BACKGROUND OF THE INVENTION Sexually transmitted diseases (STDs), referring to diseases that are most often transmitted by direct sexual contact, remain an increasingly serious public and veterinary heath problems in the United States, as well as other countries. Indeed, these diseases represent a major health problem of pandemic proportions (See e. g., Herold et al., Antimicrob. Agent. Chemother., 41: 2776-278 [1997]). The World Health Organization has estimated that 125 million new cases of major bacterial and viral STDs occur each year (See, Herold, supra, at p. 2776). In terms of human cost, the World Bank has estimated that for adults of 15 to 44 years of age, STDs other than human immunodeficiency virus (HIV) infection are the second leading cause of healthy life lost in women (See, Herold, supra, at p.

2776). Women are especially at risk, as many STDs are asymptomatic and there is a high morbidity rate associated with untreated disease (See e. g., Biro et al., Clin. Pediatr., 33: 601- 605 [1994]). Of these diseases, the primary etiologic agents are HIV, herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Chlamydia trachomatis, Chlamydia trachomatis serovars LI, L2, and L3, Calmatobacterium granulomatis, Treponema pallidum, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Haemophilus ducreyi, Gardnerella vaginalis, Trichomona vaginalis, and human papillomavirus (HPV).

In the U. S., STDs have been characterized as"hidden epidemics of enormous health and economic consequence" (See, Division of STD Prevention, Sexually Transmitted Disease Surveillance, 1996, U. S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, September, 1997; hereinafter"CDC Surveillance Report"). The following statistics may be found in this CDC Surveillance Report. For C. trachomatis, the reported number of cases in the U. S. for 1996 was 490,000 (i. e., a rate of 194.5 per 100,000 persons; this rate was based on reports from 49 states, and the District of Columbia, although only cases from New York City are included in the figures for New

York). This rate exceeds that of all other notifiable infectious diseases in the U. S. This represents an increase from 47.8 per 100,000 to 194.5, for the time period of 1987 to 1996.

For the period of 1995 to 1996, the rates increased 2% (i. e., from 313.2 cases per 100,000 to 318.6). For women, the rate (321.5 per 100,000) was over five times that reported in men.

This differential in reporting rates may be attributable to screening strategies that focus on women, as the severe sequelae primarily occur in women and the asymptomatic nature of most cases. For example, C. trachomatis infections often result pelvic inflammatory disease (PID), which can lead to infertility, ectopic pregnancy, and chronic pelvic pain. In terms of the female population infected, the highest rates of chlamydial infection occur in adolescents.

The rates are highest in the 15 to 19 year old population with 2,068.6 cases per 100,000, followed by the 20 to 24 year old age group, with approximately 1,485 cases. In addition, the prevalence of C. trachomatis infection is highest in economically disadvantaged young women.

Although the number of gonorrhea cases has steadily decreased since the establishment of gonorrhea control programs in the mid-1970s, the problem is not solved. While the 1996 rate of 124 per 100,000 is the lowest rate since national reporting began, it is greater than the objective of 100 established by the"Healthy People 2000"program (325,883 cases were reported). Gonorrhea remains a significant cause of morbidity. As with C. trachomatis, infection with N. gonorrhoeae remains a major case of PID, tubal infertility, ectopic pregnancy, and chronic pelvic pain. Furthermore, epidemiologic evidence strongly suggests that gonococcal infections facilitate HIV transmission. As with chlamydial infections, the rates of gonorrhea in women are particularly high in adolescents, with the highest rates observed in 15 to 19 year olds. For men, the highest rate was observed in the 20 to 24 year old age group. In addition, the percentage of men with repeated infection within a one year period has increased from a low of 13.8% in 1994, to 5.7% in 1996. Of additional concern is the growing number of N. gonorrhoeae isolates with decreased susceptibility to penicillin and/or tetracycline, and ciprofloxacin.

Fortunately, the 11,387 cases of primary and secondary syphilis reported in 1996 represents the lowest number of cases reported since 1959. However, only specific areas of the country continue to report syphilis cases. Thus, it is possible that the disease is under- reported. As with N. gonorrhoeae, syphilis facilitates transmission of HIV and may be particularly significant in areas of the country where both infection rates are high. Infection with Treponema pallidum, the etiologic agent of syphilis is of particular concern during

pregnancy, as untreated early syphilis results in perinatal death in up to 40% of cases. If the syphilitic infection is acquired during the four years prior to pregnancy, the fetus is infected in over 70% of cases.

In addition to chlamydial, gonococcal, and syphilitic diseases, other STD agents are of importance. For example, herpes simplex (HSV) causes a significant amount of morbidity and in some cases, mortality. While HSV type 2 (HSV-2) has been traditionally more common as an STD, HSV type 1 (HSV-1) may also be sexually transmitted. The prevalence of HSV-1 gradually increases from childhood, reaching 70-80% in later adult years, while the incidence of HSV-2 begins to increase at adolescence, with infection rates of 15 to more than 50% in some adult populations (See e. g., Arvin and Prober,"Herpes Simplex Viruses,"in Murray et al. (eds.), Manual of Clinical Microbiology, (6th ed.), ASM Press, Washington, D. C., pages 876-883 [1995]; Rosenthal et al., Clin. Infect. Dis., 24: 135-139 [1997]; and Stanberry, Understanding Herpes, University Press of Mississippi, Jackson, MS [1998]).

Many cases of primary HSV-1 and HSV-2 infection are subclinical. Indeed, primary infections are often entirely asymptomatic. In addition, despite the apparently universal establishment of latency following infection with either virus, many individuals with past HSV infections do not experience symptomatic recurrences. However, asymptomatic recurrences do occur, making prevention of the transmission of HSV-1 and HSV-2 in the population very difficult. While many cases are asymptomatic, HSV disease can be very serious, including sporadic encephalitis due to HSV-1, with an untreated mortality rate of approximately 70%. HSV also presents dangers to the newborn (See e. g., Connelly and Stanberry, Curr. Opin. Pediatr., 7: 19-23 [1995]). Unfortunately, the majority (approximately 70%) of mothers who infect their neonates with HSV during delivery are experiencing asymptomatic genital infections. For mothers experiencing recurrent infection, the attack rate is relatively low (i. e., probably less than 5%). However, if the mother is experiencing primary infection at the time of delivery, the attack rate is probably greater than 50% (See e. g., Arvin and Prober, supra). Infected neonates may present with localized infections of the skin, eyes, and mucosa, or the central nervous system, or disseminated infection. The mortality rate for untreated disseminated infection in these neonates exceeds 70%; early treatment greatly reduces morbidity and mortality in these infants.

In addition to neonates, immunocompromised individuals (e. g., AIDS patients) are at great risk for serious HSV disease. For example, recurrent or primary HSV infection in these patients can by locally invasive, with considerable mucocutaneous necrosis and/or spread to

contiguous organs (e. g., causing esophagitis or proctitis). Viremia with dissemination to multiple organs may result in meningoencephalitis, pneumonitis, hepatitis, and coagulopathy, as well as Kaposi's varicelliform eruption. Thus, prevention of HSV transmission to these immunocompromised individuals and neonates is of great importance. Nonetheless, efforts to prevent transmission have been largely ineffective, as infected individuals who do not know that they are infected may unknowingly transmit it to their partners during unprotected sexual activities.

Since its recognition in 1981, the acquired immunodeficiency syndrome (AIDS) has become a major pandemic. The worldwide prevalence of the human immunodeficiency virus (HIV) infection has been estimated at more than 18,500,000 cases, with an additional estimate of 1.5 million infected children (Famighetti, 1996 World Almanac and Book of Facts, World Almanac Books, Mahwah, New Jersey, [1995], p. 840). In 1996, the incidence of AIDS- opportunistic illnesses in the United States, was approximately 6,390 per 100,00 population for those 50 years of age and older; for those 13-49 years of age, the incidence was approximately 50,340 per 100,000 ("AIDS Among Persons Aged : 50 Years--United States, 1991-1996,"Morbidity and Mortality Weekly Report, January 23,1998). The AIDS pandemic is a serious public health concern. Individuals who are at high risk of HIV infection are also at risk of infection by other sexually transmitted pathogens. The two most common serious STDs are Neisseria gonorrhoea and Chlamydia trachomatis. In the United States alone, conservative estimates suggest that each of these pathogens infect about one million people per year. Worldwide, there is an estimated annual incidence of 25 million cases of gonorrhea and 50 million cases of chlamydia (Crotchfelt et al., J. Clin. Microbiol., 35: 1536-1540 [1997]). An additional problem with STDs, other than HIV, is that they are a cofactor in HIV infection. For instance, although all genital ulcerative diseases appear to significantly increase the chances of HIV infection, infection is particularly easy with Haemophilus ducreyi, the etiological agent of chancroid.

The explosive rise in the incidence of sexually transmitted diseases has evoked public desire for preventive measures. At present, the global strategy for preventing AIDS consists of three interrelated strategies: i) encouraging people to reduce the number of partners they have; ii) promoting widespread use of condoms; and iii) treating concurrent sexually transmitted diseases in people at risk of HIV infection. As in the case with HIV, condoms and behavior modification are the only prevention strategies for Neisseria gonorrhoea and Cl11amydia trachomatis. Although there has been considerable work on vaccines, there are

none available. In the case of gonorrhoea, it is unlikely that a vaccine will be easily developed because of the rapid and effective antigenic modulation which is one of the hallmarks of N. gonorrhoea (Phillips, Perspect. Drug Disc. Design 5: 213-224 [1996]).

Unfortunately, no vaccines have been developed to control any STDs at present, and some pathogenic organisms are becoming resistant to the commonly used antimicrobials.

Therefore. there is a pressing need for alternate strategies to prevent infection by HSV, C.'hlamydia, and HIV, as well as the other organisms associated with STDs.

SUMMARY OF THE INVENTION The present invention is related to the prevention and treatment of sexually transmitted diseases (STDs). In particular, the present invention relates to novel compositions and uses of triclosan as an intravaginal microbicide, as a method of preventing and treating the spread of STDs.

The present invention provides compositions comprising a halogenated-2-hydroxy- diphenyl ether suitable for intravaginal use. In preferred embodiments, the compositions are suitable for intravaginal administration in mammals. In particularly preferred embodiments, the compositions are suitable for intravaginal administration in humans. However, it is not intended that the present invention be limited to use in humans nor any particular non-human animal. For example, it is contemplated that the present invention will find use in both the large and small animal, as well as exotic animal fields. In preferred embodiments, the halogenated-2-hydroxy-diphenyl ether is triclosan, while in other preferred embodiments, the halogenated-2-hydroxy-diphenyl ether is a derivative of triclosan. In yet other embodiments, the composition comprising triclosan is an aqueous solution. In still further, particularly preferred embodiments, the composition is comprised of triclosan in a solution comprised of approximately 78% glycerol, approximately 10% of phosphate buffered saline (PBS), approximately 10% ethanol (EtOH), and approximately 2% sodium hydroxide (NaOH).

However, it is not intended that the present invention be limited to halogenated-2-hydroxy- diphenyl ether present in a solution or associated with any particular components. Thus, it is also intended that the present invention encompass halogenated-2-hydroxy-diphenyl ether- containing compositions that also contain additional components, including but not limited to additional antimicrobials (i. e., combinations of triclosan with other antimicrobials), spermicidals, other pharmaceutical and/or medicinal compounds, lubricants, etc. It is further intended that the present invention encompass halogenated-2-hydroxy-diphenyl ether in any

form, including but not limited to solids, semi-solids, gels, creams, ointments, capsules, suppositories, foams, and aqueous solutions comprising any suitable buffers and other components.

The present invention also provides methods for the prevention of sexually transmitted diseases, comprising the steps of providing a female subject and a preparation comprising a halogenated-2-hydroxy-diphenyl ether suitable for intravaginal use, and introducing the preparation into the vagina of the female subject prior to sexual intercourse. In particularly preferred embodiments, the halogenated-2-hydroxy-diphenyl ether is triclosan. In yet other, particularly preferred embodiments, the composition is comprised of a triclosan composition in a solution comprised of approximately 78% glycerol, approximately 10% of phosphate buffered saline (PBS), approximately 10% ethanol (EtOH), and approximately 2% sodium hydroxide (NaOH). However, it is not intended that the present invention be limited to halogenated-2-hydroxy-diphenyl ether present in a solution or associated with any particular components. Thus, it is also intended that the present invention encompass halogenated-2- hydroxy-diphenyl ether-containing compositions that also contain additional components, including but not limited to additional antimicrobials (i. e., combinations of triclosan with other antimicrobials), spermicidals, other pharmaceutical and/or medicinal compounds, lubricants, etc. It is further intended that the present invention encompass halogenated-2- hydroxy-diphenyl ether in any form, including but not limited to solids, semi-solids, gels, creams, ointments, capsules, suppositories, foams, and aqueous solutions comprising any suitable buffers and other components.

The present invention also provides methods for prevention of sexually transmitted diseases, comprising the steps of providing a female subject and preparation comprising a halogenated-2-hydroxy-diphenyl ether suitable for intravaginal use, and introducing the preparation into the vagina of the female subject after sexual intercourse. In particularly preferred embodiments, the halogenated-2-hydroxy-diphenyl ether is triclosan. In yet other embodiments, the composition is comprised of a triclosan composition in a solution comprised of approximately 78% glycerol, approximately 10% of phosphate buffered saline (PBS), approximately 10% ethanol (EtOH), and approximately 2% sodium hydroxide (NaOH). However, it is not intended that the present invention be limited to halogenated-2- hydroxy-diphenyl ether present in a solution or associated with any particular components.

Thus, it is also intended that the present invention encompass halogenated-2-hydroxy-diphenyl ether-containing preparations that also contain additional components, including but not

limited to additional antimicrobials (i. e., combinations of triclosan with other antimicrobials), spermicidals, other pharmaceutical and/or medicinal compounds, lubricants, etc. It is further intended that the present invention encompass halogenated-2-hydroxy-diphenyl ether in any form, including but not limited to solids, semi-solids, gels, creams, ointments, capsules, suppositories, foams, and aqueous solutions comprising any suitable buffers and other components.

The present invention also provides methods for prevention of sexually transmitted diseases, comprising the steps of providing a female subject and preparation comprising triclosan suitable for intravaginal use, and introducing the aid preparation into the vagina of the female subject prior to and after sexual intercourse. In particularly preferred embodiments, the halogenated-2-hydroxy-diphenyl ether is triclosan. In yet other embodiments, the composition is comprised of a triclosan composition in a solution comprised of approximately 78% glycerol, approximately 10% of phosphate buffered saline (PBS), approximately 10% ethanol (EtOH), and approximately 2% sodium hydroxide (NaOH). However, it is not intended that the present invention be limited to halogenated-2- hydroxy-diphenyl ether present in a solution or associated with any particular components.

Thus, it is also intended that the present invention encompass halogenated-2-hydroxy-diphenyl ether-containing preparations that also contain additional components, including but not limited to additional antimicrobials (i. e., combinations of triclosan with other antimicrobials), spermicidals, other pharmaceutical and/or medicinal compounds, lubricants, etc. It is further intended that the present invention encompass halogenated-2-hydroxy-diphenyl ether in any form, including but not limited to solids, semi-solids, gels, creams, ointments, capsules, suppositories, foams, and aqueous solutions comprising any suitable buffers and other components.

In particularly preferred embodiments of the invention, the etiologic agents of sexually transmitted diseases prevented by the compositions and methods of the present invention include human immunodeficiency virus (HIV), herpes simplex virus (HSV), Chlamydia trachomatis, Neisseria gonorrhoeae, Calmatobacterium granulomatis, Treponema pallidum, Ureaplasma urealyticum, Mycoplasma hominis, Haemophilus ducreyi, Gardnerella vaginalis, Trichomona vaginalis, and human papillomavirus. However, it is not intended that the present invention be limited to any particular organism or STD. Indeed, it is contemplated that the present invention will find use in treatment and/or prevention of various other diseases of humans and other animals.

DESCRIPTION OF THE FIGURES Figure 1 shows the structure of triclosan (2,4,4'-trichloro-2'-hydroxyphenyl ether).

Figure 2 shows the general structure of halogenated-2-hydroxy-diphenyl ethers triclosan derivatives.

Figure 3 shows the percent of animal survival as a measure of days post-inoculation for treatment with a triclosan formulation prepared with 100 mg/mL of triclosan at pH 8 in 78% glycerol, 100 pL, 10% PBS, 10% EtOH, and 2% NaOH, compared with the control (i. e., administration of PBS).

Figure 4 shows the percent of animal survival as a measure of days post-inoculation for treatment with various triclosan formulations prepared with 100 mg/mL of triclosan at pH 8 in 780 uL (78%) glycerol, 100 L (10%) of PBS, 100 pL (10%) EtOH, and 20tL (2%) NaOH.

Figure 5 shows the growth of N. gonorrhoeae in the presence of various concentrations of triclosan and controls.

Figure 6 shows the percent of animal survival as a measure of days post-inoculation for treatment with various triclosan formulations and controls.

Figure 7 shows the vaginal swab titers of guinea pigs treated with triclosan or PBS before inoculation with 109 IFUs GPIC.

Figure 8 shows the tissue titers of guinea pigs treated with triclosan or PBS before inoculation with 109 IFUs GPIC.

DESCRIPTION OF THE INVENTION The present invention provides methods and compositions useful in the prevention and/or treatment of STDs. In particular, the present invention provides therapeutically effective compositions comprising triclosan that are suitable for use either before or after sexual intercourse to prevent the establishment of STD infection and/or disease.

A number of agents which could have some specific inhibitory effect on HIV or on the HIV infective process have been put forward as candidates for testing. These include sulfated polysaccharides, defensins, and other antimicrobial peptides, nucleoside inhibitors, gramicidin, and immunoglobulins. In addition to interest in identifying and testing active microbicidal agents, there has been considerable interest in developing a practical formulation (e. g., with an appropriate pH) to inactivate pathogens, and appropriate incipients to facilitate retention in the vagina. Despite recent developments in the development of a vaginal product

for the prevention of STDs, however, there remains a need for an intravaginal microbicide that provides protection from as many pathogens as possible, without disturbing the natural microbial environment within the reproductive tract (e. g., the vagina).

The present invention provides methods and compositions comprising a halogenated-2- hydroxy-diphenyl ether suitable for intravaginal use in animals, including humans. In a preferred embodiment, the halogenated-2-hydroxy-diphenyl ether is triclosan, an antimicrobial with a wide spectrum of antimicrobial action, including gram positive and gram negative bacteria. Triclosan is also a substantive antimicrobial and is currently incorporated into many cosmetic and drug-type cosmetic products used topically on the skin. Triclosan was formulated at various drug concentrations, and tested for efficacy as an intravaginal microbicide. Preferably, the concentration of triclosan is 100 mg/mL in a solution comprised of glycerol (approximately 78%), phosphate buffer saline (PBS) (approximately 10%), ethanol (EtOH) (approximately 10%) and sodium hydroxide (approximately 2%).

Triclosan Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether; 5-chloro-2- (2,4- dichlorophenyoxy) phenol; Irgasan DP 300) is a halogenated-2-hydroxy-diphenyl ether with the formula represented in Figure 1, and has been described by Model and Bindler (U. S.

Patent Nos. 3,506,720 and 3,629,477, both of which are herein incorporated by reference).

Derivatives of triclosan include, but are not limited to halogenenated-2-hydroxy-diphenyl ethers. In particularly preferred embodiments, triclosan derivatives include halogenenated-2- hydroxy-diphenyl ethers in which one or both of the phenyl groups is/are substituted by one or more substituent groups already present on the phenyl rings. Suitable substituents include, but are not limited to alkyl groups containing 1 to 4 carbon atoms, haloalkyl groups containing 1 to 4 carbon atoms, alkoxy groups containing 1 to 4 carbon atoms, cyano, allyl, amino, and acetyl groups. Examples of substituents are methyl, methoxy and trifluoromethyl groups. Triclosan dissolves readily in most organic solvents and is soluble to the extent of 2.35% in an excess of 0.1 N sodium hydroxide; in water, however, it is virtually insoluble.

Derivatives of triclosan also include halogenated-2-hydroxy-diphenyl ethers having the general formula shown in Figure 2, where"p"is a whole number ranging from 1 to 5,"Hal" is a halogen atom; and when"p"is greater'than 1', the halogen atoms may be identical or different, and one or both of the phenyl groups contain one or more substituent groups selected from alkyl groups containing from 1 to 4 carbon atoms, haloalkyl groups containing

from 1 to 4 carbon atoms, alkoxy groups containing 1 to 4 carbon atoms, cyano, alkyl, amino and acetyl groups. Such derivatives are described in WO 96/00569, herein incorporated by reference.

Triclosan is a broad-spectrum antimicrobial agent that has been used against various gram-positive and gram-negative bacteria, and against various pathogenic fungi, including those associated with gastrointestinal and urinary tract infections and disease. For instance, this compound has growth-inhibiting action on Staphylococcus aureus, S. lactis, Escherichia coli, Bacillus pumilus, B. subtilis, Corynebacterium diphtheriae, Clostridium botulinum, C. butyricu7X1, C. welchii, C tetani, Klebsiellapneumoniae, Alcaligenesfaecalis, Salmonella pullorum, S. typhi, S. paratyphi A and B, S. typhimurium, S. enteritidis, Shigella dysenteriae, <BR> <BR> <BR> <BR> S. flexneri, Brucella abortus, Proteus mirabilis, Achromobacter, Serratia marcescens, Sarcina uriae, Pseudomonas pyocyaea, P. aeruginosa, Pasteurella pseudotuberculosis, Trichophyton mentagrophytes, T. rubrum, T. tonsurans var. sabouraudi, T. schonleini, T. quinckranum, Microsporon canis, M. gypseum, Blastomyces dermatitidis, Sporotrichium schenckii (Sporothrix schenkii), Epidermophytonfloccusum, Alternaria tenuis, and Botrytis cenerea.

Triclosan has also been used for the treatment of gastrointestinal disorders associated with Helicobacter pylori infections. (See, U. S. Patent No. 3,506,720 to Model and Bindler; Vischer and Regos, Zbl. Bakt. Hyg., 1. Abt. Orig. A 226: 376-389 [1974]; Regros et al., Dermatologica 158: 72-79 [1979]; PCT Publication WO 96/00569 to Dettmar et al. ; all of which are hereby incorporated by reference). Regros and Hitz (Regos and Hitz, Zbl. Bact.

Hyg., 1. Ab. Orig. A 226: 390-401 [1974]) provide a description of the mode of action of triclosan, as determined in assays involving uptake of labelled phenylalanine, thymine, and uracil by E. coli. While an understanding of the mechanism involved is not necessary in order to use the present invention, these authors suggest that the cytoplasmic membrane is the primary site of triclosan's antimicrobial activity.

Foot powders and sprays, hosiery and insoles of shoes with incorporated triclosan are called"odor-eaters,"because of triclosan's ability to suppress odor produced by microorganisms present on the foot. Triclosan has also been used for the prevention of plaque, gingivitis and/or caries (See, U. S. Patent Nos. 5,525,330 and 5,605,676 to Gaffar et al., and U. S. Patent No. 5,578,295 to Francis et al., all of which are herein incorporated by reference); for providing soaps, detergents and cosmetics with germicidal activity (See e. g., U. S. Patent No. 3,968,210 to Schenkel, herein incorporated by reference); for treatments for eczema and dermatomycoses (See e. g., U. K. Patent Appln. No. 2 148 116A, herein

incorporated by reference); for topical treatment of inflammatory and allergic diseases, such as psoriasis and bronchial asthma (See e. g., U. S. Patent No. 5,185,377 to Schewe et al., herein incorporated by reference); and for disinfectants in personal and household linen, and bacteriostatic agents in washing agents (See, U. S. Patent No. 3,629,477 to Model and Bindler; U. S. Patent No. 5,403,864 to Bruch et al., both of which are herein incorporated by reference).

During the development of the present invention, various concentrations of triclosan were tested in order to determine their efficacy against organisms associated with STDs. In these experiments, in vitro as well as in vivo tests were used, including recognized animal models of STDs. In many experiments, the in vitro tests were conducted using a plaque reduction assay with HeLa cells and HSV-2 strain MS. In these experiments, it was determined that the concentration of triclosan needed to inhibit 50% of the virus was 0.071 mg/ml.

In addition, triclosan concentrations of 100 mg/mL, 50 mg/mL, and 10 mg/mL at pH 8. were formulated in buffers containing glycerol, phosphate buffer saline (PBS), ethanol (EtOH), and sodium hydroxide (NaOH) and tested in animal models. In some experiments, agar was also used in the triclosan preparations. In the mouse tests, the results show that treatment with triclosan composition prepared with 100 mg/mL triclosan provided a 100% survival rate after 21 days of post-inoculation. These results are in contrast to other formulations, where the percentage of surviving animals decreased corresponding to the increase in the number of days of post-inoculation.

In addition to the mouse in vivo efficacy tests, the toxicity of triclosan was tested using a neutral red dye uptake assay. The method for toxicology-neutral red dye uptake assay has been described in a combination of two papers (See, Borenfreund and Puerner, Toxicol.

Lett., 24: 119-124 [1985]; Borenfreund and Puerner, Toxicol. Lett., 39: 121-134 [1986]). As discussed in greater detail in the Examples, these results indicated that triclosan was sufficiently non-toxic for use in animals such as humans.

In addition to the compositions described above, the present invention also provides methods for prevention of sexually transmitted diseases (STDs). In one embodiment, the present invention provides methods comprising the steps of : providing: i) a female subject not suspected of being infected with a sexually transmitted disease; and ii) a composition having antimicrobial activity, wherein the composition comprises triclosan or a derivative thereof suitable for intravaginal use; intravaginally administering the composition either prior

to or after sexual intercourse (or in some cases both prior to and following intercourse). In some embodiments, the intravaginal microbicidal activity of the triclosan-containing preparations is observed and/or monitored to determine the effectiveness of the preparations in preventing STD (s).

Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions which may comprise triclosan alone, as well in combination at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Any of these compounds can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with suitable excipient (s), adjuvants, and/or pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

Administration Of Pharmaceutical Compositions In preferred embodiments, administration of pharmaceutical compositions is accomplished intravaginally, although it is not intended that the present invention be limited to intravaginal administration. It is contemplated that in some embodiments, other routes of administration (e. g., rectally, orally, and/or parenterally) will also find use in the present invention. Methods of parenteral delivery include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, and intranasal administration. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and other compounds that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of"Remington's Pharmaceutical Sciences" (Maack Publishing Co, Easton PA).

Pharmaceutical compositions for administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for the desired route of administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, foams, suppositories, aqueous suspensions, etc., suitable for administration to the subject.

Pharmaceutical preparations can be obtained through combination of active compounds with solid excipient (s), optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired. Suitable excipients include carbohydrate or protein fillers, including but not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added for product identification or to characterize the quantity of active compound (i. e., dosage).

Pharmaceutical preparations include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For example, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For some routes of administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Manufacture And Storage The pharmaceutical compositions of the present invention may be manufactured in a manner that known in the art (e. g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than the corresponding free base forms. After pharmaceutical compositions comprising a compound of the invention formulated in a acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of triclosan preparations, such labeling would include amount, frequency and method of administration.

Therapeutically Effective Dose Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in any appropriate animal model. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A"therapeutically effective dose"refers to that amount of the composition which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e. g., ED50, the dose therapeutically effective in 50% of the population; and LD50, the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.

The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending

upon the dosage form employed, sensitivity of the patient, and the route of administration (i. e., clinically safe and effective treatment).

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include numerous considerations, including the age, weight and gender of the patient; diet, time and frequency of administration, drug combination (s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Guidance as to particular dosages and methods of delivery is provided in the literature (See, US Patent Nos. 4,657,760; 5,206,344; and 5, 225,212, herein incorporated by reference).

DEFINITIONS To facilitate understanding of the invention, a number of terms are defined below.

As used herein, the term"triclosan"refers to 2,4,4'-trichloro-2'-hydroxydiphenyl ether, whose structure is represented in Figure 1. The compound is also known by the names 5- chloro-2- (2,4-dichlorophenoxy) phenol; CH 3635; Irgasan CH 3635; and Irgasan DP 300.

As used herein, the term"triclosan derivatives"refers broadly to halogenated-2- hydroxy-diphenyl ethers, wherein one or both of the phenyl groups is/are substituted by one or more substituent groups already present on the phenyl rings. It is also used to refer to halogenated-2-hydroxy-diphenyl ethers having the general formula shown in Figure 2, where "p"is a whole number ranging from 1 to 5, and"hal"is a halogen atom.

As used herein, the term"halogenated"refers broadly to a compound containing one or more substituent selected from the group of fluorine, chlorine, bromine and iodine. In preferred embodiments, the term is used in reference to chlorine.

As used herein, the term"intravaginal microbicide"is used to denote vaginal preparations (e. g., compositions) to be used within the vagina to protect an individual from infection by sexually transmitted pathogens. In preferred embodiments, the term is used in reference to triclosan as an intravaginal microbicide.

As used herein, the term"sexually transmitted diseases (STDs)"refers to diseases that are most often transmitted by direct sexual contact, including kissing and vaginal intercourse, as well as anal intercourse, and other sexual activities. It also encompasses the commonly

used lay terminology of"venereal disease"or"VD."The term encompasses, but is not limited to, diseases with etiologic agents such as immunodeficiency viruses (e. g., human immunodeficiency virus [HIV]), herpes simplex viruses (e. g., types 1 and 2, [HSV-1 and HSV-2]), Chlamydia trachomatis, C. trachomatis serovars LI, L2, and L3, Calmatobacterium granulomatis, Treponema pallidum, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Haemophilus ducreyi, Gardnerella vaginalis, Trichomona vaginalis, and papillomaviruses (e. g., human papillomaviruses [HPV]). It is not intended that the present invention be limited to any particular organism or host. For example, it is contemplated that the present invention will find use in the veterinary area, as well as human health fields.

As used herein, the term"culture"refers to any sample or specimen which is suspected of containing one or more microorganisms."Pure cultures"are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to"mixed cultures,"which are cultures in which more than one genus, species, and/or strain of microorganism are present.

As used herein, the term"organism"is used to refer to any species or type of microorganism, including but not limited to, bacteria, yeasts and fungi. In preferred embodiments, the organism of interest are pathogenic organisms that cause sexually transmitted diseases. In particularly preferred embodiments, the organisms of interest are HIV-1, HSV II, Chlamydia trachomatis and Neisseria gonorrhoeae. Thus, the term"etiologic agent"refers to an organism that is associated with or recognized as causing a particular disease or pathologic condition.

As used herein, the terms"antibacterial"or"antimicrobial,"used interchangeably, are used in reference to any compound, substance, or molecule capable of inhibiting the growth of, or of killing microorganisms. It is intended that the term be used in its broadest sense, and includes, but is not limited to compounds such as antibiotics which are produced naturally or synthetically. It is also intended that the term include compounds (e. g., triclosan) and elements that are useful for inhibiting the growth of, or killing microorganisms. It is contemplated that compositions containing multiple compounds will find use in the present invention. For example, it is intended that the term encompass antimicrobial compositions in which triclosan is included as a component in addition to other compounds. It is not intended that the present invention be limited to any particular antimicrobial composition.

As used herein, the term"culture media,"and"media"refers to any substrate for the growth and reproduction of microorganisms."Media"may be used in reference to solid

plated media which support the growth of microorganisms. Also included within this definition are semi-solid and liquid microbial growth systems including those that incorporate living host organisms, as well as any type of media.

As used herein, the terms"sample"and"specimen"are used in their broadest sense.

On the one hand, they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, vaginal discharge, and saliva, as well as solid tissue. These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms.

As used herein, the term"female subject"refers to any female animal, including but not limited to humans. Indeed, it is contemplated that the present invention will find use in various species, including humans, as well as domestic animals (e. g., livestock, laboratory animals, and/or companion animals, including, but not limited to equines, bovines, caprines, ovines, canines, felines, lagomorphs, murines, primates, reptiles, amphibians, avians, piscines, etc.), as well as wild, exotic, and feral animals, including, but not limited to animals housed in zoos, aquaria, oceanaria, etc. In particularly preferred embodiments, the female subject is a human.

The phrases"pharmaceutical preparation suitable for intravaginal administration," "pharmaceutical composition suitable for intravaginal use,"and combinations thereof, refer to a any composition containing a desired compound in a pharmaceutically acceptable form for intravaginal administration. The pharmaceutical preparation may contain diluents, adjuvants and excipients, among other components, provided that those additional components neither <BR> <BR> <BR> adversely effect the preparation (e. g, they do not cause degradation of the compound) nor the recipient (e. g., they do not cause a hypersensitivity reaction). It is also contemplated that the pharmaceutical preparation of the present invention will be formulated so as to be clinically safe and effective. It is not intended that the composition or preparation be limited to any particular format (i. e., the term encompasses, but is not limited to solids, semi-solids, gels, creams, ointments, capsules, suppositories, foams, and aqueous solutions comprising any suitable buffers and other components).

EXPERIMENTAL The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. Although embodiments have been described with some particularity, many modifications and variations of the preferred embodiment are possible without deviating from the invention.

In the experimental disclosure which follows, the following abbreviations apply: M (Molar); u. M (micromolar); N (Normal); mol (moles); mmol (millimoles); p. mol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); ig (micrograms); ng (nanograms); 1 or L (liters); ml and mL (milliliters); ul (microliters); cm (centimeters); mm (millimeters); pm (micrometers); nm (nanometers); MgCl2 (magnesium chloride); NaCI (sodium chloride); Na2CO3 (sodium carbonate); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); SDS (sodium dodecyl sulfate); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); Tris (tris (hydroxymethyl) aminomethane); w/v (weight to volume); v/v (volume to volume); °C (degrees Centigrade); PBS (phosphate buffered saline); HEPES (N-2- hydroxyethyl-piperazine-N'-2-ethanesulfonic acid); PEG (polyethylene glycol); EtOH (ethanol); NaOH (sodium hydroxide); H2O (water); ddH20 (double distilled water); CaCl (calcium chloride); AcOH (acetic acid); L (microliters); mL (milliliters); M% (mole percent); BME (Basal Medium Eagle); MEM (Minimal Essential Medium); ED50 (50% effective dose); PFU (plaque forming unit); IFU (infection forming unit or infectious forming unit); CFU (colony forming unit); HSV (herpes simplex virus); HIV (human immunodeficiency virus) ; DIFCO (Difco Laboratories, Detroit, MI); U. S. Biochemical (U. S. Biochemical Corp., Cleveland, OH); Fisher (Fisher Scientific, Pittsburgh, PA); Sigma (Sigma Chemical Co., St.

Louis, MO); ATCC (American Type Culture Collection, Rockville, Maryland); Ciba-Geigy (Ciba-Geigy, Greensboro, NC); Amicon (Amicon, Inc., Beverly, MA); BBL (Baltimore Biologies Laboratory, (a division of Becton Dickinson), Cockeysville, MD); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, NJ); BioRad (BioRad, Richmond, CA); Harlan (Harlan. Indianapolis, IN); Charles River (Charles River Laboratories, Wilmington, MA); Hazelton (Hazelton Research Products, Denver, PA); Falcon (e. g. Baxter Healthcare Corp., McGaw Park, IL and Becton Dickinson); Fisher Biotech (Fisher Biotech, Springfield, NJ); GIBCO and Gibco (Grand Island Biologic Company/BRL, Grand Island, NY); Mallinckrodt (a division of Baxter Healthcare Corp., McGaw Park, IL); Millipore (Millipore Corp.,

Marlborough, MA); New England BioLabs (New England BioLabs, Inc., Beverly, MA); Pharmacia (Pharmacia, Inc., Piscataway, NJ); Upjohn (Pharmacia and Upjohn Company, Kalamazoo, MI); Sanofi (Sanofi Diagnostic, Redmond, WA); and Sigma (Sigma Chemical Co., St. Louis, MO).

EXAMPLE 1 Virus Preparations Two different HSV-2 virus strains were used in the following Examples. The HSV-2 MS strain (ATCC #VR-540) was used for in vitro evaluation of triclosan, while HSV-2 strain 186 (a gift from Dr. Rick Thompson of the University of Cincinnati, Cincinnati, Ohio) was used in experiments to determine the in vivo evaluation of triclosan.

The HSV-2 MS strain was grown in HeLa cells (ATCC #CCL 2), while the HSV-2 186 strain was grown in young rabbit kidney cells (YRK). These primary rabbit kidney ells were prepared from New Zealand white rabbits (Hazelton); Stanberry et al., J. Infect. Dis., 155: 914-920 [1987]). The cells were maintained in Basal Medium Eagle (BME, Gibco) supplemented with L-glutamine (2 mM), 10% FBS, and penicillin/streptomycin (50,000 U/ml and 50,000 g/ml, respectively), and equilibrated with 0.5% 1 M HEPES and 1.5% of a 7.5% sodium bicarbonate solution.

EXAMPLE 2 Triclosan Preparations Triclosan (Irgasan DB300, not less than 99% 5-chloro-2- (2,4-dichlorophenoxy) phenol) was obtained from Ciba-Geigy. For in vitro experiments, the highest concentration used was 10 mg/ml, in a vehicle comprised of 78% glycerol, 10% PBS, 10% EtOH (100%), and 2% 10 N NaOH. For in vivo experiments, the highest concentration used were 100 mg/ml to 1 g/ml solution in a number of vehicles, including a glycerol/PBS vehicle comprised of 78% glycerol, 10% PBS, 10% EtOH (100%), and 2% 10 N NaOH. In some experiments, the glycerol/PBS vehicle also contained either 10 mg/ml carrageenan type IV, 10 mg/ml agar, or 10 mg/ml carboxymethylcellulose. Triclosan was also formulated in peanut oil, a PEG vehicle comprised of 80% PEG 1000,10% PEG 6000 and 10% dHO, as well as a PEG vehicle containing 10% propylene glycol.

EXAMPLE 3 In Vitro Testing of Triclosan As the effects of triclosan had not been previously tested for anti-viral activity, it was necessary to determine these effects. Thus, in this Example, the effects of triclosan on a HSV-2, a common STD pathogen were determined. In these experiments, HeLa cells were seeded into a 24-well tissue culture plate (1 mL of HeLa cells at a concentration of 4x105 cells/mL in BME with 10% FBS and antibiotics as described in Example 1, above, and incubated overnight at 37°C, with 5% CO,. This level of seeding typically yielded a 100% confluent monolayer in a 24-hour period.

Controls were prepared by combining 0.5 mL undiluted HSV-2 MS strain virus (ATCC #YR-540) and 0.5 mL BME with 10% FBS and antibiotics. This suspension (0.2 mL) was used to inoculate control wells of the 24-well tissue culture plate containing HeLa cells. Virus suspensions were prepared by combining 0.5 mL undiluted HSV-2 MS strain virus and 0.5 ml BME with 10% FBS and antibiotics, combined with triclosan at each of the various concentrations tested. Serial 1: 10 dilutions of triclosan were prepared. These assays were run in quadruplicate.

The virus suspensions were used to inoculate (0.2 mL) the HeLa cells in the 24-well tissue culture plate. The tests and controls were incubated for one hour at 37°C, at 5% Cl,,.

At approximately every 15-20 minutes, the plates were gently shaken (manually) to expose the cells to the virus or control suspension. After one hour of incubation, the virus suspension and control suspension were aspirated from the wells. The cells were then fed with 1 mL/well BME with 10% FBS and antibiotics, and incubated for 72 hours at 37°C in 5% CO,.

The wells of the 24-well plates were stained by aspirating off the medium and covering the bottoms of the wells with either 0.07% crystal violet solution or ACCUSTAIN (Sigma), and incubating at 15-30 minutes at room temperature. Following incubation, the stain was decanted, and the wells were carefully rinsed with approximately 500 mL distilled HO. The plates were then inverted for drying. Once they were dry, the number of plaques in each of the wells was counted. The results were also plotted as a percent of controls compared to the triclosan concentration. The ED50 (effective dose, 50%) was determined as the triclosan concentration at which the plaque numbers were 50% of the controls. The concentration of triclosan at which the ED50 was obtained was 0.71 mg/ml.

The cytotoxicity of triclosan was determined using the neutral red dye uptake assay described in the following Example.

EXAMPLE 4 Assessment of Triclosan Cytotoxicity In this Example, the cytotoxicity of triclosan was determined using the neutral red dye uptake assay described by Borenfreund and Puerner (Borenfreund and Puerner, Toxicol. Lett., 24: 119-124 [1985]; and Borenfreund and Puerner, Toxicol., 39: 121-134 [1986]).

Briefly, HeLa cells were seeded into a 96-well tissue culture plate by dispensing 200 l of cells to yield a cell density of 60-70% confluent monolayer at 24 hours. Then, the cells were exposed to various concentrations of triclosan in solution and incubated for one hour at 37°C. The triclosan was aspirated from the wells, and 200 pl of neutral red dye (0.2%) was dispensed into the test wells. Following incubation for 3 hours at 37°C, the neutral red dye was aspirated and 200 ti of a solution of 4% formaldehyde/1% calcium chloride was dispensed into the wells, in order to remove unincorporated dye and simultaneously provide adhesion of the cells to the substratum. The solution was left on the cells for 1-2 minutes and then aspirated. Next, 200 ul of 1% acetic acid/50% ethanol was dispensed into the wells, and allowed to incubate for 20 minutes at room temperature. After this incubation period, the plate was covered with plate sealer and shaken vigorously on a plate shaker for 15 seconds, and then read on a Whittaker plate reader with a 550 nm filter.

These results indicated that the dose of triclosan that caused injury to 50% of the HeLa cells was 0.045 mg/ml.

EXAMPLE 5 In Vivo Testing of Triclosan Against HSV-2 In this Example, mice were used to test the efficacy of triclosan in preventing infection with HSV-2 in a mouse model of genital herpes. This is a recognized lethal challenge model, in which less than 10% of unprotected (control) Swiss-Webster mice inoculated intravaginally with 104 PFUs of HSV-2 186 strain survive the infection (See e. g., Bourne et al., Vaccine 14: 1230-1234 [1996]).

In these experiments, Swiss-Webster mice (Harlan), primed with medroxyprogesterone (Pharmacia) administered subcutaneously at 3 mg/mouse at seven days and one day prior to the experiments. Each group of 15-16 mice received 15 PI of various concentrations of

triclosan diluted in different vehicles. These concentrations were administered intravaginally either immediately or after administering virus to the mice. The triclosan preparations tested included 10 mg/ml, 50 mg/ml, and 100 mg/ml in a glycerol/PBS (78% glycerol, 10% PBS, 10% EtOH [100%], and 2% 10 N NaOH) vehicle, in order to determine the lowest concentration of triclosan at which anti-viral activity could be observed. In addition to glycerol/PBS, other carriers, including glycerol/PBS with 10 mg/ml carrageenan type IV, 10 mg/ml agar, or 10 mg/ml carboxymethylcellulose, were also tested as was a PEG vehicle composed of 80% PEG 1000,10% PEG 6000, and 10% dH, O, the PEG vehicle with 10% propylene glycol, and a vehicle composed of peanut oil.

The virus was used at a concentration of 5x 104 PFUs, a dose that was previously determined to cause mortality in approximately 90% of the mice treated. Control mice received 15 ul of vehicle alone (i. e., without triclosan). The mice were observed daily over a 21 day period for the development of symptoms and death. The symptoms observed included hair loss and erythema around the perineum, hind limb paralysis, and urinary incontinence.

For mice that did not exhibit symptoms, infection was confirmed by culturing vaginal swabs using methods known in the art for the presence of virus (i. e., standard methods for detection of virus).

The results are shown in the following tables. Varying doses of triclosan formulated in glycerol and PBS administered immediately prior to HSV-2 challenge (i. e., time 0) resulted in a 93% survival rate with 10 mg/ml triclosan, and 100% survival was obtained with 50 and 100 mg/ml triclosan. In these experiments, it was determined that <10% of the unprotected (i. control) mice survived the infection. At 10 mg/ml, 6% of the asymptomatic animals were HSV-2 positive, while 13% were HSV-2 positive at 50 mg/ml, and 0% were HSV-2 positive at 100 mg/ml. Triclosan at a concentration of 100 mg/ml formulated in glycerol and PBS was found to be less effective when treatment was given five minutes before challenge (53% survival was observed). Survival rates were decreased to as low as 25%, when triclosan treatment (1 g/ml in peanut oil) was given prior to virus inoculation. However, the efficacy was increased by using formulations of triclosan with 10 mg/ml agar.

Table 1. Protective Effects of Triclosan: In Vivo Survival/Infection Rates with Time 0 Treatments with Various Triclosan Concentrations Followed Immediately by HSV-2 Inoculation Triclosan Concentration% Survival% Infected* 10 mg/ml 93 6 50 mg/ml 100 13 100 mg/ml 100 0 * As determined by vaginal swab cultures.

Table 2. Protective Effects of Triclosan: Lz Vivo Survival Rates with Treatment with Triclosan in Various Vehicles Five Minutes Prior to HSV-2 Inoculation Formulation Triclosan Concentration % Survival Glycerol/PBS 100 mg/ml 53 Glycerol/PBS with 100 mg/ml 40 ,.,, , lOOmg/ml 40 10 mg/ml Carrageenan Type IV Glycerol/PBS with 100 mg/ml 73 10 mg/ml Agar 10 mg/ml Agar Glycerol/PBS with 10 mg/ml 100 mg/ml 53 Carboxymethylcellulose PEG 100 mg/ml 53 PEG with 10% Propylene Glycol 100 mg/ml 53 Peanut Oil 1 g/ml 25

Table 3. Effect of Triclosan Pre-Treatment in Prevention of Genital HSV-2 Infection in Mice Vehicle Timing Relative Triclosan (mg/ml) % Survival to Viral Challenge Glycerol/PBS Immediately Prior 100 100 Glycerol/PBS Immediately Prior 50 100 Glycerol/PBS Immediately Prior 10 93 Glycerol/PBS Immediately Prior 0 < 10 Glycerol/PBS 5 Minutes Prior 100 53 Glycerol/PBS with 5 Minutes Prior 100 40 Carrageenan Type IV _ Glycerol/PBS with Agar 5 Minutes Prior 100 73 Glycerol/PBS with 5 Minutes Prior 100 53 Carboxymethylcellulose PEG 5 Minutes Prior 100 53 PEG with Propylene 5 Minutes Prior 100 53 Glycol Peanut Oil 5 Minutes Prior 100 25 Glycerol/PBS 5 Minutes Prior 0 < 10 Figure 3 provides a graph comparing the survival of mice treated with 100 mg/ml triclosan, pH 8,78% glycerol, 10% PBS, 10% EtOH, and 2% NaOH, with control mice (PBS was administered to these animals. Figure 4 provides a graph comparing the survival of mice treated with 100 mg/ml, 30 mg/ml, and 10 mg/ml triclosan, all of which were prepared with 78% glycerol, 10% PBS, 10% EtOH, and 2% NaOH, with control mice (controls received a solution of 80% glycerol, 10% PBS, and 10% EtOH). These results clearly show that triclosan is effective in providing protection against HSV-2.

EXAMPLE 6 Effectiveness of Triclosan In Vivo In this Example, additional experiments were conducted using the guinea pig mode of genital HSV infection. This model has been used extensively to study the pathogenesis of

genital herpes, as well as to evaluate vaccines and anti-viral drugs (See e. g., Steinberry, Rev.

Infect. Dis., 13 (Suppl. 11): S720-S723 [1991], and Steinberry, Sem. Virol., 5: 213-219 [1994]).

In this model, Hartley guinea pigs intravaginally inoculated with 105 7 PFUs HSV-2 strain MS develop a self-limited vesiculoulcerative genital skin disease that is similar to that observed in humans. In two experiments, Hartley guinea pigs (Charles River) were treated intravaginally with 100 mg/ml triclosan or PBS (i. e., controls), immediately prior to <BR> <BR> intravaginal challenge with HSV-2 (10s7 PFUs). In these experiments there were 12 animals in each group (i. e., 12 in the control group and 12 in the test group). The animals were observed over 14 days for evidence of disease. As indicated in the following table, 54% of the control animals developed genital herpes, but only one of 24 (4%) of the triclosan-pre- treated guinea pigs developed disease.

Table 4. Effect of Triclosan Pre-Treatment on Prevention of Genital HSV-2 Infection in Guinea Pigs Experiment TrIeatment % Animals with Primary Exper1ment Treatment : :Genital Herpes Genital Herpes 1 PBS Control 58 Triclosan 0 2 PBS Control 50 Triclosan 8 1 These results clearly show that triclosan is effective in preventing primary HSV disease.

EXAMPLE 7 Effects of Triclosan on N. gonorrlzoeae In this Example, the effect of triclosan on N. gonorrhoeae was determined.

In these experiments, various concentrations of triclosan were added to agar containing Isovitalex and glucose (i. e., media commonly used to cultivate N. gonorrhoeae, such as chocolate agar with supplements). Plates were inoculated with 1.5 x 105 CFU of N<BR> gnnor°hneae, and incubated at 37°C, in 5% CO2, and examined for growth of N. gonorrhoeae at 24 hours post-incubation. The results for plates containing no triclosan (control), as well

as 1,1,10, and 100 tg/nil are shown in Figure 5. As indicated, organisms were only capable of growing on the control agar and agar containing only 0.01 g/ml triclosan. These results clearly show that triclosan is highly effective against a common STD pathogen, N. gonorrhoeae.

EXAMPLE 8 Effects of Triclosan on C. trachomatis In this Example, the effects of triclosan on C. trachomatis were determined. In these experiments, assays for anti-chlamydial activity were performed using HeLa cells and CI trachonzatis serotype E (UW-5/CX; a gift from the Centers for Disease Control). The assay involved mixing C. trachomatis elementary bodies (EBs) (10', 104, and 102 per ml) with various dilutions of triclosan for 4 hours at 4°C, prior to adding the mixture to a confluent HeLa monolayer. Following addition of the triclosan/EB mixture to the HeLa monolayer, the cells were incubated for 4 hours at 4°C. The infected cell monolayer was subsequently washed and incubated in 37°C, in 5% CO2 for 48 hours. Following incubation, the monolayers were fixed in methanol, and stained for 10 minutes at room temperature with Syva Microtrak, a fluorescein isothiocyanate-labelled monoclonal antibody directed against the major outer membrane protein (MOMP). Monolayers were viewed using epifluorescence at 400X, and the number of fluorescent inclusions in a minimum of 100 grid fields (approximately 700 cells) were counted. The results of two experiments performed in triplicate were combined and are shown in Figure 6. As shown in this Figure, at concentrations of 0.5 and 5 mg/ml, no infectious forming units (IFUs) were observed. At a concentration of 0.05 mg/ml, triclosan inhibited 97% of the C. trachomatis used to infect the culture.

EXAMPLE 9 Effectiveness of Triclosan Against C. trachomatis In this Example, the efficacy of triclosan against C. trachomatis was determined. In these experiments, C. trachomatis strain E/UW-5/CX (a gift from the Centers for Disease Control) was used. The cultures were passaged in HeLa 229 cells.

Human fallopian tube organ cultures (HFTOC) were established as previously described. Briefly, fallopian tube tissue was incubated overnight in HEPES-MEM supplemented with 50 ug/ml gentamycin sulfate and 10-'° M/l estrogen. A suspension of 10"

IFUs/ml of C. trachomatis was prepared and pretreated with the test compounds (i. e., various triclosan-containing compositions) for four hours at 4°C. After incubation of infected HFTOCs for 48 hours, the weighed tissues were homogenized and added to the confluent HeLa cells on the coverslips. After incubation, the cultures were fixed and stained to observe the presence of inclusions with fluorescein-conjugated antibody directed against the lipopolysaccharide genus antigen (using the Pathfinder Chlamydia Culture Confirmation System, Sanofi, as per the manufacturer's instructions). The number of inclusions per well were counted using epifluorescence.

These results indicated that at triclosan concentrations of 5 and 0.5 mg/ml, no IFUs were observed; at a concentration of 0.05% triclosan, there was an 80% inhibition of C. trachonzatiz growth. These results show that triclosan has anti-chlamydial activity in vitro.

EXAMPLE 10 Effectiveness of Triclosan Against Chlannydial Infection An In Vivo Guinea Pig Model In this Example, the effectiveness of triclosan pre-treatment to prevent genital chlamydial infection was investigated. In these experiments, female Hartley guinea pigs were used. Other researchers have shown that intravaginal inoculation of guinea pigs with C. psittaci, the etiologic agent of guinea pig inclusion conjunctivitis (GPIC) produces lower genital tract infection, which progresses to upper genital tract disease (e. g., endometritis and salpingitis) in the majority of animals (See e. g., Rank et al., Am. J. Pathol., 142: 1291-1296<BR> [1993]; and Rank et al., Infect. Immun., 38: 699-705 [1982]).

Two experiments were conducted using C. psittaci associated with GPIC ("GPIC organisms") obtained from Dr. Rank (University of Arkansas) and 100 mg/ml triclosan in PBS. In these experiments, the animals were treated with 100 mg/ml triclosan in PBS or PBS (controls) immediately prior to intravaginal inoculation with 109 IFU GPIC organisms.

The animals were observed for 10 days, and vaginal swab samples were obtained on selected days. Figure 7 shows the results for the triclosan and PBS control animals. As shown in this Figure, These results indicated that triclosan had an inhibitory effect on chlamydial replication. On day 1 post-inoculation, the mean vaginal swab title in triclosan-pre-treated GPIC-inoculated guinea pigs was 0.83 log, o IFUs (1.44), as compared to 5.35 log, IFUs (+ 2. 3) in PBS-treated control guinea pigs. Chlamydia was not isolated from the triclosan- pre-treated animals on days 3,5, or 10, while Chlamydia was isolated from the control

animals on each of these days. In the second experiment, the guinea pigs were sacrificed on day 10, and their reproductive tracts removed. Tissue was collected from the cervico-vaginal region, and the middle-left (LF) and middle-right (RF) fallopian tubes, and assayed to determine GPIC titer. Figure 8 provides the tissue titers for the cultures obtained from cervico-vaginal tissue (CV), left fallopian tube tissue (LF), and right fallopian tube tissue (RF) of animals inoculated with 109 IFUs GPIC. As shown in this Figure, the animals pretreated with triclosan had significantly lower (p < 0.05) GPIC titers in cervico-vaginal and fallopian tube tissue samples, as compared to the PBS controls.

In the second experiment, triclosan pretreatment was shown to have a less dramatic effect on GPIC infection of the lower genital tract, but on all days tested, the mean GPIC titers obtained from the vaginal swab samples from the triclosan-pre-treated animals were significantly lower (p < 0.05), than from the PBS controls.

EXAMPLE 11 Effect of Triclosan Against C trachomatis In An In Vivo Mouse Model In this Example, the effectiveness of triclosan against genital chlamydial infection in mice was determined. Studies by others have indicated that intravaginal inoculation of mice with the mouse pneumonitis biovar of C. trachomatis (MoPn), causes lower and upper genital tract infection. The mouse model of C. trachomatis (MoPn) infection has been described in such references as Barron et al., J. Infect. Dis., 143: 63-66 [1981]; and Tuffrey and Taylor- Robinson, FEMS Microbiol. Lett., 12: 111-115 [1981]).

In these experiments, female Balb/c mice (Harlan) and Swiss Webster mice were pre- treated with Depo-Provera (Pharmacia) seven days and again one day prior to intravaginal inoculation with 107 IFU MoPn. The mice were pre-treated with triclosan (100 mg/ml) or PBS immediately prior to intravaginal MoPn challenge. The mice were observed for at least six days after inoculation, and vaginal swab samples were collected on days 1, 3, and 6. in order to determine the MoPn titer. Samples were also obtained from the upper genital tracts of these animals (i. e., the uterine horns). The samples (swabs and tissues) were suspended in 500 nl Hepes-Saccharo-Calcium buffer (HSC). Then, 10-fold dilutions were prepared and inoculated into McCoy cells (ATCC#CRL1696). The McCoy cells were incubated and observed using methods known in the art to determine whether C. trachomatis (MoPn) was present in the samples. As indicated in the tables below, the results indicated that triclosan

did not prevent C. trachomatis (MoPn) infection in mice, but did significantly reduce the titre of MoPn present on days 1 and 3 of the infection. Thus, the results indicate that triclosan pretreatment can significantly impact genital chlamydial infection although in these experiments pre-treatment with triclosan did not prevent infection.

Table 5. Assessment of Infectivity (Day 3) in Balb/c Mice Inoculated with MoPn Inoculum Concentration Number of Positive Results/Total in Group (%) Cervico-Vaginal Swabs Upper Tract Tissue 10'4/4 (100%) 4/4 (100%) 10'12/12 (100%) 11/12 (92%) 10"12/12 (100%) 5/12 (42%) 10"10/12 (83%) 2/12 (17%) 03 5/12 (42%) (0%) Table 6. Assessment of Infectivity (Day 3) in Swiss Webster Mice Inoculated with MoPn Inoculum Concentration Number of Positive Results/Total in Group (%) Cervico-Vaginiil Swabs Upper Tract Tissue io, 4/4 (100%) 4/4 (100%) 10, 12/12 (100%) 12/12 (100%) 105 12/12 (100%) 0/12 (0%) 104 3/12 (25%) 0/12 (0%) 10'0/12 (0%) 0/12 (0%) However, these experiments used very high inoculum titers (i. e., 109 IFU GPIC in the guinea pig tests, and 10'IFU MoPn), which may have exceeded the capacity of the triclosan dose administered to the animals.

EXAMPLE 12 Effect of Triclosan on HIV In this Example, the effect of triclosan on HIV-1 and HIV-2 is tested. In particular, these experiments provide an indication of the efficacy of triclosan in blocking infection of peripheral blood mononuclear cells with various HIV strains.

From the above, it is clear that the present invention provide compositions and methods for the prevention of sexually transmitted diseases. In particular, the present invention provides an intravaginal microbicide comprised of triclosan or a triclosan derivative.

In particular, the present invention provides triclosan composition effective in the prevention of human immunodeficiency virus (HIV), herpes simplex virus (HSV-II), Chlamydia rachon1atis and Neisseria gonorrhoeae.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the medicinal chemistry or related fields are intended to be within the scope of the following claims.