Methods and compositions for inhibiting enveloped viruses using low-molecular-weight hydrophobically modified polymers

Abstract

FIELD: virology.SUBSTANCE: method for inhibiting the penetration of enveloped viruses into cells, comprising bringing said viruses into contact with an antiviral composition containing at least one low-molecular hydrophobically modified polymer in an effective amount for inhibiting the penetration of viruses into cells, wherein said composition is free of a surfactant having a hydrophilic-lipophilic balance (HLB) of more than 12, wherein said low-molecular hydrophobically modified polymer contains a polymer obtained from at least one first monomer component selected from the group consisting of (meth)acrylic acid, and at least one second monomer component selected from the group consisting of one or more C1-C9 alkyl (meth)acrylates, wherein the low-molecular polymer has a number-average molecular weight of 100,000 or less and methods for inhibiting the transmission of viruses by inhibiting the penetration of enveloped viruses into cells.EFFECT: reliable methods for reducing the number of infectious virions of a wide range of viral types and strains, while maintaining a gentle effect and not causing irritation to human tissue.14 cl, 3 dwg, 8 tbl, 7 ex

Description

[0001] Field of application of the invention

[0002] The method of the present invention relates to the use of low molecular weight hydrophobically modified polymers for inhibiting the transmission of viruses known as enveloped viruses. It also relates to compositions containing said low molecular weight hydrophobically modified polymers that are capable of inhibiting the transmission of said viruses. Background of the invention

[0003] Infections caused by enveloped viruses cause common diseases such as herpes simplex, HIV / AIDS, hepatitis B, influenza, chickenpox, shingles, smallpox, and respiratory infections. Although the severity of these diseases can range from mildly bothersome to life-threatening, these infections adversely affect the quality of life of the host, as well as the personal, institutional, and economic spheres of society. As a result, significant efforts have been devoted to developing means to prevent viral infection and its spread. These efforts are complicated by viral diversity and the multiple routes of transmission of viruses, including direct contact, exchange of bodily fluids (e.g., through saliva, sexual transmission, breastfeeding), and airborne transmission (e.g., through coughing, sneezing, etc.).), as well as highly developed measures by which viruses evade detection and / or destruction by host organisms. Numerous advances have been made in the discovery and commercialization of antiviral agents prescribed to patients infected with the virus. However, these treatments often require a physician's prescription, have undesirable side effects, are only suitable for a narrow range of virus types / strains, and / or have limited efficacy. Topical antiviral drugs must also not irritate tissues upon application or increase the risk of infection.

[0004] Therefore, there is an unmet need for antiviral agents that could be addressed by inexpensive and gentle agents with potent broad-spectrum antiviral activity that can significantly reduce the spread of viruses and prevent the spread of viral infections. This is especially important if the gentle properties of such agents can facilitate and encourage widespread and frequent use, given their excellent compatibility with skin, eyes, and other mucous membranes.

[0005] Viruses have high mutation and replication rates; these properties allow them to rapidly evolve in response to external selective pressures (i.e., drugs), often leading to treatment resistance and relapse. The problem of resistance is particularly acute when an antiviral compound targets a specific epitope on the virion surface. Due to high levels of viral genetic diversity, this narrow specificity also typically limits the range of viruses susceptible to the compound. Alternatively, other topical antiviral agents, such as surfactants, target nonspecific regions of the virus and are generally effective in neutralizing a variety of viruses; however, they often cause irritation and are toxic to human cells.Treatments that cause tissue irritation can increase the rate of infection; damage to cell membranes increases their permeability to certain types of viral particles. Therefore, it is highly desirable to provide non-irritating yet highly effective means of killing viruses and significantly reducing their ability to spread.

[0006] At some stage of their life cycle (transfer between host cells), most viruses (e.g., HIV and many animal viruses) are enclosed in a viral envelope, which functions as their outer layer. Robertson et al. (March 1995). Recombination in AIDS viruses. Journal of Molecular Evolution. 40 (3): 249–59. Some enveloped viruses also have a protein layer, called a capsid, which is located between the envelope and their genome. Ibid. Envelopes are usually formed from fragments of the host cell membrane (phospholipids and proteins), but include some viral glycoproteins. In this way, viruses can evade the host immune system. Glycoproteins on the envelope surface serve to identify and bind receptor sites on the host membrane. The viral envelope then fuses with the host membrane, allowing the capsid and viral genome to enter the host, causing infection.

[0007] The cell from which the virus itself buds often dies or weakens and releases large numbers of viral particles over a prolonged period. The lipid bilayer envelope of these viruses is relatively sensitive to dehydration, heat, and detergents; thus, these viruses are easier to sterilize than non-enveloped viruses. They have limited survival outside the host environment and are typically transmitted directly between hosts. Enveloped viruses are highly adaptable and can change quickly to evade the immune system. Enveloped viruses can cause chronic infections.

[0008] The classes of enveloped viruses that contain human pathogens include, for example, DNA viruses such as herpesviruses, poxviruses, hepadnaviruses, asfarviruses; RNA viruses such as flavivirus, alphavirus, togavirus, coronavirus, hepatitis D virus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus; and retroviruses such as HIV.

[0009] COVID-19

[0010] Coronaviruses (CoVs) are relatively large viruses containing a single-stranded positive-sense RNA genome encapsulated in a membrane envelope. The viral membrane is studded with glycoprotein spikes, which give coronaviruses a crown-like appearance. (See Fig. 1, taken from Liu et al., Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases, ACS Cent. Sci. 2020, 6, 315–331.) Although coronaviruses infect both humans and animals, some animal species, such as bats, which are hosts for a wide variety of coronaviruses, appear to be immune to coronavirus-induced disease. There are four classes of coronaviruses, called alpha, beta, gamma, and delta. The class of betacoronaviruses includes severe acute respiratory syndrome (SARS) virus (SARS-CoV), Middle East respiratory syndrome (MERS) virus (MERS-CoV), and the agent that causes COVID-19 (SARS-CoV-2).Like SARS-CoV and MERS-CoV, SARS-CoV-2 infects the lower respiratory tract, causing viral pneumonia, but can also affect the gastrointestinal tract, heart, kidneys, liver, and central nervous system, leading to multiple organ failure. Currently available information indicates that SARS-CoV-2 is more contagious than SARS-CoV.

[0011] A number of studies have been devoted to elucidating the virus's structure, mechanisms and dynamics of transmission, as well as identifying antiviral agents and accurate diagnostics for virus detection. These trends reflect the enormous interest and desire of the scientific community, including both academic and industrial organizations, as well as healthcare professionals, to find new methods to halt the progression of this epidemic disease and prevent future infection and transmission. COVID-19 is caused by SARS-CoV-2, a novel coronavirus of the same genus as SARS-CoV and MERS-CoV. The viral proteins responsible for SARS-CoV-2 entry into host cells and replication are structurally similar to those associated with SARS-CoV. Therefore, research and development in SARS and MERS may provide information useful for the development of therapeutic and prophylactic drugs against COVID-19.

[0013] Arbidol, CAS No. 131707-23-8, which targets the S / ACE2 protein, is an inhibitor that can disrupt the binding of the enveloped virus protein to host cells and prevent viral entry into the target cell, and has been included in clinical trials for the treatment of COVID-19. See Liu et al. above and Fig. 2 below, taken from Blasting et al., Arbidol as a broad-spectrum antiviral: An update, Antiviral Research, 107 (2014) 84–94. See also Kadam et al., Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol, PNAS January 10, 2017 114 (2) 206–214.

[0014] The emergence of severe acute respiratory disease coronavirus (SARS-CoV) in 2003 demonstrated that CoVs are capable of causing outbreaks of severe infections in humans. A second severe CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), emerged in 2012 in Saudi Arabia. More recently, COVID-19, identified in Wuhan, China, in December 2019, has proven particularly dangerous.

[0015] Given that the polymers of the invention exhibit activity against enveloped viruses, it is expected that the polymers of the invention may also exhibit activity against COVID-19 by inhibiting viral entry into the host cell. See Fig. 3.

[0016] RetroVirox, based in San Diego, California, USA, has developed cell-based assays that can be used to evaluate experimental therapies for coronaviruses, including SARS-CoV-2. To evaluate the activity of entry inhibitors against the novel coronavirus that causes COVID-19, the company is conducting trials using SARS-CoV-2 pseudoviruses. The pseudovirus assay utilizes HIV pseudoviruses coated with the SARS-CoV-2 spike (S) protein (a Wuhan isolate). The assay, which mimics the novel coronavirus's entry mode, can be used, for example, to evaluate small-molecule entry inhibitors targeting the viral S protein, the viral ACE-2 receptor, host proteases, and other targets involved in SARS-CoV-2 entry.

[0017] U.S. Patents 7,803,403 and 8,025,902, to Johnson & Johnson Consumer Inc., disclose personal care compositions that contain a low molecular weight, non-crosslinked linear acrylic copolymer and at least one surfactant; and a method of cleansing using said personal care compositions.

[0018] U.S. Patents 8,343,902 and 8,329,626, to Johnson & Johnson Consumer Inc., disclose a skin cleansing composition that comprises a low molecular weight, non-crosslinked linear acrylic copolymer and a non-ethoxylated anionic surfactant.

[0019] U.S. Patent No. 8,329,627 to Johnson & Johnson Consumer Inc. describes a skin cleansing composition that comprises a low molecular weight, non-crosslinked linear acrylic copolymer and a mixture of at least two amphoteric surfactants.

[0020] U.S. Patent No. 8,293,845 to Lubrizol Corp. describes a method for increasing the critical micelle concentration of a surfactant composition comprising incorporating into said composition a linear hydrophobically modified (meth)acrylic polymer.

[0021] U.S. Patent No. 7,892,525 to Lubrizol Advanced Materials, Inc., discloses antiperspirant compositions that comprise a cationic hydrophobically modified polymeric gelling agent and an acidic antiperspirant compound.

[0022] U.S. Patent No. 9,068,148 to Lubrizol Advanced Materials Inc. describes a mixture of acrylic polymers that contains at least one crosslinked acrylic copolymer and at least one linear non-crosslinked acrylic polymer; a method for producing a mixture of acrylic polymers; and a method for thickening an aqueous composition containing a mixture of acrylic polymers.

[0023] U.S. Patent No. 9,931,290 to Lubrizol Advanced Materials Inc. discloses a surfactant composition that comprises a surfactant and a crosslinked acrylic copolymer; and a personal care cleansing composition comprising the surfactant composition.

[0024] U.S. Patent No. 10,517,806 to Ecolab USA Inc. claims a foaming antimicrobial skin cleanser comprising a cationic active ingredient; a compatible cationic surfactant; a foam enhancer; a foam structure-improving agent; a skin conditioning agent; and water. The reference claims a method for reducing a bacterial, microbial, fungicidal, or viral population on mammalian skin tissue, comprising contacting the skin tissue with the foaming antimicrobial skin cleanser. The reference also states that the cationic active ingredients are the antimicrobial agents used in the present invention, and that the foam structure-improving agent can be polyethylene glycol. The reference describes the use of S. aureus and Escherichia coli as test microbial cultures to test the antimicrobial efficacy of the formulations they contain.

[0025] U.S. Patent No. 10,435,308 to Ecolab USA, Inc., claims a composition for improved oil removal from an oil / water phase solution by foam fractionation, comprising an associated thickening agent; a surfactant comprising a sorbitan ester; and a viscoelastic surfactant that is betaine, an amine oxide, and / or an ethoxylated fatty amine.The reference describes that the composition can be used, for example, in cleaning agents, cosmetics, blackhead removers, water-based pigment pastes, automotive paint coatings, industrial paint coatings, printing inks, greases, plaster paints and wall paints, textile coatings, pharmaceuticals, crop protection compositions, filler dispersions, adhesives, detergents, wax dispersions, polishes, auxiliary substances for the production of mineral oils by tertiary methods, etc.

[0026] U.S. Patent Application Publication No. 20160262999 by Ecolab USA, Inc. claims an antimicrobial dermal concentrate that contains a cationic active ingredient; a surfactant foam booster; a foam-enhancing copolymer; a foam-stabilizing structure; and water. The reference states that the concentrate can be used to reduce bacterial, microbial, fungicidal, or viral populations on mammalian skin tissue. The reference states that the "cationic active ingredient" is an ingredient that provides antimicrobial activity. The reference states that the concentrate may contain a skin conditioner, such as polyethylene glycol.

[0027] Menachery et al., Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses, American Society of Microbiology, 2014, 5(3): 1-11, described that influenza viruses and coronaviruses differ in terms of replication, immune stimulation, and overall lethality.

[0028] Li, Structure, Function and Evolution of Coronavirus Spike Proteins, Annu. Rev. Virul. 2016, 3(1):237-261, describes the evolution of two critical functions of coronavirus spike proteins: receptor recognition and membrane fusion, in the context of corresponding functions in other viruses and host cells.

[0029] Neutrogena Corp., Los Angeles, California, USA, markets Neutrogena® Ultra Gentle Daily Cleanser, which contains potassium acrylates copolymer as a viscosity-enhancing agent.

[0030] Johnson & Johnson Consumer Inc. markets products including Johnson's Head to Toe Baby Wash; Johnson's Baby Moisture Wash; and Johnson's Baby Wipes, which contain potassium acrylates copolymer as a viscosity-enhancing agent.

[0031] Hand sanitizers are commonly used to reduce the number of infectious agents on hands. They come in liquid, gel, and foam forms. Alcohol-based and alcohol-free options are available. Alcohol-based options typically contain some combination of isopropyl alcohol, ethanol (ethyl alcohol), or n-propanol, with those containing 60% to 95% alcohol being the most effective. Caution is necessary as they are flammable. Alcohol-based hand sanitizer is effective against a wide range of microorganisms. Alcohol-free options, which typically contain benzalkonium chloride or triclosan, are less effective than alcohol-based options.

[0032] In 2020, BluillWillow Biologics, Inc. launched NanoBio Project, a nasal antiseptic solution containing over-the-counter, pharmaceutical-grade benzalkonium chloride. The product is applied by thoroughly wiping the skin inside each nostril.

[0033] STATEMENT OF THE ESSENCE OF THE INVENTION

[0034] The present invention relates to a method for inhibiting the entry of enveloped viruses into cells, comprising, essentially consisting of and consisting of bringing said viruses into contact with an antiviral composition containing at least one low molecular weight hydrophobically modified polymer in an effective amount for inhibiting the entry of these viruses into cells, wherein said composition is substantially free of a surfactant having a hydrophilic-lipophilic balance (hereinafter HLB) value of more than 12.

[0035] It was unexpectedly found that low concentrations of certain low molecular weight hydrophobically modified polymers, known for their mild effects, were able to successfully inhibit the entry of enveloped viruses into host cells and thus inhibit the transmission of viruses to host cells.

[0036] The present inventors believe that these polymers will not experience or cause some of the known problems associated with antiviral drugs, such as drug resistance, narrow neutralization window, and host cell toxicity. The low-molecular-weight hydrophobically modified polymers used in the methods and compositions of the present invention are widely used against several viral types and multiple viral strains. Furthermore, these polymers act through a non-specific penetration inhibition mechanism, thereby increasing their chances of successful inhibition and reducing the likelihood of resistance. Furthermore, because these polymers are exceptionally gentle on mucosal tissues, they are virtually non-toxic to human tissues.

[0037] Human bodies are exposed to viruses daily, and the human immune system, including the skin barrier, is designed to minimize the number of viruses that reach infectious surfaces. Low-molecular hydrophobically modified polymers used in the methods and compositions of the present invention block the ability of viruses to bind to and / or penetrate cells, thereby reducing the likelihood that an infectious virus can reach target cells and cause a systemic infection. Viral infection is partly the result of a stochastic process—the more viruses that come into contact with infectable cells, the higher the likelihood of tissue infection. Therefore, the use of these polymers to block infectious viruses supports the immune system, further reducing the likelihood of infection, and promoting overall health.The methods and compositions of the present invention, which utilize low molecular weight hydrophobically modified polymers, have unexpectedly proven effective in reducing the number of infectious virions in a wide range of virus types and strains, while providing a gentle, non-irritating effect on human tissue.

[0038] DETAILED DESCRIPTION OF PREFERRED IMPLEMENTATION OPTIONS

[0039] As used herein, the term "infectious surface" refers to the surface of a living organism, including mammals such as humans, whose cells can be infected by a virus. Examples of such infectious surfaces include the outer tissues of the skin and mucous membranes. Mucous membranes include those of the mouth, eyes, nose, vagina, and rectum.

[0040] The term "edible product surface" as used herein refers to the surface of food products, including the surface of fruits and vegetables. The term "hard surface" as used herein refers to surfaces found in the environment, such as tables, chairs, walls, and other inanimate surfaces that may come into contact with skin and / or mucosal tissues and that may harbor viruses. The term "internal surface" refers to the surfaces of internal organs and internal tissues and fluids within the body of a living organism.

[0041] As used herein, the term "virus" means a small infectious agent that can replicate only inside living cells or organisms. Viral particles contain the following parts: genetic material, consisting of RNA or DNA, and a protein coat that protects the genetic material. In some cases, when virus particles are outside the cell, the virus particles are surrounded by a lipid membrane located around the protein coat. Viral particles that contain such a lipid membrane are referred to herein as "enveloped viruses." Enveloped viruses may include the following microorganisms: poxviruses, including, but not limited to, molluscum contagiosum, chickenpox, smallpox, and other pox viruses; coronaviruses; flaviviruses; herpes viruses, including herpes simplex virus 1 and herpes simplex virus 2; Retroviruses, including lentiviruses, including human immunodeficiency virus.

[0042] In this document, the term "surfactant" means a surface-active agent or substance that, when dissolved in water or an aqueous solution, reduces its surface tension or the interfacial tension between itself and another liquid.

[0043] As used herein, the term "inhibition of transmission" means one or more of the following: (i) blocking entry of a virus into a host cell; (ii) substantially stopping the entry of a virus from one distinct infectious surface or contact surface to another; and / or (iii) reducing damage to mucous membranes such that the membranes maintain their integrity and protect against infection by the virus.

[0044] As used herein, the term "hydrophilic-lipophilic balance" (HLB) is a measure of the degree of hydrophilicity or lipophilicity of a surfactant, determined by calculating values ​​for various sites on the surfactant molecule in accordance with methods known to those skilled in the art.

[0045] Preferably, the method of the present invention relates to a method for inhibiting the penetration of enveloped viruses into cells, comprising, essentially consisting of and consisting of bringing said viruses into contact with an antiviral composition comprising, essentially consisting of and consisting of at least one low molecular weight hydrophobically modified polymer in an amount effective to inhibit the penetration of viruses into cells, wherein said composition is substantially free of a surfactant with an HLB greater than 12. The methods of the present invention further comprise applying the compositions described herein to surfaces to be infected, as well as to surfaces of edible products. The methods further comprise bringing the viruses into contact with the antiviral compositions of the present invention.

[0046] The methods of the present invention also include applying the compositions of the present invention to the surfaces of edible products, such as food, as well as to hard surfaces that may come into contact with skin and mucous membrane tissues. Thus, the presence of the compositions of the present invention will help inhibit the penetration of viruses present on the surfaces of edible products and hard surfaces into cells of the skin, mucous membranes, internal tissues, and fluids.

[0047] Preferably, the compositions of the present invention contain at least about 50%, and preferably at least about 55% water. Alternatively, the compositions of the present invention may contain at least 50% of a protic solvent, which can be selected from the following solvents: water, glycerol, urea, alkanols, acetic acid, formic acid, and the like. More preferably, the protic solvents used in the compositions of the present invention include formic acid, acetic acid, n-butanol, isopropanol, n-propanol, ethanol, methanol, and the like.

[0048] Most preferably, the compositions of the present invention are substantially free of a surfactant having an HLB greater than about 12. Notwithstanding the above, the compositions of the present invention may further comprise surfactants with an HLB less than 12. Surfactants having an HLB greater than 12 can disrupt the cell membranes of infected surfaces, thereby facilitating the penetration and infection of cells by viruses.

[0049] Preferably, the compositions used in the methods of the present invention have a transepithelial permeability (hereinafter referred to as TEP), as described below, of at least 6.

[0050] The compositions of the present invention can be applied to the infectious surfaces of living organisms, including mammals, reptiles, birds, fish, bacteria, and the like. The infectious surfaces of these living organisms can include, but are not limited to, skin tissue, mucous membranes, and internal tissues. Mucosal tissue includes, but is not limited to, oral tissue, ocular tissue, nasal tissue, vaginal tissue, rectal tissue, or a combination thereof. It is important to note that the compositions and methods of the present invention do not destroy these biological surfaces or cause significant irritation to these surfaces.

[0051] POLYMER MATERIAL

[0052] Examples of polymeric materials used in the compositions and methods of the present invention include low molecular weight acrylics, other ethylenically unsaturated polymers, polyesters, polycarbonates, polyanhydrides, polyamides, polyurethanes, polyureas, polyimides, polysulfones, polysulfides, combinations of two or more of them, and the like. Examples of suitable low molecular weight acrylic polymers include hydrophobically modified acrylic, polysaccharide, cellulose and starch polymers, combinations of two or more of them, and the like. Suitable low molecular weight acrylic polymers include hydrophobically modified acrylic polymers, as well as other acrylic polymers, any of which can be formed as a solution, suspension, precipitate, dispersion, emulsion, inverse emulsion, microemulsion, by micellar polymerization methods and a combination of two or more of these methods.Acrylic polymers for use in the present invention can be prepared from any one or more monomers selected from the group consisting of (meth)acrylates, (meth)acrylamides, vinyl ethers, esters and amides, allyl ethers, esters, amines and amides, itaconates, crotonates, styrenes, and olefins. The acrylic polymers can be a non-ionic hydrophilic, a non-ionic hydrophobic, anionic, cationic, zwitterionic, non-associative macromer, associative macromer, or a multifunctional / crosslinked polymer.

[0053] In this document, the term “low molecular weight polymer” refers to a polymer with a number average molecular weight (M n) of about 100,000 or less, as measured by gel permeation chromatography (GPC) on a column calibrated against a poly(methyl methacrylate) (PMMA) standard. In certain preferred embodiments, the low molecular weight polymers are polymers with a molecular weight M n which is in the range of from about 5,000 to about 80,000, more preferably from about 10,000 to about 50,000, and most preferably from about 15,000 to 40,000.

[0054] Certain hydrophobically modified polymers and methods for preparing such polymers are described in U.S. Patent No. 6,433,061 to Marchant et al., incorporated herein by reference. The polymeric materials used in the composition of the present invention are preferably non-crosslinked linear acrylic copolymers that are very mild to the skin and mucous membrane. These non-crosslinked linear polymers are preferably low molecular weight polymers with a number average molecular weight of 100,000 or less, measured by gel permeation chromatography (GPC) on a column calibrated with a polymethyl methacrylate (PMMA) standard sample (unless otherwise indicated, all values ​​of number average molecular weight (M n) refer to the molecular weight measured in this way). Thus, the polymeric material functions as a copolymer compound. The copolymer compound is obtained by polymerizing at least two monomer components. The first monomer component is selected from one or more α,β-ethylenically unsaturated monomers containing at least one carboxyl group. This acid group can be obtained from monobasic or dibasic acids, dicarboxylic acid anhydrides, monoesters of dibasic acids and their salts. The second monomer component is hydrophobically modified (compared to the first monomer component) and is selected from one or more α,β-ethylenically unsaturated non-acidic monomers containing a C1-C9 alkyl group, including linear and branched C1-C9 alkyl esters of (meth)acrylic acid, vinyl esters of linear and branched C1-C 10carboxylic acids and mixtures thereof. In one aspect of the present invention, the second monomer component is represented by the formula:

[0055] СН2=CRX,

[0056] where R is hydrogen or methyl; X is -C(O)OR 1 or -OC(O)R 2 ; R 1 is a linear or branched C1-C9 alkyl; a R 2 represents hydrogen or linear or branched C1-C9 alkyl. In another aspect of the present invention, R 1 and R 2 are linear or branched C1-C8 alkyl, in a further aspect R 1 and R 2 are linear or branched C2-C5 alkyl.

[0057] Thus, preferably, the hydrophobically modified polymers used in the compositions and methods of the present invention comprise, consist essentially of, or consist of a low molecular weight, non-crosslinked linear acrylic copolymer formed from at least one first monomer component selected from the group consisting of (meth)acrylic acid and at least one second monomer component selected from the group consisting of one or more C1-C9 alkyl (meth)acrylates, wherein the number average molecular weight of the low molecular weight copolymer is about 100,000 or less.

[0058] Examples of the first monomer components include (meth)acrylic acid, itaconic acid, citraconic acid, maleic acid, fumaric acid, crotonic acid, aconitic acid, and mixtures thereof. Examples of the second monomer components include ethyl (meth)acrylate, butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, vinyl formate, vinyl acetate, 1-methylvinyl acetate, vinyl propionate, vinyl butyrate, vinyl-2-ethylhexanoate, vinyl pivalate, vinyl neodecanoate, and mixtures thereof. As used herein, the terms "(meth)acrylic acid" and "(meth)acrylate" refer to the corresponding methyl derivatives of acrylic acid and the corresponding alkyl acrylates. For example, the term "(meth)acrylic acid" refers to acrylic acid and / or methacrylic acid, and the term "(meth)acrylate" refers to alkyl acrylate and / or alkyl methacrylate.

[0059] More preferably, said first monomer component is selected from the group consisting of (meth)acrylic acid, and said second monomer component is selected from the group consisting of at least one C1-C9 alkyl (meth)acrylate.

[0060] The non-crosslinked linear acrylic copolymer compounds used in the compositions and methods of the present invention can be synthesized by free radical polymerization technology known in the art. In one aspect of the present invention, the weight ratio of the first monomer component to the second monomer component is in the range of from about 20:80 wt.% to about 50:50 wt.%, based on the total weight of all monomers in the polymerization medium. In another aspect, the weight ratio of the first monomer component to the second monomer component is about 35:65 wt.%, and in a further aspect, the weight ratio of the first monomer component to the second monomer component is about 25:75 wt.%, all values ​​​​are indicated based on the total weight of all monomers in the polymerization medium.

[0061] Methods for synthesizing polymers used in the compositions and methods of the present invention can be found in U.S. Patent No. 6,433,061, incorporated herein by reference.

[0062] The methods and compositions of the present invention can use linear copolymer materials that preferably have a viscosity of 500 mPa⋅s or less (measured on a Brookfield RVT series viscometer, 20 rpm, spindle No. 1), at a concentration of 5 wt.% polymer solids in deionized water neutralized to pH 7 with a NaOH solution with a concentration of 18 wt.%. In another aspect, the viscosity value can be in the range of from about 1 to about 500 mPa⋅s, in a further aspect - from about 10 to about 250 mPa⋅s, and in yet another further aspect - from about 15 to about 150 mPa⋅s.

[0063] Preferably, the low molecular weight non-crosslinked linear acrylic copolymer present in the compositions and methods of the present invention is a potassium acrylates copolymer.

[0064] The low molecular weight hydrophobically modified polymers used in the compositions and methods of the present invention are preferably present in said compositions in effective amounts to significantly inhibit the entry of enveloped viruses into cells and / or inhibit the transmission of the virus into cells. Accordingly, the compositions and methods of the present invention inhibit the entry of the virus into said cells and lead to a decrease in the likelihood of viral infection. Preferably, they should be present in the compositions of the present invention in an amount of from about 0.00005 to about 10% by weight of the composition. Even more preferably, they should be present in an amount of from about 0.00005 to about 3% by weight of the composition. More preferably, the low molecular weight hydrophobically modified polymers are present in an amount of from about 0.00005 to about 0.5% by weight, based on the weight of the composition.Most preferably, the low molecular weight hydrophobically modified polymers are present in an amount of from about 0.00005 to about 0.01 wt.% based on the weight of the composition.

[0065] The compositions of the present invention can be presented in the form of a lotion or liquid suitable for application to a skin surface or an inanimate surface that may contain viruses or bacteria. This composition can also be applied to a mucosal surface, such as the nasal or vaginal cavity, and can be used as a vaginal microbicide. These types of compositions can be more viscous and can be prepared based on a gel formulation. The compositions of the present invention can be applied to an absorbent article, such as a vaginal or nasal tampon, for contact with mucosal surfaces to inhibit viruses in such biological environments. The compositions of the present invention can also be prepared in a delivery form such that they can be administered to appropriate sites on the internal surfaces of the body that may harbor viruses.

[0066] Compositions of the present compound can be formulated into various types of products, including, but not limited to, liquids, lotions, creams, gels, sticks, sprays, shaving creams, ointments, liquid cleansers and bar soaps, shampoos, pastes, powders, mousses, wipes, plasters, wound dressings, adhesive dressings, hydrogels, and films. These types of products can contain several types of cosmetically acceptable topical carriers, including, but not limited to, solutions, emulsions (e.g., microemulsions and nanoemulsions), gels, solids, and liposomes. Non-limiting examples of such carriers are provided below. Those skilled in the art of formulating these types of products can incorporate other carriers into their formulation.

[0067] Preferred compositions of the present invention include polymer-containing gels; polymer-containing drops, including, for example, eye drops; polymer-containing contact lens solutions; polymer-containing sprays, such as face / body sprays, nasal sprays, and mouth and throat sprays; and polymer-containing inhalants.

[0068] The compositions of the invention can also be used as a coating on or inside personal protective equipment. Personal protective equipment, commonly referred to as PPE, is any equipment worn to minimize exposure to various hazards. Examples of PPE include coveralls, gloves, gowns, masks, respirators, and eye and foot protection.

[0069] Topical compositions used in the methods of the present invention can be prepared in the form of solutions. The solutions preferably contain an aqueous solvent (e.g., in an amount of from about 50% to about 99.99% or from about 90% to about 99% of a cosmetically acceptable aqueous solvent).

[0070] Topical compositions used in the methods of the present invention can be prepared in the form of a solution containing an emollient. Such compositions preferably contain from about 2% to about 50% of the emollient(s). As used herein, the term "emollients" refers to materials used to prevent or reduce dryness, as well as to protect the skin. A wide variety of suitable emollients are known and can be used herein. Sagarin, Cosmetics, Science and Technology, 2 ndEdition, Vol. 1, pp. 32-43 (1972) and International Cosmetic Ingredient Dictionary and Handbook, eds. Wenninger and McEwen, pp. 1656-61, 1626 and 1654-55 (The Cosmetic, Toiletry, and Fragrance Assoc., Washington, DC, 7 th Edition, 1997) (hereinafter referred to as the ICI Handbook) contains many examples of materials for use in the compositions and methods of the present invention.

[0071] Lotion can also be prepared from such a solution. Lotions preferably contain from about 1% to about 20% (more preferably from about 5% to about 10%) of emollient(s) and from about 50% to about 90% (more preferably from about 60% to about 80%) of water.

[0072] Another type of product that can be obtained from the solution is a cream. The cream preferably contains from about 5% to about 50% (more preferably from about 10% to about 20%) of the emollient(s) and from about 45% to about 85% (more preferably from about 50% to about 75%) of water.

[0073] Another type of product that can be obtained from the solution is an ointment. The ointment may contain a simple base of animal or vegetable oils or semi-solid hydrocarbons. The ointment may preferably contain from about 2% to about 10% emollient(s) and from about 0.1% to about 2% thickener(s). A more complete description of thickeners or viscosity-enhancing agents that can be used within the framework of this document can be found in Sagarin, Cosmetics, Science and Technology, 2 nd Edition, Vol. 1, pp. 72-73 (1972) and ICI Handbook pp. 1693-1697.

[0074] The topical compositions used in the methods of the present invention can also be prepared as emulsions. If the carrier is an emulsion, it preferably contains from about 1% to about 10% (e.g., from about 2% to about 5%) of emulsifier(s). Emulsifiers can be nonionic, anionic, or cationic. Suitable emulsifiers are described, for example, in U.S. Patent No. 3,755,560, U.S. Patent No. 4,421,769, McCutcheon's Detergents and Emulsifiers, North American Edition, pp. 317-324 (1986), and ICI Handbook, pp. 1673-1686, incorporated herein by reference.

[0075] Lotions and creams can also be prepared in the form of emulsions. Preferably, such lotions contain from 0.5% to about 5% emulsifier(s). Such creams typically contain from about 1% to about 20% (more preferably from about 5% to about 10%) emollient(s); from about 20% to about 80% (more preferably from 30% to about 70%) water; and from about 1% to about 10% (more preferably from about 2% to about 5%) emulsifier(s).

[0076] Other compositions used in the methods of the present invention include gels and liquid compositions that can be applied to mucosal surfaces to suppress the transmission of viruses. Mucosal surfaces include, but are not limited to, the vagina, rectum, nasal passages, oral cavity and throat. Preferably, such compositions should include at least one polyhydric alcohol, including glycerin, polyethylene glycol, propylene glycol, sorbitol, or a combination thereof. Other polyhydric alcohols known to those skilled in the art can be used in the compositions and methods of the present invention, including polyethylene glycols with a molecular weight in the range of about 300 to about 1450. Preferably, they should contain from about 0.1 to about 50% by weight glycerin and from about 2 to about 40% by weight propylene glycol.

[0077] The compositions of the present invention for mucosal surfaces should also contain one or more water-soluble cellulose-derived polymers. Preferably, such polymers should be cellulose gum, for example, one or more hydroxyalkyl cellulose polymers. More preferably, the hydroxyalkyl cellulose polymer should be one or more of hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like. Preferably, the cellulose-derived polymer should be present in the compositions of the present invention in an amount of from about 0.1 to about 2% by weight of the composition.

[0078] The compositions of the present invention intended for vaginal use may also contain one or more spermicides, including, but not limited to, nonoxynol-9, etc. Although such spermicides can be classified as surfactants, they generally have an HLB of less than 16 and are not suitable for use as or in cleansing compositions, and do not form foam.

[0079] To adjust the pH of the composition to ensure compatibility with vaginal, oral, or rectal mucosal surfaces, an inorganic base can preferably be used. Potassium hydroxide or another alkali or alkaline earth metal hydroxide can be used to maintain the appropriate pH. Of course, any other physiologically acceptable base can also be used in this manner. Preferably, from about 0.05 to about 5% by weight of the inorganic base is used.

[0080] The compositions of the present invention can be prepared according to methods and processes known to those skilled in the art, or according to the production methods of the present invention. For example, water-soluble components such as glycerin, propylene glycol, sorbitol, an inorganic base, preservatives, and the like can be dissolved in water, and cellulose-derived polymers can be added to the resulting combination. Another production method is to mix all the ingredients into an anhydrous suspension and then add the suspension to water.

[0081] The composition is preferably substantially free of surfactant, including anionic, cationic, amphoteric or nonionic surfactants.

[0082] The composition in the form of a liquid or lotion may include water, oils, preservatives, emulsifiers, viscosity enhancers, emollients, electrolytes, fragrances, buffers, pH modifiers, skin protectants, agents that enhance the excretion of metal ions, etc.

[0083] The compositions of the present invention can be used in the manufacture of hand and / or body washes, fruit and / or vegetable washes, oral compositions, suppositories, nasal sprays, postoperative tampons, and the like, which can be applied to surfaces or inserted into the body to inhibit the transmission of viruses. The compositions of the present invention can be applied to an absorbent article, such as a vaginal tampon or nasal tampon, for contact with mucosal surfaces to inhibit viruses in such biological environments.

[0084] METHODS

[0085] Various testing methods were used herein to evaluate various aspects of the methods and compositions of the present invention and their effects on skin, mucous membranes, and viruses when exposed to the compositions of the invention. The transepithelial permeability (TEP) test is used in the rapid methods and in the following examples. The TEP test is used to determine the degree of skin or mucous membrane irritation caused by the composition.

[0086] Transepithelial permeability test (“TEP test”)

[0087] The expected eye and / or skin irritation of a given formulation is measured according to Invittox Protocol #86 (May 1994), "Transepithelial Permeability (TEP) Assay," set forth in U.S. Patent #7,157,414, which is incorporated herein by reference. In general, the potential for eye and / or skin irritation of a product can be assessed by determining its effect on the permeability of a cell layer by assessing the leakage of fluorescein through it. Monolayers of Madin-Darby canine kidney (MDCK) cells are grown to confluence on microporous inserts in a 24-well plate, with the lower wells containing medium or the assay buffer. The irritation potential of the product is assessed by measuring the disruption of the impermeable barrier in the cell monolayer after 15 minutes of exposure to weak dilutions of the product. Barrier disruption is assessed by the amount of sodium fluorescein that leaks into the lower well after 30 minutes, which is determined spectrophotometrically.Fluorescein leakage is plotted against the concentration of the test material to determine the EC. 50 (the concentration of the test material that causes 50% of the maximum dye leakage, i.e., 50% of the barrier's permeability is compromised). Higher values ​​correspond to more gentle formulas.

[0088] Exposure of a test sample to a layer of MDCK cells grown on a microporous membrane simulates the first event when an irritant comes into contact with the eye. In vivo, the outer layers of the corneal epithelium form a selectively permeable barrier due to tight junctions between cells. When exposed to an irritant, the tight junctions separate, leading to the disappearance of the impermeable barrier. Fluid is absorbed into the underlying epithelial layers and the stroma, causing separation of the collagen lamellae, resulting in opacity. The TEP assay measures the effect of an irritant on the disruption of tight junctions between cells in a layer of MDCK cells grown on a microporous insert. The disruption is assessed spectrophotometrically by measuring the amount of marker dye (sodium fluorescein) that leaks through the cell layer and the microporous membrane into the lower well.

[0089] Virucidal suspension test

[0090] Brief description of the experiment. To evaluate the virucidal properties of the hydrophobically modified polymers used in the compositions and methods of the present invention, a virucidal suspension test (in-vitro time-of-death study) can be used under conditions of infection with herpes simplex virus type 1 (HSV-1) strain HF (ATCC#VR-260).

[0091] The starting material, such as potassium acrylates copolymer, is available from the supplier as a 30% stock solution. The inoculating viral strain for the HSV-1 test is herpes simplex virus type 1 strain HF (ATCC#VR-260). Host cells should be prepared as follows. Vero cells (ATCC#CCL-81) are incubated as monolayers in disposable cell culture vessels and used for the virucidal suspension test on the HSV-1 strain HF. Prior to testing, host cell cultures are plated on suitable cell culture plates. Cell monolayers should be 100% confluent and less than 48 hours old prior to virus inoculation. The growth medium (GM) and maintenance medium (MM) were 1× minimum essential medium (MEM) with appropriate supplements.

[0092] The test virus was prepared as follows. The HF HSV-1 strain from a high-titer BSL1 virus stock solution can be used for this study. On the day of use, aliquots of the virus stock solution are removed from a 70°C freezer and thawed before use in the test. The compositions of the test products of the present invention can be prepared as follows. 5.0 ml of a 30% test product solution is added to 4.5 ml of a phosphate buffer solution with 0.5 ml of sodium hydroxide. The mixture is shaken, and the pH is measured using pH strips. The pH of the solution (15% v / v test product) should be between 6.0 and 6.5. The test product is diluted to a concentration of 90% (v / v) due to virus inoculation and mock virus inoculation. The test product is evaluated at a concentration of 13.5% (v / v). Determine the percentage and logarithmic log 10Reduction of the initial viral strain population after exposure to the test product for 15 minutes, 30 minutes, and 1 hour. Applications are performed in quadruplicate. Testing is performed in accordance with good laboratory practice as specified in 21 CFR Part 58.

[0093] Neutralization Test

[0094] The neutralization test can be performed before the virucidal suspension test. Maintenance medium (MM) was added to a sample of the test product to simulate the viral inoculum, achieving a concentration of 90% (v / v) of the test product. The mixture was added to the appropriate amount of neutralizer and mixed thoroughly. An aliquot of neutralizer / MM / product was discarded and replaced with the test virus. Serial 10-fold dilutions of the neutralized product in MM were prepared. The dilutions were plated in quadruplicate.

[0095] To conduct the cytotoxicity test, MM is added to the test product sample to simulate a viral inoculum. The mixture is added to the appropriate amount of neutralizer and mixed thoroughly. Serial 10-fold dilutions of the neutralized product in MM are obtained. The dilutions are applied to the plate in quadruplicate.

[0096] The toxicity assessment of the neutralizer for the test virus can be performed as follows.

[0097] Virus Control #1 is used to determine whether the neutralizer had a significant inhibitory effect on the test virus and to determine the titer of the test virus subject to neutralizer treatment, as in the neutralization test. The test virus is diluted with the neutralizer, and subsequent dilutions are carried out in MM. Each dilution is applied to the plate in quadruplicate. Virus Control #2 is used to determine the titer of the test virus in the absence of neutralizer treatment. 10-fold dilutions of the test virus are prepared in MM. Each dilution is applied to the plate in quadruplicate. Cell Culture Control. Intact cell culture monolayers serve as a control for cell culture viability. GM medium is replaced with MM in all control wells for cell culture.

[0098] The plates are incubated in a CO2 incubator for 5-14 days at 37°C ± 2°C. Cytopathic / cytotoxic effects are monitored using an inverted biological microscope.

[0099] Virucidal test. An appropriate amount of test virus is added to a sample of the test product and mixed thoroughly to obtain a concentration of 90% (v / v) of the product. The test virus is exposed to the test product for 15 minutes, 30 minutes, and 1 hour, timed using a calibrated minute / second timer. Immediately after each exposure, the test virus / product is neutralized and diluted 1:10 in MM. Each dilution is applied to the plate in quadruplicate.

[0100] Virus control. An appropriate amount of test virus is treated with a neutralizer similar to the virucidal test and then diluted 1:10 in MM. Each dilution is applied to the plate in quadruplicate.

[0101] Cytotoxicity control. Aliquots of MM are added to a sample of the test product to simulate viral inoculum. The MM / product mixture is neutralized and diluted 1:10 in MM. Each dilution is plated in quadruplicate. Neutralization control. Aliquots of MM are added to a sample of the test and reference products to simulate viral inoculum. The MM / product mixture is neutralized and diluted 1:10 in MM. Approximately 100-1000 infectious units of virus are added to the neutralized product sample. Dilutions were plated in quadruplicate.

[0102] Cell Culture Control. Intact cell culture monolayers serve as a cell culture viability control. GM is replaced with MM in all cell culture control wells. Plates are incubated in a CO2 incubator for 5-14 days at 37°C ± 2°C. Cytopathic / cytotoxic effects are monitored using an inverted biological microscope.

[0103] Test Acceptance Criteria

[0104] For the test to be valid, the following requirements apply: 1) the virus control must show at least 4 log 10 TCID doses 50 (tissue culture infective dose); 2) cells in the cell culture control should remain viable and attached to the bottom of the well; 3) the medium should be free of contamination in all wells of the plate; 4) if cytotoxicity occurs, a titer reduction of at least 3 log is required. 10 beyond the cytotoxic level; and 5) the test product must be completely neutralized immediately after the measured exposure time so that the infectivity of the virus is not altered.

[0105] The following protocols can be used to determine the activity of embodiments of the present invention against HIV-1, hepatitis B, influenza, adenovirus, and rhinovirus.

[0106] Evaluation of anti-HIV-1 activity IIIB in CEM-SS cells

[0107] Fifty microliters (50 µl) of CEM-SS cells at a density of 2.5×10 3 cells / well in 10% Roswell Park Memorial Institute Complete Medium (RPMD-1640 (10% FBS with 1% L-glutamine and 1% penicillin / streptomycin, available from Invitrogen, Carlsbad, CA, USA) were placed in a 96-well round-bottomed plate. One hundred microliters (100 μl) of each polymer at 6 concentrations in triplicate were added, followed by 50 μl of HIV-1 IIIB with a predetermined titer. The cultures were incubated for 6 days at 37°C, 5% CO2. After incubation, the cells were stained with XTT to assess the efficacy of the chemical compounds and cellular toxicity, as described below. AZT was also evaluated as a positive control compound.

[0108] XTT staining for cell viability and compound cytotoxicity:

[0109] TC values 50for the test materials were obtained by measuring the reduction of the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT is metabolized in metabolically active cells by the mitochondrial enzyme nicotinamide adenine dinucleotide phosphate oxidase (NADPH) to a soluble formazan product. XTT solution was prepared daily as a 1 mg / mL stock solution in RPMI-1640 without additives. Phenazine metasulfate (PMS) solution was prepared at a concentration of 0.15 mg / mL in DPBS and stored in the dark at -20°C. The XTT / PMS stock solution was prepared immediately before use by adding 40 µL of PMS per mL of XTT solution. Fifty microliters (50 µl) of XTT / PMS were added to each well of the plate, and the plate was incubated for 4 hours at 37°C. The 4-hour incubation period was determined empirically to be within the linear range of sensitivity for the decline in XTT staining with the specified cell numbers for each assay.The plates were sealed and inverted several times to mix the soluble formazan product, then the plate was scanned at 450 nm (reference wavelength 650 nm) using a Molecular Devices SpectraMax Plus 384 96 spectrophotometer.

[0110] Assay of HIV-1 activity in human peripheral blood mononuclear cells (PBMCs)

[0111] Leukophoretized blood cells are washed several times with Dulbecco's phosphate-buffered saline (DPBS). After washing, the leukophoretized blood is diluted 1:1 with DPBS and layered onto 15 ml of Ficoll-Hypaque density gradient in a 50 ml conical centrifuge tube. These tubes are then centrifuged for 30 min at 600 x g. The PBMC layer is carefully aspirated from the resulting interface and then washed three times with DPBS by centrifugation at low speed. After the final wash, the cells are counted by trypan blue dye exclusion and resuspended at a concentration of 1 × 10 6cells / ml in RPMI 1640 with 15% fetal bovine serum (FBS), 2 mmol / l L-glutamine, 2 μg / ml PHA-P, 100 units / ml penicillin, and 100 μg / ml streptomycin and incubated for 48-72 h at 37°C. After incubation, PBMCs were centrifuged again and resuspended in tissue culture medium (RPMI 1640 with 15% FBS, 2 mmol / l L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, and 3.6 ng / ml recombinant human IL-2). Cultures were then maintained until use by replacing half the volume of the culture medium with fresh IL-2-containing tissue culture medium every 3 days. The assay begins with induction of PBMC proliferation with RNA-P for 72 hours. For the PBMC assay, RNA-P-stimulated PBMC from two donors are pooled to minimize variability that occurs when using cells from individual donors and resuspended in fresh tissue culture medium at a concentration of 1 x 10 6cells / ml and seeded into the inner wells of a 96-well round-bottomed microtiter plate at a volume of 50 μl / well. One hundred microliters (100 μl) of a two-fold concentration of polymer-containing medium are transferred to the labeled wells of a round-bottomed 96-well plate containing cells, in triplicate. Immediately after adding the polymer, 50 μl of a predetermined virus dilution are added to the wells and mixed well. After 7 days of culturing at 5% CO2 / 37°C, HIV-1 replication is quantified by measuring cell-free HIV-1 reverse transcriptase (RT) activity in tissue culture supernatant as described below. Cytotoxicity is assessed using XTT tetrazolium dye as described above.

[0112] Assessment of chronic inhibition of HIV-1 replication

[0113] CEM-SS cells chronically infected with HIV-1 IIIB , added to a 96-well microtiter plate at a density of 2.5×10 3cells per well in a volume of 100 µl. Serial dilutions of the compound(s) are prepared so that a total of six concentrations are assessed. One hundred microliters (100 µl) of each concentration are added to the cells in triplicate. The plates are incubated at 37°C / 5% CO2 for 6 days. After incubation, cell-free supernatant samples are collected from each well of a 96-well plate and assayed for reverse transcriptase (RT) activity. The plate is stained with XTT tetrazolium dye to measure cell viability. Reverse transcriptase activity assay

[0114] Reverse transcriptase was measured in cell-free supernatants using a standard polymerization radioincorporation assay. Tritiated thymidine triphosphate (TTP) was purchased at a concentration of 1 Ci / mL, and 1 μL was used per enzymatic reaction. Poly rA and oligo dT were prepared at concentrations of 0.5 mg / mL and 1.7 units / mL, respectively, from a stock solution stored at -20°C. Fresh RT reaction buffer was prepared daily and consisted of 125 μL 1 M EGTA, 125 μL dH2O, 125 μL 20% Triton X-100, 50 μL 1 M Tris (pH 7.4), 50 μL 1 M DTT, and 40 μL 1 M MgCl2. For each reaction, mix 1 µl of TTP, 4 µl of dH2O, 2.5 µl of rAdT, and 2.5 µl of reaction buffer. Ten microliters (10 µl) of this reaction mixture are added to a round-bottomed microtiter plate containing 15 µl of virus-containing supernatant. The plate is incubated at 37°C in a humidified incubator for 60-90 minutes.After incubation, 10 µl of the reaction mixture are applied to a DEAE filter plate in the appropriate format. Each filter plate is washed five times for 5 minutes in 5% sodium phosphate buffer, twice for 1 minute in distilled water, twice for 1 minute in 70% alcohol, and then air-dried. The dried filter plate is placed in a plastic sleeve, and 4 ml of Opti-Fluor O reagent is added to each sleeve. The incorporated radioactivity is quantified using a Wallac 1450 Microbeta Trilux liquid scintillation counter.

[0115] Assay for inhibition of cell-to-cell cell transmission

[0116] Uninfected CEM-SS cells were seeded in a 96-well flat-bottomed plate at a density of 1×10 5 cells per well in a total volume of 50 µl. Chronically infected with the HIV-1 virus IIIB CEM-SS cells are added at cell densities ranging from 1×10 5 cells per well up to 1×10 0cells per well in a volume of 50 µl. The test compounds are serially diluted to obtain test concentrations and added to the wells in a volume of 100 µl in triplicate. The plate is incubated at 5% CO2 / 37°C for 48 hours. After incubation, the number of syncytia per well is counted. After an additional 24-hour incubation, cell-free supernatant samples from each well of the 96-well plates are assayed for reverse transcriptase (RT) activity as described above.

[0117] Assay for inhibition of HIV-1 virus entry BaL into TZM-bl-FcRI cells

[0118] Twenty-four hours before compound exposure, TZM-bl-FcRI cells were seeded in a 96-well flat-bottomed plate at a density of 1×10 4 cells per well in a volume of 100 μl. After incubation at 5% CO2 / 37°C, 50 μl of serial dilutions of the compound are added to the cells in triplicate 10-15 minutes before the addition of HIV-1 BaL . HIV-1 BaLThe cultures are diluted to a predetermined titer and added to potency plates in a volume of 50 µl. The same volume of medium is added to the toxicity plates. After a two-hour incubation at 5% CO2 / 37°C, the cultures are washed to remove residual virus and compound. The plates are incubated at 5% CO2 / 37°C for an additional 48 hours, during which time the potency plates are assessed using a chemiluminescent substrate (Gal Screen, Tropix), and toxicity is assessed using XTT, as described above.

[0119] Evaluation of Compound Efficacy Using Chemiluminescent Detection

[0120] All medium is removed from the efficiency plates and replaced with 50 ml of DPBS. Fifty microliters (50 µl) of Gal-Screen substrate, diluted 1:25 in Gal Screen Buffer, are added to each well. The plate is incubated for 90 minutes at room temperature. After incubation, the well contents are transferred to a clear-bottomed plate. The plate is covered, and chemiluminescence is measured using a Microbeta scintillation counter.

[0121] Assay of TZM-bl-FcRI cell-cell fusion inhibition

[0122] HeLa-CD4-LTR-β-Gal cells were seeded at a density of 5×10 3 cells per well in a volume of 50 µl, adding 50 µl of six-fold serial dilutions log 10Compounds were mixed in triplicate and incubated for one hour at 37°C / 5% CO2. After incubation, 100 µl of HL2 / 3 cells were added to the plates. The cultures were incubated for another 48 hours at 37°C / 5% CO2. After incubation, potency plates were assayed for β-galactosidase production using a chemiluminescent substrate, and toxicity plates were stained with XTT to assess cell viability, as described above.

[0123] ASSESSMENT OF HIV-1 ENZYME INHIBITION

[0124] HIV-1 reverse transcriptase inhibition assay

[0125] The HIV-1 reverse transcriptase (RT) inhibition assay used HIV-1 reverse transcriptase (RT) enzyme provided by ChimerX. Six logarithmic serial dilution concentrations in water were added to a 96-well U-bottom plate containing 50 μl of a reaction mixture of 2M Tris-HCl, pH 8.0, 3M KCl, 1M MgCl2, 2M DTT, 10 mM dGTP, 25 U / ml rC:dG template, 10 μl [ 32P]-α-dGTP (800 Ci / mmol) and 20 μl of the enzyme reaction mixture containing 5 μl of HIV-1 reverse transcriptase, BSA, and Triton X-100. The reaction plate is incubated at 37°C for 50 min. After incubation, 10 μg / ml of sonicated salmon milt DNA and 150 μl of 10% TCA are added to the wells to facilitate DNA precipitation and extraction and the wells are left to incubate at room temperature for 15 min. The well contents are then transferred to DEAE anion exchange paper and washed by aspirating through the filter using a vacuum manifold. The plate is then washed once with 200 μl of 10% TCA as above. Fifteen microliters (15 µl) of Wallac Supermix Scintillant reagent are added to each well and the plate is counted using a Wallac Micro Beta scintillation counter.

[0126] HIV-1 protease inhibition assay

[0127] HIV-1 protease activity is determined using the Sensolyte® 520 HIV-1 Protease Assay Kit, which contains the Hilyte Fluor™ 488 / QXL™ 520 FRET peptide. In the FRET peptide, the fluorescence of HiLyte Flour™ 488 is quenched by QXL™ 520 until the peptide is cleaved into two separate fragments by HIV-1 protease. After cleavage, the fluorescence of Hilyte Fluor™ 488 is detected and monitored at excitation / emission wavelengths of 490 nm / 520 nm. Recombinant HIV-1 protease is diluted to a concentration of 2.5 ng / mL in protease assay buffer, and 40 µL of the diluted protease is added to all but two wells of a 96-well black flat-bottomed plate for NUNC fluorescence detection. Six serial dilutions are prepared. -log 10Test compounds and saquinavir (positive control) at 10-fold the final well concentration in protease assay buffer. Ten microliters (10 μl) of the diluted compounds are added to the wells of the assay plate containing protease in triplicate. Ten microliters (10 μl) of buffer alone are also added to six wells containing protease to provide a compound-free positive control. The plate is incubated at 37°C for 15 minutes. Fifty microliters (50 μl) of fluorescent protease substrate (diluted 1:500 in assay buffer) is added to each well, and the plate is incubated at room temperature for 60 minutes. Fifty microliters (50 μl) of stop solution is added to each well. Fluorescence intensity is measured using excitation / emission wavelengths = 490 nm / 520 nm EU 50 calculated from the fluorescence endpoint data for each of the compounds.

[0128] HIV-1 integrase inhibition assay

[0129] HIV-1 integrase activity is assessed using the HIV-1 Integrase Assay Kit (XpressBio Life Science). Streptavidin-coated 96-well plates are coated with donor substrate (DS) DNA—the double-stranded HIV-1 LTR U5 containing a terminal biotin label. One hundred microliters (100 μl) of 1× DS DNA solution are added to each well, and the plate is incubated for 30 minutes at 37°C. After incubation, the plate is washed three times with wash buffer, 200 μl of blocking buffer are added to each well, and the plate is incubated for 30 minutes at 37°C. One hundred microliters (100 μl) of reaction buffer (negative control) or integrase enzyme solution (positive control) are added to the designated wells and incubated for 30 minutes at 37°C.The plate is washed as described above, and 50 μl of each test product diluted in reaction buffer (for positive and negative controls, only reaction buffer) are added to the designated wells and incubated for 5 minutes at room temperature. Fifty microliters (50 μl) of IX TS DNA solution are added to the plate, mixed, and the plate is incubated for 30 minutes at 37°C. After washing the plate five times with wash buffer, 100 μl of the HRP antibody solution is added, and the plate is incubated for 30 minutes at 37°C. After an additional 5 washes, 100 μl of TMB peroxidase substrate solution is added, and the plate is incubated for 10 minutes at room temperature. One hundred microliters (100 μl) of TMB stop solution are directly added to the wells containing the TMB substrate. The plate is read at an absorbance of 450 nM.

[0130] Evaluation of compound addition time in HeLa-CD4-LTR-β-Gal cells

[0131] HeLa-CD4-LTR-Gal cells were seeded at a density of 1×10 4 cells / well in a volume of 100 µl 24 hours before the start of the assay and incubated at 37°C / 5% CO2. After incubation, the compound is serially diluted to the indicated concentrations and added to the cells in triplicate at time points of 30 minutes, 0, 1, 2, 4, 8, and 24 hours before or after the addition of the virus in a volume of 100 µl. HIV-1 IIIB added to cells with a predetermined titer. The cultures are incubated at 37°C / 5% CO2 for 48 hours, after which the efficacy is assessed using a chemiluminescent substrate (Gal-Screen) and toxicity is assessed (three time points: 0, 4, and 24 hours) using the tetrazolium dye XTT.

[0132] Assay of anti-HBV activity on AD38 cells

[0133] AD38 cells contain a stably transfected HBV gene with transcriptional control by the tet operon. HBV expression is suppressed when cells are cultured in the presence of tetracycline and can be induced by removing tetracycline from the culture medium. AD38 cells are seeded in 96-well flat-bottomed plates at a density of 1 x 10 5 cells per well and cultured in the presence of 0.3 μg / ml tetracycline for 2 days at 37°C / 5% CO2. After incubation, the medium was removed and the cells were washed to remove residual tetracycline. Six concentrations of serially diluted polymer (1 / 2 log increments) were added to the cells and incubated for 6 days at 37°C / 5% CO2, with the medium changed on day 3 (polymer was added again). On day 6, 100 μl of supernatant from each well was removed for viral DNA analysis by qPCR, and the cell monolayers were restained with XTT to assess cytotoxicity as described above.

[0134] qPCR Method

[0135] One hundred microliters (100 μl) of cell culture supernatant is diluted with 90 μl of dilution buffer containing 10 μM Tris and 40 μg / ml salmon DNA. The samples are heated at 102°C for 15 minutes. Then six microliters (6 μl) of the sample are mixed with 12.5 μl of 2X Platinum PCR supermix with ROX, 0.5 μl of HBV forward primer (10 μM AD38 qF1), 0.5 μl of HBV reverse primer (10 μM AD38qR1), 0.5 μl of Taqman probe (AD38 qP1), and 6 μl of molecular biology grade water. The polymerase chain reaction (PCR) is carried out under the following conditions: 50 cycles of 95°C for 15 seconds, then 60°C for 1 minute.

[0136] Evaluation of activity against influenza A virus and influenza B virus

[0137] MDCK cells cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 1 mM sodium pyruvate and 0.1 mM NEAA were seeded in a 96-well flat-bottomed plate at a cell density of 1×10 4cells per well in a volume of 100 µl. The plates are incubated at 37°C and 5% CO2 for 24 hours. After incubation, the polymers are sequentially diluted in half-log increments (6 concentrations in total) and 100 µl of each concentration is added to the cells in triplicate. Influenza A virus CA / 27 / 07 or influenza B virus Allen The virus is diluted to a predetermined titer in the assay medium and added to the cultures in a volume of 100 µl. This viral titer is the amount determined to kill 80% of the cells 4 days after infection. The cultures are incubated for 4 days at 37°C / 5% CO2. After incubation, the test plates are stained with XTT tetrazolium dye as described above.

[0138] Assessment of activity against adenovirus and rhinovirus

[0139] Inhibition of virus-induced cytopathic effects (CPE) and cell viability after adenovirus replication in HeLa cells or human rhinovirus replication in MRC-5 cells were measured using XTT tetrazolium dye. Cells (1×10 4 Cells (cells per well) are seeded into 96-well flat-bottomed tissue culture plates and allowed to attach overnight at 37°C / 5% CO2. After incubation, the medium is removed from the cell monolayers, and serial dilutions of the polymer (6 concentrations) and virus diluted to a predetermined titer leading to 85 to 95% cell killing at 6 days post-infection are applied to the plate. Ribavirin is also assessed in parallel as a positive control compound. After incubation at 37°C, 5% CO2 for six days, cell viability is measured by XTT staining as described below.

[0140] Toxicity assessment for Ca Ski and ME180 cells

[0141] Twenty-four hours before exposure to the compound, 100 μl of cells are plated at a density of 5×10 4 cells per well in a flat-bottomed plate. After 24 hours of incubation at 5% CO2 / 37°C, serial dilutions of the compound and medium are added to the cells in triplicate. The cultures are incubated for an additional 24 hours at 5% CO2 / 37°C and then washed to remove residual compound. The plates are incubated at 5% CO2 / 37°C for an additional 24 hours and then assessed using XTT as described above.

[0142] Toxicity assessment of Lactobacillus species from normal vaginal flora

[0143] Frozen glycerol-coated stock strains of Lactobacillus jensenii and Lactobacillus crispatus were grown in MRS broth for 48 hours prior to compound exposure. Six (6) concentrations of each compound were prepared by serial dilution in MRS broth, and each was plated in triplicate on a 96-well round-bottomed plate. Each Lactobacillus species was diluted to OD 625 =0.06 in MRS broth and added to the appropriate wells of the plate. The cultures are incubated anaerobically for 24 hours, after which bacterial growth is assessed spectrophotometrically at 490 nm. Penicillin / streptomycin solution is used as a control for the analysis.

[0144] Vaginal-ectocervical tissue toxicity assessment

[0145] On the day of testing, warm the MatTek assay medium and add 900 µl to each well of a 6-well plate. One hour before dosing, remove the epivaginal tissue from the refrigerator and place it in the 6-well plate. The plate is then incubated for 1 hour at 37°C / 5% CO2. After incubation, remove the medium from the 6-well plate and add 900 µl of fresh medium. One hundred microliters (100 µl) of each concentration are added to the epivaginal tissue in duplicate.

[0146] The plate is then incubated for an additional 24 hours at 37°C / 5% CO2. One hour before the end of the incubation, a 1 mg / ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (hereinafter MTT, manufactured by Invitrogen Carlsbad, California, USA) in DMEM is prepared. Fifteen minutes before the end of the incubation, 300 μl of MTT is added to each well of a 24-well plate. After incubation, residual fluid is removed from the tissue, and the tissue is then carefully washed twice with PBS. Each epivaginal tissue insert is then transferred to a separate well of a 24-well plate containing the MTT solution. The plate is then incubated for 3 hours at 37°C / 5% CO2. Each insert is then transferred to a pre-labeled extraction plate, and 2.0 ml of the extraction solution is added to the well so that the tissue insert is completely immersed.Extraction is carried out for two hours at room temperature, after which the extraction solution is thoroughly mixed and 200 µl is transferred to a 96-well round-bottomed plate. The optical density of each sample is determined at 570 nm, background at 650 nm. % viability is determined using the formula 100× [OD(sample) / OD(negative control)]. Triton™ X-100 and N-9 are used as controls for the analysis.

[0147] Materials

[0148] In the compositions of the present invention, a low molecular weight hydrophobically modified polymer, a potassium acrylate copolymer (Lubrizol, Brecksville, Ohio, USA), was used as the low molecular weight hydrophobically modified polymer.

[0149] Example 1

[0150] Invention Examples E1-E3 and Comparative Examples C1-C3: Preparation of Compositions for Testing

[0151] Compositions E1-E3, A1, and C1-C3 were prepared according to the descriptions below using the amounts of materials listed in Table 1. Compositions E1-E3 correspond to the compositions and methods of the present invention. Composition A1 corresponds to the compositions and methods set forth in copending patent application, Attorney Docket No. JCO6079USNP, filed concurrently with this document. Compositions C1-C3 are comparative compositions.

[0152]

[0153] Each of the compositions in Table 1 was independently prepared as follows:

[0154] E1 - 1.7 g of potassium acrylates copolymer (30% activity) was mixed with 98.3 g of deionized water and the pH was adjusted to 6.5 using 20% ​​sodium hydroxide solution;

[0155] A1 - 0.5g PEG6000 was dissolved in water under gentle heating, and the measured pH value was 6.65.

[0156] C1 - 17.4 g cocamidopropyl betaine, 23.4 g sodium laureth sulfate and 8.3 g PEG 80 sorbitan laurate were added to 150.9 g deionized water and the pH was measured at 6.8.

[0157] C2 - 3.4 g of potassium acrylates copolymer, 17.4 g of cocamidopropyl betaine, 23.4 g of sodium laureth sulfate, and 8.3 g of PEG 80 sorbitan laurate were added to 150.9 g of deionized water, and the potassium acrylates copolymer was neutralized with 20% sodium hydroxide. The final measured pH was 6.5.

[0158] E2 - 10.0 g of potassium acrylates copolymer (30% activity) was mixed with 88.56 g of deionized water and the pH was adjusted to 6.7 using 1.44 g of 20% sodium hydroxide solution.

[0159] E3 - 3 g of nonoxynol-9 was mixed with 86.24 g of deionized water and stirred on a mixing plate until nonoxynol-9 was completely dissolved. 10 g of potassium acrylates copolymer was added to the mixture, and the pH was adjusted to 6.4-6.6 using a 20% sodium hydroxide solution.

[0160] C3 - 3g of nonoxynol 9 was mixed with 97g of deionized water, stirred on a mixing plate until nonoxynol was completely dissolved, and the measured pH value was 5.2-5.4.

[0161] Example 2

[0162] Inventive Examples E4-E11: Preparation of Illustrative Embodiments of the Compositions of the Present Invention

[0163] Stable antiviral compositions E4-E11 were prepared according to the materials and amounts listed in Table 2 and the methods described below.

[0164]

[0165]

[0166] Each of the compositions of the embodiments in Table 2 was independently prepared as follows.

[0167] E4-E5. For E4, water was measured into the main beaker. Carbomer was poured into the water with stirring. The carbomer was allowed to disperse evenly. Potassium acrylates copolymer was added, and heating was started until a temperature of 65°C was reached. Potassium cetyl phosphate (and) hydrogenated palm oil glycerides and mineral oil were added to the mixture. After 10 minutes, the heating was turned off and the mixture was cooled. At room temperature, phenoxyethanol and ethylhexylglycerin were added, and the pH of the composition was adjusted to 6.7-7.00 to the desired volume and placed into storage. The pH of the lotion was recorded as 6.8.

[0168] For E5, the same procedure was used as for E5, except that after adding mineral oil, polyglyceryl-10 laurate and stearyl alcohol were added to the mixture.

[0169] E6. Cross-linked acrylates polymer and water were added to a beaker. Potassium acrylates copolymer was added and neutralized with 20% sodium hydroxide solution. A clear gel was obtained. The pH was recorded as 7.2.

[0170] E7. Water was added to the beaker, hydroxyethyl cellulose was added to the water while stirring, and the mixture was left to stir for 45 minutes. The mixture was heated to 50°C. Potassium acrylates copolymer, cocoglucoside (and) glyceryl oleate, lauryl glucoside (and) polyglyceryl-2-depolyhydroxystearate (and) glycerin were added to the beaker. The heat was turned off, and when the composition reached a temperature of approximately 30°C, glycerin, phenoxyethanol, ethylhexylglycerin, and sodium benzoate were added to the beaker. The pH was adjusted to 7, and the cross-linked acrylates polymer was added.

[0171] E8. Water was added to the beaker and stirred. Carbomer was poured in while stirring. The carbomer was allowed to disperse evenly. Potassium acrylates copolymer was added to the composition, and heating was initiated until a temperature of 65°C was reached. Potassium cetyl phosphate (and) hydrogenated palm oil glycerides, stearyl alcohol, and mineral oil were added to the composition. Heating was turned off after 10 minutes, and the mixture was allowed to cool to room temperature. Phenoxyethanol was added; ethylhexylglycerin was added, and the pH of the composition was adjusted to 6.7-7.00, adjusted to the desired volume, and stored.

[0172] E9. Water and propylene glycol were added to the main beaker. Potassium acrylates copolymer was added to the beaker while stirring. Sodium hydroxide was added to neutralize the potassium acrylates copolymer (until a clear solution and a pH of 6.5 to 7 were obtained). Sodium benzoate was added, and the mixture was stirred for 30 minutes. The mixture was heated to 55-60°C. Hydroxyethyl cellulose was slowly added, and the mixture was stirred until a homogeneous gel was formed. The mixture was cooled to room temperature, and the pH of the gel was recorded at 4.5.

[0173] E10. Water and carbomer were added to a beaker with stirring. They were mixed with hydroxyethyl cellulose and left to stir for 45 minutes while heating to 50°C. The heating was turned off, and when the composition reached a temperature of about 30°C, potassium acrylates copolymer was added. Sodium hydroxide was added to neutralize the potassium acrylates copolymer (until a clear solution and a pH of about 6.5 to about 7 were obtained). Glycerin, phenoxyethanol, ethylhexylglycerin, and sodium benzoate were added to the desired volume and the pH was adjusted to 6.5-7.0.

[0174] E11. Carbomer and water were added together to a beaker. Kathon CG was then added. Potassium acrylates copolymer was then added to the composition and neutralized with a 20% sodium hydroxide solution while stirring. A clear gel was obtained, and a pH of 7.2 was recorded.

[0175] Example 3

[0176] Test of mildness of action using transepithelial permeability test (TEP)

[0177] Samples E2, E3 and C3 were tested for TEP according to the method described in detail above.

[0178]

[0179] C3, which contains nonoxynol N9 and water, exhibited significant leakage compared to E2 and E3, both of which contained potassium acrylates copolymer (E3 also contained nonoxynol N9). Both E2 and E3 formulations demonstrated superior gentleness in the TEP assay, while C3 exhibited membrane damage, which could lead to agent penetration through mucosal tissue.

[0180] Example 4

[0181] Virucidal effect of potassium acrylates copolymer (hydrophobically modified polymer) against HSV-1 using the in vitro time-kill study method.

[0182] Using the protocol described above, E1 neutralization studies were conducted on the input viral strain to verify that the neutralizing solution (De Engle [D / E] Neutralizing Broth) effectively neutralized the virucidal activity of the product. The neutralizing solution (D / E) effectively neutralized the virucidal activity of the test product and was shown to be nontoxic to the virus and cell cultures.

[0183]

[0184]

[0185] The virus population recovered from virus control #1 was 6.50 log 10 , from virus control No. 2 - 6.25 log 10 , and from the neutralization control - 6.25 log 10 The observed differences did not exceed 1 log. 10 , and thus the infectivity of the test virus did not change.

[0186] It was unexpectedly found that the E1 composition containing potassium acrylates copolymer reduced the infectivity of the HF strain of HSV-1 virus (ATCC#VR-260) by 4.00 log 10 (99.99%) after 15-minute, 30-minute and 1-hour exposure (Table 4).

[0187] Example 5

[0188] Activity of embodiments against HIV-1, hepatitis B virus, influenza virus, adenovirus and rhinovirus

[0189] Using the protocol described above, embodiments E1, A1, C1, and C2 were tested against a wide range of HIV-1 subtypes and tropisms, representing the broad spectrum of global HIV-1 diversity (Table 5).

[0190]

[0191]

[0192] Embodiment E1 also demonstrated similar activity against several other HIV-1 strains representing additional geographic and genetic diversity, including 92UG029, 92HT599, 98IN017, 92UG024, 92RW020, 92BR003, 97ZA003, 92UG035, and 93TH073, but not against influenza. Surprisingly, embodiment A1 was found to exhibit activity against influenza virus but no activity against the other enveloped viruses tested, as reflected in co-pending patent application with Attorney Docket Number JCO6079USNP. The inventors theoretically expect that compositions containing polyalkylene glycols will be effective in inhibiting influenza viruses, more preferably polyethylene glycols and polypropylene glycols, most preferably polyethylene glycols having a higher molecular weight, for example about 6000.In addition, the authors of the invention suggest that compositions containing both low-molecular hydrophobically modified polymers and polyalkylene glycols should have a broad spectrum of activity in terms of inhibiting enveloped viruses, including influenza viruses.

[0193] As shown, embodiments of the present invention exhibit activity against an unexpectedly wide range of HIV-1 subtypes and tropisms at very low concentrations. These data demonstrate the broad spectrum of activity of E1 embodiments against HIV-1.

[0194] Example 6. Mechanism of activity against HIV-1

[0195] The E1 embodiment was evaluated in several in vitro HIV-1 mechanism of activity assays to evaluate and determine the mechanism of action against HIV. Results

[0196] As shown in Table 6, E1 had no effect when treating cells chronically infected with HIV-1. In addition, embodiment E1 had no activity in preventing cell-to-cell transmission of HIV-1 or preventing the fusion of HIV-1-infected cells. Embodiment E1 demonstrated efficacy in TZM-bl-FcRI cell viral entry inhibition assays, indicating that the polymer is a weak entry inhibitor. E1 could inhibit the HIV-1 enzymes integrase, protease, and reverse transcriptase; however, this activity is likely nonspecific and is not expected to contribute to the efficacy against the virus in cell culture. According to the data in Tables 6 and 7, embodiment E1 likely blocks an early step of HIV replication, such as viral attachment, fusion, or entry into target cells, but before reverse transcription.The polymer must be present within the first 1-2 hours after infection, which is consistent with other active substances that block early stages of viral replication.

[0197]

[0198]

[0199] Example 7. Toxicity test to mucosal cells

[0200] Example E1 was tested for toxicity to mucosal cells using the protocol described above. E1 was compared with the broad-spectrum antiviral surfactant N-9. In all tested cases, E1 was less toxic to cells and tissue than nonoxynol-9, as shown in Table 8. Furthermore, the toxic concentration of E1 was 100-1000 times higher than the concentrations of E1 that demonstrated efficacy against HIV-1 (Table 7). These data demonstrate the mild effects of E1 and similar embodiments on mucosal tissue.

[0201]

[0202] Embodiment E1 also demonstrated no toxicity to Lactobacillus populations at biologically significant concentrations (TSC) in the above-described tests. 50 >1450 mcg / ml). This further demonstrates its compatibility with mucosal surfaces.

Claims

1. A method for inhibiting the penetration of enveloped viruses into cells, comprising bringing said viruses into contact with an antiviral composition containing at least one low molecular weight hydrophobically modified polymer in an effective amount for inhibiting the penetration of viruses into cells, wherein said composition is free of a surfactant having a hydrophilic-lipophilic balance (HLB) of greater than 12, wherein said low molecular weight hydrophobically modified polymer comprises a polymer obtained from at least one first monomer component selected from the group consisting of (meth)acrylic acid and at least one second monomer component selected from the group consisting of one or more C1-C9 alkyl (meth)acrylates, wherein the low molecular weight polymer has a number average molecular weight of 100,000 or less.

2. The method according to claim 1, further comprising applying said antiviral composition to the infected surfaces of the subject.

3. The method of claim 2, wherein said infectable surfaces comprise one or more of the group consisting of skin tissue and mucous membrane of the subject.

4. The method of claim 3, wherein said mucosal tissue comprises tissue selected from the group consisting of oral tissue, ocular tissue, nasal tissue, vaginal tissue, or rectal tissue, and combinations thereof.

5. The method of claim 1, wherein said low molecular weight hydrophobically modified polymer is present in said composition in an amount of from 0.00005 to 10% by weight of the composition.

6. The method of claim 1, wherein said composition further comprises at least 50 wt.% of a protic solvent.

7. The method of claim 1, wherein said composition further comprises at least 97% by weight of water.

8. The method according to claim 1, wherein said viruses are selected from the group consisting of poxviruses, herpesviruses, retroviruses of the genus lentiviruses and combinations thereof.

9. The method according to claim 8, wherein said virus selected from the herpesvirus family is herpes simplex virus type 1, herpes simplex virus type 2, and combinations thereof.

10. The method according to claim 8, wherein said virus, selected from the family of retroviruses of the genus lentivirus, is human immunodeficiency virus type 1.

11. The method according to claim 1, wherein said inhibition of virus penetration into said cells results in a reduction in the likelihood of developing a viral infection.

12. The method of claim 1, wherein the antiviral composition does not substantially destroy biological surfaces.

13. A method for inhibiting the transmission of viruses by inhibiting the penetration of enveloped viruses into cells, comprising applying to non-biological surfaces an antiviral composition containing at least one low molecular weight hydrophobically modified polymer in an amount effective to inhibit the penetration of viruses into cells, wherein said composition is free of a surfactant having an HLB greater than 12, wherein said low molecular weight hydrophobically modified polymer comprises a polymer obtained from at least one first monomer component selected from the group consisting of (meth)acrylic acid and at least one second monomer component selected from the group consisting of one or more C1-C9 alkyl (meth)acrylates, wherein the low molecular weight polymer has a number average molecular weight of 100,000 or less.

14. A method for inhibiting the transmission of viruses by inhibiting the penetration of enveloped viruses into cells, comprising applying to the surfaces of edible products an antiviral composition containing at least one low molecular weight hydrophobically modified polymer in an amount effective to inhibit the penetration of viruses into cells, wherein said composition is free of a surfactant having an HLB greater than 12, wherein said low molecular weight hydrophobically modified polymer comprises a polymer obtained from at least one first monomer component selected from the group consisting of (meth)acrylic acid and at least one second monomer component selected from the group consisting of one or more C1-C9 alkyl (meth)acrylates, wherein the low molecular weight polymer has a number average molecular weight of 100,000 or less.

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