AU783242B2 - Methods and devices for preventing transmission of sexually transmitted diseases - Google Patents

Description

PAOPERpnAlwI2OO4-XZS AmclsUU558760 Omd spe doc.24MSM -1- METHODS AND DEVICES FOR PREVENTING TRANSMISSION OF SEXUALLY TRANSMITTED DISEASES FIELD OF THE INVENTION This invention relates generally to methods for prophylaxis against diseases S*i. transmittable by sexual contact and more particularly to methods for prophylaxis of HIV transmission. The invention also relates to combinations of compositions and devices used with such methods.

BACKGROUND INFORMATION The need to develop simple, effective and low cost methods to prevent HIV transmission between individuals is an important strategy to manage HIV infection.

20 Recent developments that have identified new more virulent strains of HIV, coupled with the suggestion that certain current therapies may be less effective than they were in the past, have heightened the need to develop methods to circumvent primary HIV infection from sexual contact.

In addition to HIV, many diverse diseases are transmitted sexually. Among the more common are condylomata acuminata (veneral warts), gonorrhea, syphilis, herpes simplex, granuloma cenereum, chancroid, granuloma inguinale, non-gonococcal urethritis, acute pelvic inflammatory disease, vaginitis and anorectal disease.

Although several methods for prophylaxis of these diseases are known, no universally satisfactory method has been developed. In theory, the best way to prevent transmission is abstinence, but in reality this is often impractical and not followed. Other WO 01/54636 PCT/US01/03043 methods involve the insertion of physical barriers to prevent the direct contact of bodily fluid between individuals. However. such methods are often inconvenient, requiring user acquiescence. Moreover, the barriers themselves sometimes fail, negating any potential prophylactic benefits.

More targeted prophylactic methods involve destroying HIV itself so that it is incapable of invading the recipient tissue. Such methods involve the administration of biologically active materials to the vagina, for example. Nonoxynol-9® is known to have bacteriocidal microbicidal) action and to be capable of destroying HIV. However, the use of Nonoxynol-9® and other spermicidal and bacteriocidal agents in vaginally inserted suppositories, creams, foams or the like, are discouraged because of the risk of causing mucosal inflammation. In addition, these agents tend to destroy the healthy bacterial flora of the vagina and often lead to yeast infections. Specifically, the use of Nonoxynol-9® has been reported to result in an increased risk of HIV infection among prostitutes. Kreiss et al., JAMA 268(4):477-482 (1992), which is incorporated by reference herein.

Other methods to destroy HIV in seminal fluid involve the use of antiseptics containing iodine, for example. (United States Patent No. 5,545,401). However, such methods often require vigilance by the user to ensure that irritation does not result.

Another method employs the use of zinc salt containing genital lubricants, in which the user spreads the lubricant on a genital surface to create an anti-viral chemical barrier. (United States Patent No. 5,624,675). However, some individuals may choose not to use such a method due to the uncertain effects that zinc may have upon entry into the bloodstream.

It has been observed that HIV is relatively rarely transmitted by oral secretions. In fact, the saliva of viremic individuals usually contains only noninfectous components of HIV indicating virus breakdown. This has been reported to be due to hypotonic disruption by which saliva kills infected mononuclear leukocytes and prevents their attachment to mucosal epithelial cells and production of infectious HIV. (Baron et al., Arch. Intern. Med.

159:303-310 (1999)). Although saliva could be used as a method for prevention of HIV transmission in oral, vaginal and rectal intercourse, the hypotonic preventative effect of saliva would be negated by the relatively larger quantity of isotonic semen.

WO 01154636 PCT/US01/03043 Thus, there exists a need for less irritating substances which prevent the transmission of sexually transmitted diseases, as well as the transmission of HIV-infected leukocytes in seminal fluid or HIV in free form. In addition, there is a need for a method in which the preventative properties of the substances remain intact relative to the volume of semen typically introduced. Finally, there is a need for methods for prophylaxis which are simple and convenient to use both vaginally and rectally. The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION The present invention provides a method for preventing the transmission of HIV vaginally or rectally or orally by contacting a bodily fluid suspected of containing HIV with a composition having at least one of polyquatemium, glycerin, and a preservative system.

An embodiment of the preservative system in this method and those enumerated below comprises methylparaben and proplyparaben.

The invention also provides a method for prophylaxis of HIV transmission vaginally or rectally or orally by contacting a bodily fluid suspected of containing HIV with a composition having at least one of polyquaternium, glycerin, and a preservative system.

The invention also provides a method for reducing the risk of transmission of HIV vaginally or rectally or orally by contacting a bodily fluid suspected of containing HIV with a composition having at least one of polyquatemium, glycerin, and a preservative system.

The invention also provides a method for inhibiting the transmission of a sexually transmitted disease caused by an agent that is present in a bodily fluid by placing a composition having at least one of polyquaternium, glycerin, and a preservative system in a vagina, rectum, oral cavity, condom or on some other barrier device that can be inserted into a vagina or rectum prior to deposition of the bodily fluid into the vagina or rectum.

Yet another aspect of the provides a method for inhibiting the transmission of a sexually transmitted disease caused by an agent that is present in a bodily fluid by placing a composition having at least component selected from the group consisting of surfactants, microbicides, anticellular agents, acid agents, alkaline agents, oxidizing agents. inhibitors, WO 01/54636 PCT/US01/03043 organic solvents, and hypotonic solutions. The composition is placed into the oral cavity prior to deposition of the bodily fluid into the oral cavity. Alternatively, the composition can be combined with a condom or other barrier device that is inserted into the vagina prior to deposition of the bodily fluid into the respective cavities.

Yet another aspect of the invention is a method for preventing the transmission of HIV by a needle and syringe in use by a plurality of persons. The needle and syringe comprise viable donor cell-free HIV and/or donor HIV infected cells as a result of contamination from one or more of the people using the syringe and needle. The method involves the step of contacting, prior to use of the syringe and needle, the donor cell free HIV and donor HIV infected cells with a composition that inhibits production of HIV by the donor cells, or inhibits the viability or infectivity of cell free HIV. The composition comprises at least one component selected from the group consisting of surfactants, microbicides, anticellular agents, acid agents, alkaline agents, oxidizing agents, inhibitors, organic solvents, and hypotonic solutions.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphical representation of the effectiveness of over the counter vaginal preparations at inhibiting VSV multiplication in CEM lymphocytes in seminal fluid.

Figure 2 represents the effectiveness of over the counter vaginal preparations at inhibiting HIV multiplication in human CEM lymphocytes in seminal fluid.

Figure 3 shows the time required for over the counter vaginal preparations to inhibit production of HIV by infected human CEM lymphocytes.

Figure 4 shows the effectiveness of over the counter vaginal preparations at inhibiting the multiplication of cell-free HIV.

Figure 5 shows the effectiveness of over the counter vaginal preparations at inhibiting the multiplication of cell-free HIV in seminal fluid.

Figure 6 shows the toxicity and inhibitory titers of components of over the counter vaginal preparations. Toxicity and inhibitory activities of the components were absent in WO 01/54636 PCT/US01/03043 concentrations below 8% glycerol, 0.2% polyquatemium 32. 0.2% methylparaben, and 0.004% propylparaben.

Figure 7 shows interruption of VSV multiplication in L-cells by surfactants, bile salts, and components of douches.

Figure 8 shows interruption of VSV multiplication in L-cells by surfactants. vinegar and vaginal jellies.

Figure 9 shows inactivation by detergents of VSV infected CEM lymphocytes suspended in seminal fluid and placed on rectal tissue.

Figure 10 shows interruption by detergents of virus (VSV) multiplication in CEM lymphocytes in seminal fluid.

Figure 11 shows decontamination of syringes containing VSV infected CEM lymphocytes.

Figure 12 shows inactivation by detergents of VSV infected CEM lymphocytes suspended in seminal fluid and placed on rectal tissue.

Figure 13 shows interruption of virus (VSV) multiplication in L-cells by detergents.

Figure 14 shows interruption of virus (VSV) multiplication in L-cells by saliva and surfactants.

Figure 15 shows interruption of virus (VSV) multiplication in L-cells by detergents.

Figure 16 shows interruption of virus (VSV) multiplication in L-cells by enemas and mouthwashes.

Figure 17 shows interruption of virus (VSV) multiplication in L-cells by detergents.

Figure 18 shows interruption of virus (VSV) multiplication in L-cells by douches.

Figure 19 shows interruption of virus (VSV) multiplication in L-cells by detergents and mouthwashes.

Figure 20 shows detergents and over the counter vaginal or oral preparations inhibit cell-free HIV.

Figure 21 shows inactivation by bile salts and saliva of HIV infected CEM lymphocytes.

Figure 22 shows decontamination of syringes containing cell-free HIV in blood using 1% ursodeoxycholate.

WO 01/54636 PCTIUS01/03043 Figure 23 shows inactivation of cell-free HIV by ursodeoxycholate but not by saliva.

This application is a continuation-in-part application of U.S. Application Serial No.

09/494,201. filed January 28, 2000. Figure 24 shows decontamination of syringes containing cell-free HIV in blood using 1% ursodeoxycholate.

Figure 25 shows inactivation by bile acids and saliva of cell-free HIV.

Figure 26 shows inactivation by bile acids of cell-free HIV.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods for preventing the transmission of HIV, methods for prophylaxis of HIV transmission, methods for reducing the risk of transmission of HIV vaginally or rectally and to methods for inhibiting the transmission of sexually transmitted diseases in general. The methods involve the use of compositions that have at least one of the following components in an aqueous solution: polyquaternium, glycerin, and a preservative system. In another aspect the compositions for use in the methods of the invention comprise at least one component selected from the group consisting of surfactants, microbicides, anticellular agents, acid agents, alkaline agents, oxidizing agents, inhibitors, organic solvents, and hypotonic solutions.

Despite extensive public awareness campaigns, the incidence of HIV infection remains high, especially in developing countries. Therefore, to prevent HIV transmission vaginally and rectally, substances are needed that are easy to use, effective and low cost.

One formulation of the present invention comprises a sexual lubricant. As such, the chances of it being used, in addition to, or if necessary, separately from barrier devices.

such as condoms, are greater. In addition, compositions of the invention are relatively inexpensive to produce.

As used herein, the term "barrier device" refers to a device that serves to physically keep one individual's bodily fluid from coming into contact with another individual.

Examples of such devices include, condoms, female condoms, and can also include cervical caps and sponges, as well as intrauterine devices, and vaginal diaphragms. Also as used 1 WO 01/54636 PCT/US01/03043 herein, the term bodily fluid refers to a fluid or exudate of an individual that emanates, is expelled or released from the body. Examples of such bodily fluids include, blood, saliva, tears, semen, vaginal discharge, pus, mucous, urine and feces. Such bodily fluids can come into contact with a different individual thereby transmitting sexually transmitted diseases.

A bodily fluid may contain HIV in free form or HIV-infected cells.

Infected mononuclear leukocytes are the major infectious component of a donorcarrier's seminal fluid. In addition, they routinely survive in the isotonic seminal fluid in the recipients vagina. The infected mononuclear leukocytes then penetrate the vaginal epithelium and transmit infection to subepithelial leukocytes or attach to and infect CD4negative epithelial cells. (Milman et al., AIDS Res. Hum. Retrov. 10:1305-1312 (1994).

which is, as are the other references and patents cited herein, incorporated herein by reference). It is believed that an infected HIV-leukocyte or a cell-free HIV particle can remain in the vagina or rectum for approximately one-half hour before penetration of the epithelial tissues takes place(Bomsel, M. Nature Medicine, 1997, 3:42-47). Accordingly, the compositions of the present invention ideally should be placed in contact with the donor bodily fluid preferably within a half hour but in any case within this one hour time period.

Most preferably, the compositions will be placed in the vagina or rectum or oral cavity, or even on the penis, before the commencement of sexual contact. Also as used herein, the term "sexual contact" refers to the deposition of a donor individual's bodily fluid onto or into a recipient individual's mucosal surface or surfaces, that is to say. from a donor to a recipient.

The compositions of the methods of the invention include at least two commercially available vaginal lubricant products: AstroGlide® (BioFilm, Vista, California) and Vagisil® (Combe Inc., White Plains, NY). The AstroGlide® composition is also sold under the brands Silken Secret®, ViAmor®, and Target®.

The AstroGlide® product includes water, glycerin, polyquatemium #33, propylene glycol, methylparaben and propylparaben. Vagisil® comprises polyquatemium #32.

Without limitation, other polyquatemium-containing vaginal lubricants, included in the compositions of the method, are Just Between Us® Personal Lubricant (Key West Aloe Co.),CVS® Personal Lubricant (Sun Mar Laboratories), and Ultra Lube® (Super Brands), all of which contain polyquatemium Enhance® Personal Lubricant (The Xandria WO 01/54636 PCT/US01/03043 Collection) contains polyquaternium Also without limitation, non-polyquaterium containing personal lubricants included in the composition of the methods disclosed herein include Replens® Vaginal Moisturizer, K-Y Liquid®, K-Y® Long Lasting Vaginal Moisturizer, K-Y Silk-E® Vaginal Moisturizer, K-Y® Jelly Personal Lubricant, Summer's Eve® Vaginal Moisturizer, Aqua Lube®, and Wet, Light®Personal Lubricant Gel.

A novel aspect of the present invention, which has not been recognized previously is that the compositions herein also prevent or reduce the risk of HIV transmission as well as the transmission of other sexually transmitted diseases. For example, the compositions of the present invention may be used to prevent or reduce the likelihood of transmission of the herpes simplex virus, for example, since its morphology and chemistry are similar to that of HIV. It is understood that viral agents morphologically or chemically similar to HIV are subject to prophylaxis or the transmission-preventative methods of the present invention. It is further understood that the selection of morphologically and chemically similar agents are routine to those skilled in the art (Baron et al., Medical Microbiology, 4th ed., Galveston TX, The University of Texas Medical Branch at Galveston,(1996)).

In addition, the inventive methods may be effective with sexually transmitted diseases involving bacterial agents. As used herein, the term "agents" refers to a viral, bacterial or other factor that is the pathogen responsible for the transmission of sexually transmitted disease from one individual to the next. For example, an agent in AIDS is HIV. and an agent in genital herpes is HSV.

Also as used herein, the term "effective amount" refers to an amount or concentration of the composition of the present invention which is used in the methods to produce the intended result. In the case of the present invention, effective amounts are generally anti-HIV effective amounts, which may include amounts of the composition which prevent, reduce or are prophylactic for the sexually transmitted spread of HIV, for example. However, effective amounts can refer to anti-HSV or anti-microbe amounts.

One skilled in the art understands that the selection of an effective amount will depend upon which agent is under consideration, and further that such a selection will be routine.

Specifically, anti-HIV effective amounts or concentrations are amounts or concentrations of the compositions herein which inhibit the replication, growth and elaboration of HIV.

For example, an effective amount of an anti-HIV composition is approximately 0.5 ml or WO 01/54636 PCT/US01/03043 more. A preferable effective amount is 1 ml, however the user would likely use much more than that. Alternatively an effective amount can be in the range of 0.5 ml to greater than ml.

Concentration ranges ofpolyquaternium useful in the present invention are from 0.1% to 10% by weight. Alternatively, any polyquaternium can be used at any concentration that is non-irritating to the mucosal epithelium. It is contemplated that the polyquaternium used herein is cationic in nature, although polyquaterniums which are anionic or non-ionic also find use n the compositions of the method. The International Cosmetic Ingredient Dictionary and Handbook Edition (1997),Vol. 2, eds. J.A.

Wenninger and G.N. McEwen, publ. The Cosmetic, Toiletry, and Fragrance Association.

Wash. discloses, without limitation, polyquatemiums (and their commercial sources) that are useful in the compositions of the methods of the invention.

Concentration ranges of glycerol useful in the present invention are from about to about 60% by weight. An example of a concentration range of a preservative system useful in the invention involves methylparaben at 0.03% to 0.3% by weight and propylbaraben at 0.015% to 0.15% by weight.

As used herein, the term "preservative system" refers to a chemical or group of chemicals that act to prevent the contamination or degradation of other components in the composition. For example, methylparaben and propylparaben are used as preservatives herein. Examples of other preservatives useful in the present invention include, but are not limited to, the following FDA approved preservative systems for food, cosmetics, and food preparations: FOOD PRESERVATIVES Sorbic acid Thiabendazole Sodium sorbate Nisin Potassium sorbate Natamycin Calcium sorbate Formic acid Benzoic acid Sodium formate Sodium benzoate Calcium formate Potassium benzoate Hexamine W001/54636 PTUO/34 PCTIUSOI/03043 Calcium benzoate Ethvl4-hydroxybenzoate Sodium salt Propybaraben Propyl4-hydroxvbenzoate Methylparaben Methy 14-hydroxybenzoate Sulphur dioxides Sodium sulphite Sodium bisulsuiphite Sodium metabisuiphite Potassium metabisuiphite Potassium sulphite Calcium sulphite Calcium hydrogen sulphite Potassium bisuiphite Biphenyl 2-Hydroxybiphenyl Sodium biphenyl-2-vl oxide Potassium nitrite Sodium nitrite Sodium nitrate Potassium nitrate Acetic acid Potassium acetate Sodium diacetate, sodium acetate Ammonium acetate Lactic acid Propionic acid Sodium propionate Calcium propionate Potassium propionate Carbon dioxide COSMETIC PRESERVATIVES Vitamin A Vitamin C Vitamin E (tocopherol) Grapefr-uit Seed Extract Gum Benzoin Pycnogenol Butyl Paraben Methyl Paraben o-phenyl-phenol Propyl Paraben Ethyl Paraben Isopropyl Myristate Isopropyl Palmitate Octvl Palmitate WO 01/54636 PTUO/34 PCTIUS01103043 Diazolidinyl Urea Imidazolidinyl Urea trisodiumn and tetrasodiumn edetate (EDTA) PHARMACEUTICAL PRESERVATIVES Ammonia Anhydrous Potassium Persulfate Ammonium Persulfate Potassium Sorbate Benzoic Acid Potassium Sulfite Benzoyl Chloride Propyl l-vdroxv'benzoate Butyl Hydroxybenzoate Propyl Paraben Butvl Paraben Propylene Glycol Calcium Ascorbate Saccharin Calcium Fluoride Salicylic Acid Ethanol Sodium Acetate Ethyl Hydroxybenzoate Sodium Ascorbate Ethyl Paraben Sodium Benzoate Formaldehyde Sodium Bifluoride Formalin Sodium Bisulfite Hydrofluosilicic Acid Sodium Chloride Isopropanol Sodium Chromate Lactic Acid Sodium Erythorbate Maleic Acid Sodium Fluoride Maleic Anhydride Sodium Nitrate Methyl Hydroxybenzoate Sodium Silicofluoride Methyl Paraben Sodium Sulfite Pine Oil Sorbic Acid Potassium Benzoate Stannous Chloride Potassium Bifluonide Stearic Acid Potassium Metabisulfite Sulfur Dioxide Potassium Nitrate Zinc Chloride WO 01/54636 PCT/US01/03043 As used herein the term "prophylaxis" refers to preventing or reducing the risk of acquiring or transmitting sexually transmitted diseases. In addition, the term refers to inhibiting the growth or replication of HIV transmitted by sexual contact, for example.

One method of the present invention involves preventing the transmission of HIV vaginally or rectally by contacting a bodily fluid suspected of containing HIV with an aqueous composition having at least one component selected from the group consisting of polyquaternium, glycerin and a preservative system. In a typical embodiment, the bodily fluid is semen, and, in some circumstances may be vaginal fluid. In addition, the method can comprise placing an effective amount of the composition on a penis or in a vagina or rectum within one hour of commencement of sexual contact. Ideally, the composition should be placed before the sexual contact has begun. In one embodiment an effective amount of the composition is 1 ml. One convenient feature of the invention is that the composition can be placed with an applicator. As used herein the term "applicator" refers to a device such as, but not limited to a douche or syringe with an elongated neck, sufficient to comfortably reach inside the vagina or rectum and deposit an effective amount of the composition. Alternatively, the composition can be first combined with a device such as a suppository. condom, sponge or other barrier device, and then inserted into the vagina or rectum or cover the penis as appropriate. Preferable compositions used in the present methods include AstroGlide®, Vagisil® or ViAmor®.

Other methods of the present invention involve methods of prophylaxis of HIV transmission vaginally or rectally by contacting a bodily fluid suspected of containing HIV with a composition having at least one component selected from the group consisting of polyquaternium, glycerin and a preservative system.

In addition, the invention provides methods for reducing the risk of transmission of HIV vaginally or rectally by contacting a bodily fluid suspected of containing HIV with a composition having at least one component selected from the group consisting of polyquaternium, glycerin and a preservative system.

The invention provides a method for inhibiting the transmission of a sexually transmitted disease caused by an agent present in a bodily fluid, by placing a composition having at least one component selected from the group consisting of polyquatemium, glycerin and a preservative system in a vagina, rectum, condom, sponge or on some other WO 01/54636 PCT/US01/03043 barrier device that can be inserted into a vagina or rectum, prior to the deposition of the bodily fluid into the vagina or rectum. In one embodiment, the agent is HIV. in another embodiment, the bodily fluid is semen or vaginal fluid. and in another the preservative system is methylparaben and propylparaben.

As illustrated in part by Examples XXIII through XXIX below, another aspect of the invention is directed to a method for preventing transmission of HIV vaginally or rectally in a recipient who receives a donor's bodily fluid as a result of sexual contact. This method involves the step of contacting donor HIV infected cells, leukocytes for example. in a bodily fluid, for example, semen, with an effective amount of a composition that inhibits production of HIV by the donor cells. In certain types of sexual contact, the donor source of infected cells may be vaginal fluid. The composition comprises at least one component selected from the following: surfactants, microbicides, anticellular agents, acid agents, alkaline agents, oxidizing agents, inhibitors, organic solvents, and hypotonic solutions.

Preferred formulations of the composition are sufficiently viscous for retention in the vagina or rectum and are miscible with seminal fluid. Non-limiting examples of such formulations are lubricants, gels, creams, pessaries, tampons, pastes, foams or spray formulation, and, in particular, vaginal or rectal lubricants, gels or creams. It is understood that a method of placing the composition in the vagina, rectum, or mouth comprises topical application of the composition. Methods of formulating the compositions into sufficiently viscous compositions are well known in the art. It is a matter of ordinary skill for those in the art of formulation to combine the composition of the method into a lubricant, gel, or cream, which could be, without limitation, selected from a commercially branded lubricant, gel or cream. Non-limiting examples include Astroglide® brand products, Vagisil brand products, KY® brand products, Walgreen® brand products, and Replens® brand products.

It should be understood that a component of the composition may be categorized in one or more of the group consisting of surfactants, microbicides, anticellular agents, acid agents, alkaline agents, oxidizing agents, inhibitors, organic solvents, and hypotonic solutions. For example, many antineoplastic (anticancer) compounds could be classified as anticellular agents and as inhibitors. Antibiotic agents may be classified as microbicides, anticellular agents, or as inhibitors.

WO 01/54636 PCT/US01/03043 In addition to the experimental findings presented herein, one skilled in the art can select components for formulating a composition for use in the method by using the assay methods disclosed herein to distinguish without undue experimentation components and compositions thereof which kill or inactivate the donor cells from those components and compositions which do not. Using the assay methods herein, one can easily determine or measure a dose required in tissue-culture killing or inactivating of HIV infected donor cells.

Cell death or inactivation are clear end points. A dead or inactivated donor cell, for example, cannot produce virus. Therefore any components or composition thereof found effective in the assays would prevent virus multiplication in the donor cells.

The surfactant for use in the composition of the method of the invention is selected from one or more of the group consisting of detergents, wetting agents, and emulsifiers. It is understood that surfactants (surface-active agents) involve any compounds that reduces surface tension when dissolved in water or water solutions, or that reduces interfacial tension between two liquids, or between a liquid and a solid. Guides to the properties and uses of surfactants in medicine, biology, and biochemistry are available to those of skill in the art A Guide to the Properties and Uses of Detergents in Biology and Biochemistry, Calbiochem Biochemicals, San Diego, CA). Another example of guidance for using and selecting surfactants is The Bile Acids, Chemistry, Physiology, and Metabolism (1971), edited by Padmanabhan Nair and David Kritchevsky (Plenum Press.

NY). One or ordinary skill in the art using the assay methods disclosed herein can distinguish without undue experimentation surfactants which kill or inactivate the donor cells from those which do not.

Microbicides which include spermatocides are understood to mean natural or synthetic compounds that otherwise kill or inactivate cells (procaryotic or eukaryotic)and/or viruses. Microbicides which are useful in the methods of the invention are those which kill or inactivate donor cells in or from semen, thereby inhibiting production of HIV by the donor cell. One or ordinary skill in the art using the assay methods disclosed herein can distinguish without undue experimentation microbicides which kill or inactivate the donor cells from those which do not. Microbicides for use in the invention can be selected from one or more of the following: antifungal agents, antiinfective agents, AIDS chemotherapeutic agents, amebicide agents, antihelmintic agents, WO 01/54636 PCT/US01/03043 antibiotics, antimalarial agents. anti-protozoan agents. antituberculosis agents. antiviral agents, leprostatic agents, quinolones. sulfonamides, and antineoplastic (anticancer) agents (see Physicians'Desk Reference, 5 3 'd ed. (1999), Medical Economics Company)).

Microbicides further comprise commercially available disinfectants and components thereof.

The term "anticelluar agent" means a broad category of natural or synthetic substances which, for use in the method of the invention, otherwise effectively kill or inactivate cells (procaryotic or eukarvotic)and/or viruses. Anticellular agents which are useful in the methods of the invention are those which kill or inactivate donor cells in or from semen, thereby inhibiting production of HIV by the donor cell. One or ordinary skill in the art using the assay methods disclosed herein can distinguish without undue experimentation anticellular agents which kill or inactivate the donor cells from those which do not. As used herein, the term "inactivate" means inhibition of HIV production by donor infected cells. Anticellular agents for use in the method of the invention include, without limitation, antineoplastic (anticancer) agents, peptides (non-limiting examples include cytokines, chemokines. defensins. and tumor necrosis factor), apoptotic agents, enzymes (non-limiting examples include lipases, proteases, carbohydrases, DNAse, RNAse), antibodies targeted to donor cells (preferably antibodies and conjugated with a toxic agent), and spermicidal agents.

Inhibitors are a diverse group of compounds which otherwise kill or inactivate donor HIV infected cells. Groups of inhibitor compounds from which a composition is selected for use in the method of the invention include DNA synthesis inhibiting agents, RNA synthesis inhibiting agents, protein synthesis inhibiting agents, metabolic inhibitors and metabolic antagonists, which include various inhibitors of mitochondria and various respiratory chain inhibitors, and antibiotics (see Inhibitor Tools in Cell Research, (1969) edited by Th. Bucher and H. Sies, Springer-Verlag, NY)).

Acid agents for use in the method of the invention are selected from the group consisting of inorganic acid agents. such as but not restricted to hydrochloric acid. nitric acid, and sulfuric acid; and organic acids, which include but are not restricted to preservative acids benzoic acid. citric acid, sorbic acid, and acetic acid (vinegar)and acidic amino acids, which includes glycine. It has been shown (Ongradi, et al. AIDS WO 01/54636 PCT/US01/03043 Res. Hum Retroviruses (1990) 6(12):1433-1436) that virus producing cells lose their ability to infect as the pH decreases.

Alkaline agents for use in the method of the invention include inorganic alkaline agents, such as but not restricted to sodium hydroxide, potassium hydroxide. and calcium hydroxide; and organic alkaline agents.

Oxidation agents for use in the method of the invention include but are not restricted to hydrogen peroxide, salts of periodate, superoxide, nitric oxide, and glutathione disulfide.

Organic solvents for use in the method of the invention are selected from the group consisting of alcohols, ethers, esters, fatty acids and derivatives (saturated or unsaturated), diglycerides, triglycerides, lipids including phospholipids, glycolipids, sphingolipids, chromophoric lipids. Examples of organic solvents for use in the compositions of the methods of the invention are available in commercial chemical catalogs, for example, Burdick Jackson Laboratories, Inc. Small molecule organic solvents include propylene glycol and chloroform.

Hypotonic solutions for use in the method of the invention include water or solutions with low solute content, e.g. salts or sugars or any physiologically compatible solute.

One or ordinary skill in the art using the assay methods disclosed herein can distinguish without undue experimentation acid agents, alkaline agents, oxidation agents, organic solvents, and hypotonic solutions which kill or inactivate the donor cells from those which do not.

As above, the composition, an effective amount of which is contained in at least a volume of 0.5 ml, is placed in a recipient's vagina or rectum or mouth or on a donor's penis before or after sexual contact has begun, and preferably within one hour of the commencement of sexual contact. An applicator may be used to place the composition in the vagina or rectum or mouth of the recipient.

Embodiments of the method involve use of the composition in combination with a device, which could be a suppository, condom, sponge or other barrier device.

As shown in the examples below, compositions that find use in the method include, without limitation, commercially branded soaps, enemas, mouthwashes, and douches.

WO 01/54636 PCT/US01/03043 Nonlimiting examples of these branded products include Jergens® soap and Ivory soap detergent, vaginal jelly by Walgreen® or by KY®, mouthwash by Walgreen® and by Listerine®, douches by Summer's Eve® or Massengil or by Walgreen®.

The anti-HIV effects of the compositions comprising at least one component selected from the group consisting of surfactants, microbicides. anticellular agents, acid agents, alkaline agents, oxidizing agents, inhibitors, organic solvents, and hypotonic solution involve both inactivating virus extracellulary and by killing or inactivating donor cells in which the virus resides.

The following examples are intended to illustrate but not limit the present invention.

EXAMPLE I PREPARATION OF REAGENTS A. Specimens Seminal fluids are collected from normal males. Samples are stored at 4 0 C for 1 to 2 days or alternatively stored at -20 0 C for up to three months before use. University of Texas Institutional Review Board Approval Guidelines, including informed consent were followed for all samples collected.

B. Viruses Stocks of 213, ACO-1 and 9H strains of HIV were propagated in human H9 lymphocytes using the standard procedure described in Baron et al., Arch. Intern. Med.

159(3):303-310 (1999) and Williams et al., Virology 184(2):723-728 (1991), each of which is incorporated herein by reference. Aliquots were stored frozen at -70°C. The Indiana strain of vesicular stomatitis virus (VSV) was propagated in murine L cells using the method in Coppenhaver et al., New Eng. J. Med. 330(18):1314-1315 (1994). which is incorporated herein by reference. VSV stocks were stored frozen at -70 0

C.

WO 01/54636 PCT/US01/03043 C. Cells Human peripheral blood mononuclear leukocytes (PBL or PBML) and the human lymphocyte cell line, CEM. were prepared and propagated in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics, as described in Baron et al.. Arch.

Intern. Med., supra. Human peripheral blood macrophages were obtained by Ficoll- Hypaque purification of normal peripheral blood leukocytes. To obtain macrophages. the purified PBL were allowed to adhere to a glass surface for three hours at 37CC.

Unattached mononuclear leukocytes were rinsed away with medium and the adherent macrophages used in the experiments involving cell viability in the presence of saliva.

Human CEM lymphocytes and THP-1 macrophages were propagated by established procedures, reported in Baron et al., Arch. Intern. Med., supra.

EXAMPLE II VIRUS MULTIPLICATION The effect of various treatments on virus multiplication was determined in virusinfected human CEM lymphocytes, THP-1 macrophages peripheral blood mononuclear leukocytes, or L929 cells. The multiplication of HIV in PBLs was determined as the yield of infectious HIV from 2x10 6 Ficoll-Hypaque purified normal peripheral blood mononuclear cells that had been cultured with phytopemagglutinin (4 ug/ml) for 2 days before treatment with interleukin 2 (40 units/ml) for an additional 2 days and then infected with 10 5 50% tissue culture infectious doses of HIV, strain 213. The infected cells were then incubated with 20u/ml interleukin 2 for five days, washed four times, and then incubated with or without samples, the various mixtures of samples, or culture medium for or 60 minutes. These leukocytes were subsequently washed and cultured for HIV production in RPMI 1640 tissue culture medium, plus 15% fetal bovine serum and 20 u/ml of interleukin 2 for 24 hours before harvesting the cell-free medium for assay of HIV yield as described according to the assays described in EXAMPLE III. In experiments using the human CEM lymphocyte cell line, described in McKeating et al., J. Gen. Virol.

WO 01/54636 PCT/US01/03043 80(12):3327-3333 (1989). which incorporated herein by reference, multiplication was determined as the yield of infectious HIV from 6xl0 5 CEM lymphocytes that were infected with 105. 50% tissue culture infectious doses of HIV strains. The cells were then incubated for four days and washed four times before 15 to 60 minute treatments with the various preparations. These infected lymphocytes were subsequently washed four times and cultured for 24 hours before harvesting for assay of HIV production as described in EXAMPLE III. For experiments requiring non-containment facilities to perform more complex procedures, a substitute safer surrogate virus, vesicular stomatitis virus (VSV) was used in place of HIV. Other surrogate viruses have been used previously, as described in Baron et al., Arch. Intern. Med., supra. VSV multiplication are measured similarly as the yield of infectious virus in the culture medium from the CEM lymphocytes. THP-1 macrophages, or murine L cells. This is done following infection with 3.000 viral plaqueforming units. Pilot experiments were done initially using VSV-infected human CEM lymphocytes, THP-1 macrophages or murine L cells. Confirmatory experiments were done using HIV-infected CEM lymphocytes and peripheral blood mononuclear leukocytes. All studies were replicated three times.

EXAMPLE III ASSAYS OF HTV AND VSV PRODUCTION The production of HIV in the experiments was determined by the standard tissue culture infectious dose 50% assay using MT-2 human lymphocytes, as described in McKeating et al., supra. Specifically, the culture fluids harvested from the HIV-infected human cells were serially diluted in 0.5 log,, increments using RPMI culture medium containing 10% fetal bovine serum. 50ul of each dilution was added to quadruplicate microtiter wells, each containing 110 ul of 2xl0 MT-2 cells in culture medium. After the serial dilutions, 120 ul of nutrient medium was added to each well. These microtiter plates were then incubated at 37°C for three days in a CO, incubator, and then re-fed culture medium containing 10% fetal bovine serum. The wells were read for HIV multinucleated WO 01/54636 PCT/US01/03043 giant cell cytopathic effect on day 5 or 6. The 50% tissue culture infections dose was determined using the Reed-Muench method, as described in Reed et al., Am. J. Hyg.

27:493-497 (1938), which is incorporated herein by reference. The production of VSV was determined as plaque-forming units in L cells.

In addition to basic descriptive statistics, e.g. the Student T Test, statistical methods such as regression analysis and nonparametric methods were used. with no adjustments for multiple testings. In the past, in every instance the pertinent trends and differences observed were large and all related significance levels (P values) were small, even with sample sizes used.

EXAMPLE IV VSV INHIBITION WITH VAGINAL PREPARATIONS IN SEMINAL FLUID This example provides evidence that over the counter vaginal preparations can inhibit multiplication of vesicular stomatitis virus (VSV) in seminal fluid. As show in Figure 1, commercially available vaginal preparations, including AstroGlide® and Vagisil®, as well as Nonoxynol-9® were tested for their effectiveness in inhibiting the multiplication of VSV. For example, VSV infected CEM lymphocytes were exposed to AstroGlide® and the log 0 c inhibition of multiplication recorded. VSV was chosen in this experiment since it has similar structural properties to HIV, but is considerably more safe and practical to handle in a laboratory. Greater than 10,000 fold inhibition was observed, both when AstroGlide® was mixed with the cells and when it was merely layered on. Nonoxynol-9® showed an almost identical degree of inhibition. Only Vagisil® showed a slightly less inhibition when it was mixed with the cells. Although Nonoxynol-9® exhibits strong inhibitory properties, its use in vivo is impractical due to its propensity to cause mucosal inflammation, which may be the cause of its ineffectiveness in human clinical trials.

These results demonstrate that the compositions of the methods of the invention, including without limitation AstroGlide@ and Vagisil®, can penetrate seminal fluid WO 01/54636 PCT/US01/03043 sufficiently to reach cells infected with the virus and can kill those cells preventing multiplication and production of the virus.

EXAMPLE V HIV INHIBITION WITH VAGINAL PREPARATIONS IN SEMINAL FLUID Following the observation that the vaginal preparations above strongly inhibit VSV multiplication in seminal fluid, an experiment under similar conditions using HIV was performed.

As shown in Figure 2, AstroGlide® and Vagisil® strongly inhibit the multiplication of HIV infected CEM lymphocytes in seminal fluid. For example, when the preparations were mixed with the seminal fluid, each of the preparations showed greater than a 1,000 fold inhibition. As expected, when the preparations were layered on, they produced greater than 30 fold inhibition. Interestingly, Nonoxynol-9® resulted in greater than 1,000 fold inhibition both when mixed and layered on.

These results confirm that the compositions of the methods of the invention, including without limitation AstroGlide® and Vagisil® are extremely effective at inhibiting the multiplication of HIV infected CEM lymphocytes in seminal fluid.

EXAMPLE VI TIME STUDIES OF VAGINAL PREPARATIONS FOR HIV INHIBITION This example identifies the time required for the compositions of the methods of the invention, and in particular, vaginal preparations to inhibit production of HIV by infected CEM lymphocytes.

As seen in Figure 3, the vaginal preparations began to act within 5 minutes and within 20 minutes of incubation they produced a maximal level of inhibition of greater than WO 01/54636 PCT/US01/03043 100 fold. This level of inhibition remained steady for at least 60 minutes. As described previously herein, it has been demonstrated that HIV infected lymphocytes and HIV in cell-free form can remain in seminal fluid inside a vagina or rectum for up to one hour before the virus enters the recipient's epithelial tissue, assuming no trauma to the surrounding epithelial tissue has occurred. Accordingly, these results show that timely placing of the compositions of the invention in the vagina or rectum after sexual contact would destroy HIV before it could be transmitted in this manner.

EXAMPLE VII INHIBITION OF CELL FREE HIV BY VAGINAL PREPARATIONS This example demonstrates the ability of the compositions of the methods of the invention to inhibit cell-free HIV. It is generally reported by the scientific literature that in seminal fluid, most HIV is found sequestered in cells, with a ratio of 10 HIV sequestered to 1 free HIV. Accordingly, the ability of the vaginal preparations to inhibit cell-free HIV was studied as well.

As shown in Figure 4, the vaginal preparations AstroGlide® and Vagisil® produced a significant inhibition of cell-free HIV. For example, Vagisil® produced an inhibition of approximately 45 fold, while ViAmor® produced approximately 15 fold inhibition and Astroglide® produced approximately 45 fold inhibition of HIV.

Accordingly, these results demonstrate that the compositions of the methods of the invention, including without limitation the over the counter vaginal preparations Astroglide® (ViAmor®),and Vagisil® are highly effective at inhibiting cell-free HIV as well.

WO 01/54636 PCT/US01/03043 EXAMPLE VIII INHIBITION OF CELL FREE HIV BY VAGINAL PREPARATIONS IN SEMINAL FLUID This example was performed to determine whether the compositions of the method of the invention would also inhibit cell-free HIV in seminal fluid.

As shown in Figure 5, each of the vaginal preparations was effective at inhibiting cell-free HIV in seminal fluid. For example. an inhibition of approximately 6 fold was observed with each of Vagisil® and AstroGlide®. Accordingly, these results demonstrate that the vaginal preparations were able to penetrate the somewhat protective seminal fluid, and strongly inhibited cell-free HIV.

EXAMPLE IX TOXICITY AND INHIBITORY TITERS OF VAGINAL PREPARATION

COMPONENTS

This example explored the toxicity and inhibitory titers of the various components of the vaginal preparations.

Solutions of the components shown in Figure 6 were diluted, and the inhibitory and toxicity characteristics of the diluted solutions were evaluated. At an 8% concentration, glycerol was not very effective at killing L-929 cells but produced greater than a 10 fold inhibition of VSV. Polyquaternium 32 at 0.2% concentration produced a greater than fold killing of L-929 cells and a greater than 100 fold inhibition in VSV. Propylene G (propylene glycol) produced almost no inhibition or killing at all. Methylparaben at only a 0.2% concentration produced a greater than 300 fold titer of L-929 cells but produced a VSV inhibition of approximately 30 fold. Proplyparaben at 0.004% produced a L-929 toxicity titer of approximately 15 fold and a VSV inhibition of approximately 55 fold.

WO 01/54636 PCT/US01/03043 Accordingly, glycerol. polyquaternium. methylparaben and proplyparaben each individually were effective at inhibiting VSV, and most likely HIV as well. Further studies will determine if similar effects are seen with HIV and whether the inhibition is cumulative or synergistic.

This example demonstrated that the protection afforded by AstroGlide® was not due to hypotonicity, but was rather due to the inactivation of infected leukocytes by one or more of the compounds comprising Astroglide®. Accordingly. the hypotonic factor of AstroGlide® was not responsible for the inhibition of HIV-infected leukocytes. Therefore.

AstroGlide® had protective chemical compound(s)independent of hypotonicity. and that it was not the lack of salts but one or more chemical compounds which accounted for the inactivation of HIV by the compositions useful in the methods of the invention. Further studies will be performed using the compositions on HIV-infected leukocytes. In further studies, salts are added to create an isotonic condition to further demonstrate that AstroGlide® and the compositions of the method of the invention retain highly protective properties even when present or dissolved in an isotonic environment.

EXAMPLE X INHIBITORY ACTION IN MULTIPLE STRAINS OF HIV This example demonstrates that compounds of the methods of the invention strongly inhibit multiple strains of HIV. Three additional HIV isolate strains, 213, AC-1 and 9H, are used to infect human CEM lymphocytes suspended in seminal fluid. Stocks of the 213, Ac-l and 9H strains of HIV are propagated in human H9 lymphocytes using the standard procedure described in Baron et al., Arch. Intern. Med., supra and Williams et al., Virology 184(2):723-728 (1991). both of which are incorporated by reference herein. Two groups of infected cells are prepared, and one group is treated with, for example, AstroGlide® and the other is not. It is determined that AstroGlide® strongly inhibits the multiplication of the three HIV strains. Cells that are not treated remain viable. The results confirm the general applicability of the protection AstroGlide® among the compounds of WO 01/54636 PCT/US01/03043 the methods of the invention provides against multiple strains of HIV, which has utility for HIV infection in humans.

EXAMPLE XI STUDIES WITH HIV-INFECTED MONOCYTES Since HIV infects monocytes as well as lymphocytes in human seminal fluid, the following experiment are performed to determine whether the compounds of the methods of the invention, including AstroGlide®. can protect against HIV-infected monocytes as well as lymphocytes. THP-1 monocytes are suspended in seminal fluid. Two groups of cells are prepared. One group is treated with AstroGlide® and the other group is not. The results show that AstroGlide® strongly inhibits HIV-infected monocytes. Therefore. the findings demonstrate that AstroGlide® can protect against all types of HIV-infected leukocytes in seminal fluid.

EXAMPLE XII STUDIES WITH PRIMARY CELLS This example is performed to demonstrate whether the compounds of the methods of the invention, including AstroGlide®, could provide protection against HIV-infected primary peripheral blood leukocytes. Human seminal fluid normally contains primary peripheral blood leukocytes rather than the cell lines used in previous studies. Normal human peripheral blood mononuclear leukocytes are purified from human blood, according to established protocols, which are known to those skilled in the art. Next, they are WO 01/54636 PCT/US01/03043 suspended in seminal fluid. infected with HIV and then treated or not with AstroGlide®.

The results demonstrate that AstroGlide@ is highly effective at inhibiting HIV-infected primary peripheral blood leukocytes. Therefore. these findings are applicable to human therapy.

EXAMPLE XIII INACTIVATION OF CELL-FREE HIV STRAINS This example is performed using the assay methods disclosed herein to determine whether compounds of the method of the invention, including AstroGlidel. inactivate four strains of cell-free HIV. Four strains of cell-free HIV suspended in seminal fluid are treated or not treated with AstroGlide®. The results demonstrate that AstroGlide® inactivates at a level greater than 90% all strains of cell-free HIV in the absence of seminal fluid and at an level in the presence of seminal fluid. Accordingly, AstroGlide® is protective against cell-free as well as HIV-infected cells.

EXAMPLE XIV PROTECTION AGAINST SEXUALLY TRANSMITTED AGENTS This example is performed to determine whether compounds of the methods of the invention, including AstroGlide®, provide protection against sexually transmitted agents, other than HIV, as well. For example, the agent of herpes is herpes simplex virus (HSV).

In order to test the protection against HSV. Astroglide® is added to free HSV. After incubation for one hour at 37*C, the surviving infectious HSV is assayed on Vero cells.

The results demonstrate that AstroGlide® strongly inhibits HSV as well. Accordingly. they provide protection against herpes transmission.

WO 01/54636 PCT/US01/03043 The compositions of the method of the invention, including AstroGlide.. are also tested for their ability to inhibit the Papilloma virus through study of its virus family.

Papoviruses. Protection against the genital strain of Papovirus is studied in infected cells and cell-free virus suspended in seminal fluid. AstroGlide® is added to the culture. The cells are then assayed according to the procedure described previously herein. The results demonstrate that AstroGlide® strongly inhibits the genital strain of papovirus.

Accordingly, they provide protection against genital wart transmission.

AstroGlide® and the other compositions are additionally tested for their ability to inhibit other sexually transmitted microbes, such as gonorrhea, syphilis, chlamydia and mycoses. To determine the protective effect against these microbes. each organism is cultured in nutrient medium according to procedures that are well known to those skilled in the art. The cultures are either treated or not treated with AstroGlide® at systematically varying concentrations, all of which can be determined by those skilled in the art. and which are further described in Baron et al., Medical Microbiology. 4 th ed., Galveston TX, The University of Texas Medical Branch at Galveston, (1996), which is incorporated herein by reference. The results demonstrate that AstroGlide® is highly effective at destroying these organisms, as indicated by the antimicrobial titer. In addition, they provide protective action.

EXAMPLE XV PERSISTENCE STUDIES This example demonstrates the duration of anti-HIV activity of the compositions of the method of the invention, including AstroGlide®. in the vagina and rectum.

Five rabbit vaginas are treated with AstroGlide® and sampled at four hours.

Additionally, five rabbit rectums are treated with AstroGlide® and sampled at four hours.

These rabbits will have been placed on a water-only diet for 12 hours prior to testing.

The recovered samples are tested for anti-HIV activity in comparison with the original WO 01/54636 PCT/US01/03043 preparations. The results demonstrate that AstroGlide® retains high anti-HIV activity even after four hours. Accordingly. AstroGlide® is useful in practice.

EXAMPLE XVI HUMAN STUDIES This example demonstrates the ability of AstroGlide® and the compositions of the methods of the invention to provide protection against HIV in humans. It further explores the duration of that protection.

The following is performed according to standard human subject protocol and is subject to Institutional Review Board approval. Women are provided with stocks of AstroGlide®, which is subsequently placed in the vagina. After four hours. aliquots are removed and tested for anti-HIV activity. In addition, either fasting men or women have AstroGlide® placed in the rectum. Samples are also removed at four hours and tested in a similar fashion. The results demonstrate that AstroGlide® retains high anti-HIV activity after four hours in the body. Accordingly, AstroGlide® is useful for human application.

EXAMPLE XVII PREPARATION OF VIRUSES AND CELLS a. Viruses Stocks of the 213 strain of HIV were propagated in human H9 lymphocytes using the standard procedure described in Baron et al., Arch. Intern. Med. 159(3):303-310 (1999) and Williams et al., Virology 184(2):723-728 (1991), each of which is incorporated herein by reference. Aliquots were stored frozen at -70 0 C. The Indiana strain of vesicular stomatitis virus (VSV) was propagated in murine L cells using the method in Coppenhaver et al., New Eng. J. Med. 330(18):1314-1315 (1994), which is incorporated herein by reference. VSV stocks were stored frozen at -70 0

C.

WO 01/54636 PCT/US01/03043 b. Cells The human lymphocyte cell line, CEM. were prepared and propagated in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics, as described in Baron et al., Arch. Intern. Med., supra. Human CEM lymphocytes and THP-1 macrophages were propagated by established procedures, reported in Baron et al., Arch. Intern. Med., supra and Baron et al., J. Infect. Dis. 181:498-504 (2000).

EXAMPLE XVIII VIRUS MULTIPLICATION The effect of various treatments on virus multiplication was determined in virusinfected human CEM lymphocytes, or L929 cells. In experiments using the human CEM lymphocyte cell line, the procedure of McKeating et al., J. Gen. Virol. 80(12):3327-3333 (1989), which incorporated herein by reference, was followed. Multiplication was determined as the yield of infectious VSV from 2x 10' CEM lymphocytes that were infected with 10', 50% tissue culture infectious doses of VSV and suspended or not in seminal fluid.

The cells were then incubated for 60 minutes and washed four times before 30 to 60 minute treatments with the various preparations. These infected lymphocytes were subsequently washed four times and cultured for 24 hours before harvesting for assay of VSV production as described in EXAMPLE III.

In experiments that used the human CEM lymphocyte cell line, described in McKeating et al., J. Gen. Virol. 70:3327-33 (1989), multiplication was determined as the yield of infectious HIV from 6x 10 CEM lymphocytes that were infected with 10 TCIDso of HIV strain 213. The cells were then incubated for 4 days and washed 4 times before a to 60-minute treatment with the various preparations. These infected lymphocytes were subsequently washed 4 times and cultured for 24 hours before being harvested for assay of HIV production as described below.

WO 01/54636 PCT/US01/03043 EXAMPLE XIX ASSAYS OF HIV AND VSV PRODUCTION The production of HIV in the experiments was determined by the standard TCID,, assay with MT-2 human lymphocytes, described in McKeating et al.. J. Gen. Virol.

70:3327-3333 (1989). Specifically, culture fluids harvested from the HIV-infected human CEM lymphocytes, as described in Baron et al., Arch. Intern. Med. 159(3):303-310 (1999).

were serially diluted in 0.5-logio increments in RPMI culture medium containing 10% fetal bovine serum. Next, 50 ml of each dilution was added to quadruplicate microtiter wells.

each containing 110 ml of 2 x 10' MT-2 cells in culture medium. After the serial dilutions.

120 ml of nutrient medium was added to each well. These microtiter plates were incubated at 370 C for 3 days in a CO, incubator and then were again fed culture medium containing fetal bovine serum. The wells were read for HIV multinucleated giant cell cytopathic effect on day 5 or 6. The TCIDso of HIV was determined by the method of Reed and Muench, as described in Reed et al. Am. J. Hyg. 27:493-7 (1938), and the production of VSV was determined as pfu in L cells, as described in Baron et al., Arch. Intern. Med.

159(3):303-310 (1999).

TITRATION OF THE VSV-INHIBITORY ACTIVITY Serial two-fold dilutions of each substance were made in murine L cells grown in 96-well microtiter plates and which were infected 2 hours previously with 10' plaqueforming units of VSV. The substances were allowed to incubate with the infected cells for 1 hour before washing three times and refeeding with nutrient medium. The cells were then incubated for 24 hours to allow virus multiplication. The culture fluids were harvested individually and titered for virus plaque yield as described. The yield of virus was calculated as pfu per 50 ml.

WO 01/54636 PCT/US01/03043 CELL TOXICITY ASSAY In the studies determining the anticellular activity of substances on L cells, the cytopathic effect of each dilution of each substance was determined microscopically. The 50% tissue culture cytopathic dose was calculated by the Reed Muench method. as described in Reed et al. Am. J. Hyv. 27:493-7 (1938).

STATISTICS

In addition to basic descriptive statistics, the Student T Test. statistical methods such as regression analysis and nonparametric methods were used. with no adjustments for multiple testing. In the past, in every instance the pertinent trends and differences observed were large and all related significance levels (P values) were small, even with sample sizes used.

EXAMPLE XX VSV INHIBITION WITH OTC PREPARATIONS OR COMPONENTS

THEREOF

This example provides evidence that over-the-counter vaginal preparations and/or their components can inhibit multiplication of vesicular stomatitis virus (VSV) in seminal fluid. As shown in the figures, commercially available vaginal preparations, or components thereof, were tested for their effectiveness and for inhibiting the multiplication of VSV.

For example, VSV-infected CEM lymphocytes were exposed to the preparations and/or components thereof and the logo inhibition of multiplication recorded. VSV was chosen for this experiment since it has similar structural properties to HIV, but is considerably more safe and practical to handle in a laboratory.

WO 01/54636 PCT/US01/03043 EXAMPLE XXI DECONTAMINATION OF SYRINGES Syringes of the sizes commonly used for drug injection (3 ml with 1.5 inch 22 gauge needles) were contaminated by drawing into the syringe 0.1 of normal blood to which was added either cell-free VSV (10' infectious units/0.1 ml), or for comparison, infected CEM cells (105 cells/0.1 ml). To estimate the volume of a composition used in the method required for inactivation of either VSV or HIV, the composition was brought into contact with either cell-free HIV or VSV infected cells. The syringes were rinsed with a total volume of 3 ml of a solution of the composition. Controls were rinsed with nutrient medium. Then residual virus or cells were collected from the syringes in 1 ml of culture medium, which was assayed for infectious virus as described.

EXAMPLE XXII INHIBITION OF CELL-FREE HIV To determine whether cell-free HIV is inhibited by the preparations, we layered 200 ml of 1.6 x 104 TCIDso cell-free HIV in 200 pI of seminal fluid or culture medium and incubated for 1 hour before titering for residual cell-free HIV by the methods presented above.

EXPERIMENTAL FINDINGS Figure 7 shows the interruption of VSV multiplication in L-cells by surfactants (bile salts) and by components of douches. For example, it was demonstrated that benzoic acid (0.3%)in dilutions up to three-fold had a 5 log inhibitory titer, and from three-fold to about 15-fold dilutions, showed a 0.5 log inhibitory titer. Beyond fifteen fold dilution, an inhibitory influence on VSV multiplication in L-cells was not demonstrated.

WO 01/54636 PCT/US01/03043 Figure 8 shows interruption of VSV multiplication in L-cells by surfactant bile salts, soap solutions, vaginal jellies. an organic solvent, an acid. and the surfactants TritonX100 combined with tributyl phosphate.

Figures 9, 10, 12. 13, 14, 15, and 17 demonstrate inactivation by detergents of VSV infected CEM lymphocytes suspended in seminal fluid by bile salts and Triton x100® detergent.

Figure 16 shows interruption of VSV multiplication in L-cells by enemas and mouthwashes, and by douches in Figure 11.

Figure 19 illustrates that detergents and mouthwashes interrupted VSV multiplication in L-cells.

Figure 20 demonstrates that over the counter vaginal preparations (douches) or oral preparations (mouthwashes) inhibited cell-free HIV.

Figure 21 shows inactivation by bile salts and saliva of HIV-infected CEM lymphocytes, while Figure 19 shows their inhibitory effects on cell-free HIV. Figure shows inactivation by bile acids of cell free HIV.

Figure 23 shows inactivation of cell-free HIV by ursodeoxycholate but not by saliva.

Figures 11, 22, and 24 show the decontamination either the inhibition of VSV production or the inhibition of cell free HIV) of syringes containing either cell-free HIV or VSV infected cells using ursodeoxycholate. colic acid, deoxycholate, Triton X100. tributyl phosphate, combination of Triton X100 and tributyl phosphate. and tap water. This finding indicates that the method of the invention prevents or inhibits HIV transmission from a needle and syringe contaminated with either free HIV or VSV-infected cells, and that the method is effective for disinfecting the needle and syringe between users by using a composition that carries less risk to the user than bleach solutions, which are in common use for disinfecting or decontaminating needles and syringes but which pose a significant health hazard if inadvertently injected into a user.

Further examples involve studies of the claimed methods. which use a composition comprising at least at least one component selected from the group consisting of surfactants, microbicides, anticellular agents, acid agents, alkaline agents. oxidizing agents, inhibitors, organic solvents. and hypotonic solutions. Similar to Examples X through XVI WO 01/54636 PCT/US01/03043 above, these studies include Examples XXIII through XXIX, and are focused upon methods of the invention which use compositions or candidate compositions comprising at least one component selected from the group consisting of surfactants. microbicides, anticellular agents, acid agents, alkaline agents. oxidizing agents. inhibitors. organic solvents, and hypotonic solutions: EXAMPLE XXI INHIBITORY ACTION IN MULTIPLE STRAINS OF HIV This example demonstrates that compounds of the methods of the invention strongly inhibit multiple strains of HIV. Three additional HIV isolate strains. 213, AC-1 and III-B, are used to infect human CEM lymphocytes suspended in seminal fluid. Stocks of the 213, Ac-I and 9H strains of HIV are propagated in human H9 lymphocytes using the standard procedure described in Baron et al., Arch. Intern. Med.. supra and Williams et al., Virology 184(2):723-728 (1991), both of which are incorporated by reference herein.

Two groups of infected cells are prepared, and one group is treated with a composition comprising at least at least one component selected from the group consisting of surfactants, microbicides, anticellular agents, acid agents. alkaline agents. oxidizing agents, inhibitors, organic solvents, and hypotonic solutions. The other group acts as a control. It is determined that the composition strongly inhibits the multiplication of the cell-associated donor cell) three HIV strains. Cells that are not treated remain viable. The results confirm the general applicability of the protection provided by a composition comprising at least at least one component selected from the group consisting of surfactants, microbicides, anticellular agents. acid agents, alkaline agents, oxidizing agents. inhibitors, organic solvents, and hypotonic solutions used in the methods of the invention provides against multiple strains of HIV, which has utility for preventing, inhibiting, or reducing the risk of HIV infection caused by sexual contact between an infected donor and a recipient.

WO 01/54636 PCT/US01/03043 EXAMPLE XXIV STUDIES WITH HIV-INFECTED MONOCYTES Since HIV infects monocytes as well as lymphocytes in human seminal fluid, the following experiment are performed to determine whether the compositions used in the methods of the invention, can protect against HIV-infected monocytes as well as lymphocytes. THP-1 monocytes are suspended in seminal fluid. Two groups of cells are prepared. One group is treated with a candidate composition and the other group is not.

The results show that the candidate composition comprising at least one component selected from the group consisting of surfactants, microbicides. anticellular agents, acid agents, alkaline agents, oxidizing agents, inhibitors, organic solvents, and hypotonic solutions strongly inhibits HIV-infected monocytes. Therefore. the findings demonstrate that the candidate composition can protect against all types of HIV-infected leukocytes in seminal fluid.

EXAMPLE XXV STUDIES WITH PRIMARY CELLS This example is performed to demonstrate whether the compounds of the methods of the invention could provide protection against HIV-infected primary peripheral blood leukocytes. Human seminal fluid normally contains primary peripheral blood leukocytes rather than the cell lines used in previous studies. Normal human peripheral blood mononuclear leukocytes are purified from human blood, according to established protocols, which are known to those skilled in the art. Next, they are suspended in seminal fluid, infected with HIV and then treated or not with a candidate composition used in the method of the invention. The results demonstrate that the candidate composition is highly effective WO 01/54636 PCT/US01/03043 at inhibiting HIV-infected primary peripheral blood leukocytes. Therefore. these findings are applicable to human therapy.

EXAMPLE XXVI INACTIVATION OF CELL-FREE HIV STRAINS This example is performed to determine whether candidate compounds of the method of the invention inactivate four strains of cell-free HIV. Four strains of cell-free HIV suspended in seminal fluid are treated or not treated with a candidate composition.

The results demonstrate that the candidate composition inactivates at a level greater than all strains of cell-free HIV and at an 80% level in seminal fluid. Accordingly. the candidate composition is protective against cell-free as well as HIV-infected cells.

EXAMPLE XXVII PROTECTION AGAINST SEXUALLY TRANSMITTED AGENTS This example is performed to determine whether candidate compounds of the methods of the invention provide protection against sexually transmitted agents, other than HIV, as well. For example, the agent of herpes is herpes simplex virus (HSV). In order to test the protection against HSV, Vero cells are suspended in seminal fluid. A candidate composition is added to the cells. The cells are assayed according to procedures described previously herein. The results demonstrate that the candidate composition strongly inhibits HSV as well. Accordingly, they provide protection against herpes transmission. The candidate compositions of the method of the invention are also tested for their ability to inhibit the Papilloma virus through study of its virus family, Papoviruses. Protection against the genital strain of Papovirus is studied in infected cells and cell-free virus suspended in seminal fluid. A candidate composition is added to the culture. The cells are WO 01/54636 PCT/US01/03043 then assayed according to the procedure described previously herein. The results demonstrate that a candidate composition strongly inhibits the genital strain of papovirus.

Accordingly, the candidate composition provides protection against genital wart transmission.

Candidate compositions are additionally tested for their ability to inhibit other sexually transmitted microbes, such as gonorrhea, syphilis, chlamydia and mycoses. To determine the protective effect against these microbes, each organism is cultured in nutrient medium according to procedures that are well known to those skilled in the art. The cultures are either treated or not treated with a candidate composition at systematically varying concentrations, all of which can be determined by those skilled in the art, and which are further described in Baron et al., Medical Microbiology, 4 t ed., Galveston TX, The University of Texas Medical Branch at Galveston, (1996), which is incorporated herein by reference. The results demonstrate that a candidate composition is highly effective at destroying these organisms, as indicated by the antimicrobial titer. In addition, they provide protective action.

EXAMPLE XXVIII PERSISTENCE STUDIES This example demonstrates the duration of anti-HIV activity of the compositions and candidate compositions of the method of the invention in the vagina and rectum.

Five rabbit vaginas are treated with a composition and sampled at four hours.

Additionally, five rabbit rectums are treated with a composition and sampled at four hours.

These rabbits will have been placed on a water-only diet for 12 hours prior to testing. The recovered samples are tested for anti-HIV activity in comparison with the original preparations. The results demonstrate that the composition retains high anti-HIV activity even after four hours. Accordingly, the composition is useful in practice.

WO 01/54636 PCT/US01/03043 EXAMPLE XXIX HUMAN STUDIES This example demonstrates the ability of the ability of the compositions of the methods of the invention to provide protection against HIV in humans. It further explores the duration of that protection.

The following is performed according to standard human subject protocol and is subject to Institutional Review Board approval. Women are provided with stocks of a composition, which is subsequently placed in the vagina. After four hours, aliquots are removed and tested for anti-HIV activity. In addition, either fasting men or women have a composition placed in the rectum. Samples are also removed at four hours and tested in a similar fashion. The results demonstrate that the composition retains high anti-HIV activity after four hours in the body. Accordingly, the compositions used in the claimed methods are useful for human application. Among the compositions used in the method are those which inactivate donor infected cells but which do not cause inflammation of the recipient's mucosa. Components and compositions thereof which are already FDA-approved for safety FDA category I safety) find use in the methods of the invention.

It will be appreciated that the methods of the invention lend themselves to femalecontrolled prevention methods, i.e. methods intended primarily, but not exclusively, for vaginal use, and includes compositions which can be used without a partner's knowledge.

Further, the methods of the invention are not limited to an exclusive focus on vaginal application. The methods and compositions disclosed herein may be used primarily for vaginal application, however, it should be recognized that they will also be applied to the penis, and used orally and rectally by both heterosexual and homosexual couples.

Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

-38A- Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

*o *•o •o* *oo o0

Claims (22)

1. A method for the prophylaxis, prevention or reducing the risk of HIV transmission vaginally or rectally comprising, contacting a bodily fluid suspected of containing HIV with an aqueous composition having at least one active microbicide component selected from the group consisting of polyquaternium and glycerin.

2. The method of claim 1 wherein the bodily fluid is semen.

3. The method of claim 1 further comprising placing an effective amount of the composition before or after sexual contact has begun in the vagina and/or rectum.

4. The method of claim 3 wherein the composition is placed in the vagina or rectum using an applicator. S:

5. The method of claim 4, wherein an effective amount is at least 1 ml.

6. The method of claim 5, wherein the composition is placed within one hour of the commencement of sexual contact.

7. The method of claim 1 wherein the composition is combined with a device selected from the group consisting of a suppository, condom, sponge or other barrier device.

8. The method of claim 1 wherein the composition is selected from the group consisting of vaginal lubricants and personal lubricants.

9. The method of claims 1-8 wherein the composition further comprises a preservative system. The method of claim 9 wherein the preservative system is methylparaben and propylparaben.

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11. Use of at least one active microbicide component selected from the group consisting of polyquaternium and glycerin for the manufacture of an aqueous composition for the prophylaxis, prevention or reducing the risk of HIV transmission vaginally or rectally.

12. The use of claim 11, wherein the bodily fluid is semen.

13. The use of claim 12 further comprising placing an effective amount of the composition before or after sexual contact has begun on a penis or in the vagina and/or rectum.

14. The use of claim 11 wherein the composition is placed in the vagina or rectum using an applicator.

15. The use of claim 11, wherein an effective amount is at least 1 ml.

16. The use of claim 11 wherein the composition is placed within one hour of the commencement of sexual contact.

17. The use of claim 11 wherein the composition is combined with a device selected from the group consisting of a suppository, condom, sponge or other barrier device.

18. The use of claim 1, wherein the composition is selected from the group consisting of vaginal lubricants and personal lubricants.

19. The use of claims 11-18 wherein the composition further comprises a preservative system. The use of claim 19 wherein the preservative system is methylparaben and propylparaben.

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21. A method according to any one of claims 1-10 substantially as hereinibefore described with reference to the examples and/or figures.

22. A use according to any one of claims 11-20 substantially as hereinbefore described with reference to the examples and/or figures. DATED this 17 1h day of August, 2005 Board of Regents, The University of Texas System by its Patent Attorneys DAVIES COLLISON CAVE