, 19 tbl

FIELD: pharmaceutics.

SUBSTANCE: the suggested composition has got viscosity being below of about 15000 cP and pH being approximately 3.0-9.0 for treating human skin diseases. He suggested composition consists of (a) therapeutically efficient quantity of, at least, one compound being useful in treating the above-mentioned disease; (b) pharmaceutically acceptable, partially bound polymer of polyacrylic acid being compatible with the compound; (c) not obligatory, a solvent being mixed with water, (d) not obligatory, a conserving agent, (e) not obligatory, a component of butyric phase and acceptable surface-active substance, and (f) water. The suggested composition is useful to treat inflammatory skin disease, acne or acne erythematosa. The composition of low viscosity has got its advantage in the fact that it is applied more accurately when in contact with a container that doses the composition in the form of drops.

EFFECT: higher efficiency of application.

23 cl, 15 ex, 19 tbl

FIELD: medicine, proctology, pharmaceutics.

SUBSTANCE: the present innovation deals with obtaining medicinal forms as suppositories applied for treating proctological diseases. Medicinal preparation as suppositories containing active substance, anesthetic, purified water and foundation could additionally contain antiseptic and local irritating substance, as an active substance - glucocorticoid at the following ratio of components, weight%: glucocorticoid 0.0277-0.0368, anesthetic 1.8-2.2, antiseptic 2.8-3.52, local irritating substance 0.288-0.352, purified water 1.8-2.2, foundation - the rest. As glucocorticoid that inhibits the release of inflammation mediators and causes pronounced antiphlogistic and antiallergic action one should apply, for example, hydrocortisone or synaflane. As anesthetic one should apply, for example, anesthesin, lidocaine or trimecaine. As antiseptic being of astringent, drying off and analgesic actions one should apply, for example, dermatol or xeroform. As local irritating substance being of reflector, venotonic, analgesic and antiphlogistic and, also, antimicrobial actions one should apply, for example, racemic menthol or essential oils.

EFFECT: higher therapeutic efficiency.

5 cl, 3 ex, 4 tbl

FIELD: medicine, ophthalmology.

SUBSTANCE: the suggested composition includes gelatin, glucose, hydroxide of alkaline metal, distilled water at certain ratios. As hydroxide of alkaline metal one should apply sodium hydroxides (NaOH), potassium hydroxides (KOH) or lithium hydroxides (LiOH). Composition should be prepared due to adding gelatin into aqueous glucose solution to obtain homogeneous solution, then one should supplement hydroxide of alkaline metal as 3 M aqueous solution, pH of composition is increased up to 10.5-11.5. The obtained composition should be heated at hot water bath for 2-3 min at 94-98 C, then it should be cooled with cold water for 3-5 min up to 20 C. Heating and cooling cycle should be repeated 4-6 times. On finishing the process composition's pH is decreased up to 7.5-8.5. Composition should be poured into vials to be dried and kept at room temperature. The process lasts for 5-7 d. The obtained curative, soft contact lens should be sterilized by placing them together with the form for 1 h into 96°-ethanol solution, to be dried by not separating against the form and stored in dry sealed vials out of dark glass. Before application lens should be impregnated in isotonic solution of sodium chloride (NaCl) for swelling to separate then against the form and rinsed 4-5 times in 0.9%-NaCl solution. The innovation provides efficient ocular protection in case of burns, traumas and chronic diseases.

EFFECT: higher efficiency of manufacturing and application.

3 cl, 3 ex

FIELD: biochemistry.

SUBSTANCE: invention relates to neutral lipopolymer of formula 1 , wherein R1 and R2 independently are alkyl or alkenyl C8-C24-chain; n = 10-300; Z is selected from group consisting of hydroxyl, alkoxyl, benzyloxy, carboxyl acid ester, sulfonic acid ester, alkyl or aryl carbonate, amino, and alkylamino; L is selected from group comprising: (i) X-(C=O)-Y-CH2-, (ii) -X-(C=O)-, and (iii) -X-CH2-, wherein X and Y independently are oxygen, NH and direct bond with the proviso, that when L is -X-(C=O)-, X is not NH. Liposomal composition and method for prolongation of liposome circulating time also are disclosed.

EFFECT: neutral lipopolymer providing elongation of liposome circulating time.

11 cl, 5 ex, 6 tbl, 10 dwg

FIELD: medicine; medical engineering.

SUBSTANCE: method involves supplying target materials and core materials, carrying out target materials ablation with washed-out particle materials being produced and coating core materials with the washed-out particle materials. The method is applied under pressure of approximately equal to 10 torr or higher. Coating of thickness from one to several nm is applied at atmospheric pressure with pseudo-fluidized particle substance state, achieved by means of pneumatic pseudo-fluidization, being used.

EFFECT: improved pharmacokinetic drug properties.

22 cl, 22 dwg

The invention relates to the pharmaceutical industry, in particular the production of medicines used for colds, relieving headaches and neuralgia
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