Broad spectrum microbicidal and spermicidal compositions and methods having activity against sexually transmitted agents including papillomaviruses

Microbicidal and spermicidal methods and topical pharmaceutical compositions containing sodium dodecyl sulfate or its derivatives as active ingredients for the prevention and control of pregnancy and sexually transmitted diseases, including human papilloma viruses. Spermicidal barriers and microbial disinfectants containing the compositions are also provided.

FIELD OF THE INVENTION
 The present invention relates to the prevention of pregnancy and the
 prevention and control of sexually transmitted diseases (STDs) with the
 use of compositions having broad spectrum microbicidal and spermicidal
 activity, including the ability to inactivate particularly resistive
 pathogens such as human papillomaviruses and other non-enveloped viruses.
 BACKGROUND OF THE INVENTION
 Sexually transmitted diseases (STDs) are among the most prevalent and
 communicable diseases, and continue to be a significant public health
 problem. It is estimated that more than 250 million people worldwide, and
 close to 3 million people in the United States, are infected annually by
 gonorrhea. Annual worldwide incidence of syphilis is estimated at 50
 million people, with 400,000 in the United States annually needing
 treatment. More recently, the human immunodeficiency virus (HIV),
 resulting in fatal acquired immunodeficiency syndrome (AIDS), has spread
 rapidly in both homosexual and heterosexual groups. Strong associations
 have now also been discovered between cervical cancer and papillomaviruses
 (PVs). It has been estimated that about 25% of women worldwide have human
 papillomavirus (HPV) genital infection.
 The human papillomaviruses (HPVs), of which there are now more than 90
 known types, cause papillomas (warts) in a variety of human epithelial
 targets including common warts of the hands (verruca vulgaris) and feet
 (plantar warts), as well as genital warts in vulvar, vaginal, cervical and
 penile epithelium. Genital warts represent a ubiquitous STD. Women with
 genital lesions containing certain HPV types, including types 16, 18, 31,
 33 and 35, are at increased risk for progression to cervical cancer. In
 the United States, 15,000 women per year are diagnosed with cervical
 cancer, and there are about 5000 deaths per year. In developing countries,
 cervical cancer is the leading cause of cancer related deaths among women.
 PVs present a unique challenge for investigators attempting to identify
 virucidal agents. PVs are inherently extremely resistive to attack by
 antimicrobial agents. In addition, PVs do not exist free in nature in the
 same manner that many non-enveloped viruses exist. Rather, PVs exist
 encased in the squames of differentiated epithelial cells. Thus, the PVs
 are not only protected by their own very difficult to penetrate capsids,
 but also by the surrounding, heavily keratinized and cross-linked squames
 of epithelial cells.
 One approach to the general control of STDs is the use of topically
 applied, female controlled microbicides that inactivate the relevant
 pathogens. Most frequently, these microbicides are spermicidal
 preparations containing NONOXYL-9 (N-9) detergent that inactivates
 enveloped viruses, such as HSV-2 and HIV-1. To date, these preparations
 have not been effective, however, against non-enveloped viruses such as
 the HPVs.
 Inability to inactivate HPVs makes N-9 an inadequate virucide against this
 STD. In addition, chronic use of N-9 was recently associated with
 increased seroconversion for positivity to HIV-1 antibodies in a group of
 prostitutes, raising the possibility that N-9 may erode vaginal
 epithelium. Frequent use of N-9 is also positively correlated with
 bacterial vaginosis, genital ulcers and vulvitis, vaginal candidiasis,
 toxic shock syndrome, and epithelial disruption of the cervix and the
 vagina. The detergent, however, is spermicidal and has been shown to
 inactivate enveloped viruses. It is present in a large number of condoms
 and other spermicidal agents.
 Other microbicides, such as octoxynol-9 (O-9), benzalkonium chloride (BZK)
 and chlorhexidine, are also surfactants that can disrupt the envelopes of
 HSV-2 and HIV-1 via surfactant/detergent properties. Like N-9, however,
 these microbicides also do not inactivate the non-enveloped PVs. Topical
 microbicides for inactivation of the PVs and prevention of animal or human
 transmission are currently not available, but would be highly desirable
 given the ubiquous nature of HPV infections.
 U.S. Pat. No. 5,004,757 is directed to a method of deactivating viruses on
 surfaces by applying a three-part composition containing gluteraldehyde.
 The composition also contains hydrogen-bonded glycol molecules to
 eliminate aldehyde odor, and an anionic surfactant such as sodium dodecyl
 sulfate (SDS) as a potentiator of the virucidal activity of the
 gluteraldehyde component. The patent indicates that SDS has limited
 virucidal activity on its own, but has a synergistic effect when combined
 with gluteraldehyde. Due to the presence of gluteraldehyde, a well-known
 mutagen, the formulation is not useful against STDs because it cannot be
 applied to human epithelium.
 What is needed are safe and effective microbicides against STDs which
 extend microbicidal activity to non-enveloped viruses and, in particular,
 to papillomaviruses.
 SUMMARY OF THE INVENTION
 The present invention provides pharmaceutical compositions, articles and
 methods for preventing pregnancy and transmission of STDs, including safe
 and effective vaginal compositions for controlling and preventing STDs.
 The microbicidal compositions of the invention contain an alkyl sulfate,
 such as SDS, lithium dodecyl sulfate, lauric acids or salts thereof, as an
 active ingredient capable of inactivating sperm and a broad spectrum of
 pathogenic microbes, including HPVs and other non-enveloped viruses.
 The present invention also provides disinfectant compositions for
 destroying pathogenic microbes on medical instruments, shower stalls,
 bathroom fixtures, exercise equipment and other inanimate surfaces, as
 well as spermicidal barriers coated or impregnated with an alkyl sulfate
 compound for combined spermicidal and microbicidal effects.
 It is interesting and surprising to note that, although SDS has been known
 for several years to have limited activity against enveloped viruses, and
 has been used as a surfactant for soaps, cosmetics and various other
 topical applications, such as shampoos and toothpastes, there have been no
 reports of its use, or the use of other topical antimicrobics, to control
 PVs. If indeed any such use occurred, it was unintended and unappreciated;
 it was an unrecognized accident. None of the reported studies or uses of
 SDS were conducted with the intent of controlling papillomavirus
 infections. Their purpose was merely as a surfactant/detergent, or at best
 as a facilitator of the antimicrobial activity of gluteraldehyde. There
 is, in fact, no known prior use of SDS for topical application which can
 be considered to have consistently achieved virucidal activity, as
 described hereinbelow.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 We have discovered that SDS and related anionic surfactants have potent
 spermicidal and virucidal activity, including virucidal activity against
 non-enveloped viruses, including the papillomaviruses, as well as against
 HSV-2 and HIV-1. As used herein, "SDS or related anionic surfactant" means
 sodium dodecyl sulfate and other members of the virucidal alkyl sulfate
 group, including but not limited to lithium dodecyl sulfate, lauric acid
 and salts or other derivatives thereof.
 In experiments conducted by the present inventors, very low concentrations
 of the detergent/sulfactant SDS completely inactivated HSV-2 and HIV-1, as
 well as three separate papillomavirus types after brief exposures to SDS
 at physiologic temperatures. In all cases, 0.1% concentrations of SDS were
 well above those exhibiting complete virus inactivation. Related anionic
 surfactants and derivatives also exhibited significant virucidal activity.
 As used herein, "virucidal" means capable of inactivating or destroying a
 virus. A susceptible virus is any virus which is inactivated or destroyed
 by SDS or related anionic surfactants. The susceptible viruses are readily
 identified in tests such as those described below, wherein the amount or
 concentration of SDS or related anionic surfactant is considered virucidal
 if the virus titer is reduced by at least 99.9% (3 log units).
 The invention can be carried out both in vitro and in vivo. In vitro means
 in or on nonliving things, especially on objects having hard or soft
 surfaces located or used where preventing viral transmission is desired.
 Hard surfaces include those of medical instruments, building interiors,
 furniture, bathroom fixtures, gym equipment and exterior fences for, e.g.,
 livestock containment. Soft surfaces include those of paper or cloth, for
 example, pre-moistened pads or tissues, dry facial tissues, hospital
 garments and bed clothing. In vivo means in or on a living person, plant
 or animal, especially on mammal skin and mucous membranes, including
 intravaginally, orally or rectally.
 To carry out the methods of the invention, SDS or a related anionic
 surfactant can be used alone or in the form of a composition containing or
 consisting essentially of a virucidally effective concentration of SDS or
 related anionic surfactant and a pharmaceutically acceptable carrier. A
 virucidal effect can be achieved whether the composition is brought into
 contact with the virus or vice versa, whenever contact occurs with a known
 or potential locus of the virus. Virucidally effective concentrations of
 SDS or related anionic surfactant are generally in the range of about 0.05
 to about 5.0 wt. percent, although a greater or lesser concentration may
 be used depending upon the particular circumstances.
 The compositions of the invention include topical virucidal uses for both
 in vitro and in vivo purposes, especially for intravaginal use. For these
 purposes the SDS or related anionic surfactant can be formulated in any
 appropriate vehicle, provided that the surfactant and the vehicle are
 compatible, that is, that the virucidal activity of the surfactant is not
 diminished by the vehicle. Thus, the compositions can be in the form of
 creams, foams, lotions, ointments, solutions or sprays. The carrier or
 vehicle diluent can be aqueous or non-aqueous, for example alcoholic or
 oleaginous, or a mixture thereof, and may additionally contain other
 surfactants, emollients, lubricants, stabilizers, dyes, perfumes,
 antimicrobial agents either as active ingredients or as preservatives, and
 acids or bases for adjustment of pH. The preferred pH is about 4 to 5.
 Conventional methods are used in preparing the compositions.
 The preferred microbicidal and spermicidal agent for the compositions,
 articles and methods of the present invention is SDS. Preferably, the
 pharmaceutically acceptable carrier or vehicle for topically applied
 compositions is in the form of a liquid, jelly, or foam containing the
 surfactant. The surfactant can be incorporated into: (a) ointments and
 jellies, (b) inserts (suppositories, sponges, and the like), (c) foams,
 and (d) douches. The composition is preferably introduced into the vagina
 of a female, at about the time of, and preferably prior to, sexual
 intercourse, but may also be administered to other mucous membranes. The
 compositions can be employed for the treatment and for protection against
 sexually transmitted diseases. The manner of administration will
 preferably be designed to obtain direct contact of the surfactant
 compositions of the invention with sexually transmitted microbes.
 For topical applications, the pharmaceutically acceptable carrier may
 additionally comprise organic solvents, emulsifiers, geling agents,
 moisturizers, stabilizers, other surfactants, wetting agents,
 preservatives, time release agents, and minor amounts of humectants,
 sequestering agents, dyes, perfumes, and other components commonly
 employed in pharmaceutical compositions for topical administration.
 With regard to the articles provided by the present invention, the
 compositions of the invention may be impregnated into absorptive substrate
 materials, such as sponges, or coated onto the surface of solid substrate
 materials, such as condoms, diaphragms or medical gloves, to deliver the
 compositions to vaginal or other potentially infectable epithelium,
 preferably before or during sexual intercourse. Other articles and
 delivery systems of this type will be readily apparent to those skilled in
 the art. The presently preferred articles are condoms, which are coated by
 spraying SDS onto the surfaces of the condoms, or by impregnating the SDS
 into the condom during manufacture by processes known in the art.
 Preferred coating compositions include silicon which provides lubricity
 and releases the surfactant in a time release manner. Bioadhesive polymers
 may also be used to prolong the time release aspects of the particular
 topical or other medicament employed.
 Solid dosage forms for topical administration include suppositories,
 powders, and granules. In solid dosage forms, the compositions may be
 admixed with at least one inert diluent such as sucrose, lactose, or
 starch, and may additionally comprise lubricating agents, buffering agents
 and other components well known to those skilled in the art.
 Actual dosage levels of the surfactant in the compositions and articles of
 the invention may be varied so as to obtain amounts at the site of
 sexually transmitted fluids to obtain the desired therapeutic or
 prophylactic response for a particular surfactant and method of
 administration. Accordingly, the selected dosage level will depend on the
 nature and site of infection, the desired therapeutic response, the route
 of administration, the desired duration of treatment and other factors.
 Generally, the preferred dosage for SDS will be in the range of about 0.05
 to 2.0 wt. percent. A preferred topical vaginal dosage form is a cream or
 suppository as described above containing from 0.05 to 2.0 wt. percent of
 the composition according to the invention. In each treatment, typically
 twice daily, from about 1 to about 5 ml of such dosage form is applied
 intravaginally, preferably high in the vaginal orifice. Greater amounts
 are generally avoided to minimize leakage.
 SDS is of low intrinsic toxicity both to skin and mucous membranes.
 Preparations, such as shampoos and detergents that contact both skin and
 mucous membranes, contain dodecyl sulfate derivatives (sodium or ammonium
 dodecyl sulfate) in concentrations exceeding 10%. In addition, products
 that are routinely used in the oral cavity, such as toothpaste, have high
 (5-8%) concentrations of these compounds and are not acutely toxic to the
 oral mucosa. In the Examples provided below, virucidally effective
 concentrations of SDS were non-toxic in rabbit skin and in human newborn
 foreskin.
 The methods and compositions of the invention can be used to prevent and
 treat a broad spectrum of infections by pathogenic microbes. As used
 herein, "pathogenic microbes" is intended to include pathogenic bacteria,
 fungi, viruses, yeast, Chlamydia, or protozoans which do not normally
 reside in the host or which are capable of causing host pathology, and
 which are capable of being killed by SDS or related anionic surfactants,
 as described in detail herein.
 The preferred pathogenic microbes for target by the compositions and
 methods of the invention are papillomaviruses (PVs), which represent a
 group of non-enveloped, icosahedral DNA viruses. The PVs induce benign
 neoplasms that can progress to cancers. Animal papillomas occur in a large
 number of species; certain viruses, such as the bovine papillomaviruses
 (BPVs) and the cottontail rabbit papillomavirus (CRPV), represent
 well-studied model systems. HPVs cause warts in epithelial target tissues.
 Verrucae vulgaris, plantar warts and genital condylomata all represent
 common clinical infections in humans. The compositions and methods of the
 invention have utility in preventing or controlling these human
 infections, and also preventing and controlling genital lesions containing
 HPV which can progress to malignancy, if left untreated.
 Because cervical cancer is the number one cause of cancer related mortality
 in women in developing countries, effective prevention of HPV transmission
 should have significant impact on world health. Accordingly, the preferred
 method of the invention comprises contacting the virucidal compositions of
 the invention with HPVs transmitted to the vagina or other mucous
 membranes during sexual activity. The preferred mode of contact is by use
 of a condom coated or impregnated with, or by the use of a topical
 pharmaceutical composition containing, SDS in sufficient quantity to
 control or prevent HPV transmission and infection. The spermicidal
 activity of the active ingredients of the condoms and other articles and
 compositions of the invention provides an added benefit where the
 prevention of pregnancy is desired.
 In addition, the SDS and related anionic surfactant compositions and
 methods of the invention may be broadly utilized as a disinfectant for
 effective inactivation of non-enveloped and enveloped, animal and human
 viruses on surfaces such as floors, medical instruments, bathroom
 surfaces, and gym equipment. The disinfectant composition containing SDS
 or related anionic surfactant is preferably incorporated into a spray-type
 dispenser whereby it can be sprayed directly onto the surface to be
 treated. An example of such use would be for a person to spray the
 composition on surfaces in public restrooms or gym equipment in order to
 kill any pathogenic microbes present from other persons who have used the
 facilities. The disinfection composition preferably contains SDS in
 solution or suspension with a diluent such as phosphate buffered saline at
 about 0.05 to 1.0 wt. % SDS.
 The experimental results which demonstrate the effectiveness of the claimed
 compositions, articles and methods are set forth in the Examples below.
 The examples described and discussed herein are intended to be
 illustrative of the present invention but not limiting. Numerous
 variations and modifications may be effected without departing from the
 scope of the novel concepts of the present invention.
 EXAMPLE 1
 Antiviral Activity of SDS
 Materials and Methods
 Chemicals
 SDS was purchased from Bio-Rad (Richmond, Calif.) and filtered sterile
 solutions were made in phosphate buffered saline (PBS). N-9 was obtained
 from Rhone-Poulenec Rorer Pharmaceuticals Inc. (Collegeville, Pa.). All
 additional detergents were purchased from Boehringer Manheim
 (Indianapolis, Ind.). The following reagent was obtained through the AIDS
 Research and Reference Reagent Program, Division of AIDS, NIAID, NIH:
 HeLa-CD4-LTR-.beta.-gal from Dr. Michael Emerman.
 HSV-2 Inactivation Assay. HSV-2 (strain 333) virus stocks were propagated
 by low multiplicity infection of African Green monkey kidney (CV-1) cells
 and subsequent preparation of cell-free supernatants from frozen and
 thawed preparations of lytically infected cultures. Virus titers were
 determined by assay in CV-1 cell monolayers. Virus stocks were maintained
 in CV-1 cell culture medium which was Dulbecco's medium supplemented with
 antibiotics and 10% fetal calf serum. The protein concentration of the
 virus stocks was also increased by serum proteins and by cellular proteins
 released by the freezing and thawing of the infected cells.
 For inactivation of HSV-2, 39 .mu.l of virus was mixed with 1 .mu.l of a
 40.times. concentrated solution of detergent and then incubated at
 37.degree. C. for 10 min. After inactivation, the 40 .mu.l of virus sample
 was diluted to 4 ml (1:100) using cell culture medium, and 1 ml of the
 diluted virus was adsorbed onto CV-1 monolayers for 1 hr at 37.degree. C.
 Following adsorption, monolayers were refed and incubated at 37.degree.
 C., 5% CO.sub.2. Between 20 and 24 hr post infection, monolayers were
 fixed, stained with crystal violet and plaques counted using a dissecting
 microscope. The numbers in Table 1 each represent an average of 2 plates.
 HIV-1 Inactivation Assay. One day prior to the assay,
 HeLa-CD4-LTR-.beta.-gal cells were seeded into 12-well culture dishes at a
 concentration of 8.times.10.sup.4 cells per well. A high titer
 (10.sup.7.17 TCID.sub.50 /ml) virus stock of HIV-1 (strain IIIB; Advanced
 Biotechnologies, Inc., Columbia, Md.) was diluted 1:10 with RPMI 1640
 supplemented with 10% FBS. To assess viral inactivation by SDS, 78 .mu.l
 of diluted virus were mixed with 2 .mu.l of detergent solution, and
 incubated for 10 min at 37.degree. C. After the inactivation period, the
 virus and detergent were diluted with 720 .mu.l R10 (1:10) supplemented
 with DEAE dextran (20 .mu.g/ml final concentration). Aliquots of treated
 virus (300 .mu.l ) were then added to duplicate wells of HeLa cells and
 incubated at 37.degree. C. for 2 hr. Following viral adsorption, 2 ml of
 fresh media (DMEM supplemented with 10% FBS, 0.1 mg/ml G418, and 0.05
 mg/ml hygromycin B) were added to each well After incubation at 37.degree.
 C. and 5% CO.sub.2 for 48 hr post-infection, cells were fixed and stained
 for .beta.-galactosidase expression.
 BPV-1 Focus Assay. Cell-free stocks of BPV-1 were prepared by extraction
 (10% w/v) of epidermal bovine warts in phosphate buffered saline (PBS). In
 order to detect the transforming ability of BPV-1, C127 mouse cells were
 seeded (3.times.10.sup.5 cells per flask) into T-25.sup.2 flasks. After 24
 hr of growth, subconfluent cells were infected with BPV-1. For the
 positive controls, stock virus (20 .mu.l) was diluted (1:1) with PBS,
 incubated at 37.degree. C. for 10 min, diluted 1:10000 and then added (100
 .mu.l) into the 5 ml of cell culture medium present on the cells. Cells
 were refed at 24 hours and subsequently 2 times weekly. The presence of
 morphologically transformed foci was counted after 2 weeks and then again
 at 3 weeks.
 Virus inactivations were carried out in vitro by addition of concentrated
 SDS solutions to the virus stocks (20 .mu.l of virus plus 20 .mu.l of
 detergent) and subsequent incubation at 37.degree. C. for 10 or 30 min as
 indicated. Following inactivation, virus was diluted 1000 fold to lower
 the detergent concentration and the preparations were immediately used for
 infection as above.
 Shope Papilloma Induction. Stocks of Shope CRPV were prepared from
 papillomas generated in wild cottontail rabbits. Virus stocks were cell
 free extracts (10% w/v) of papillomas in PBS. Shaved dorsal skin was
 lightly scarified with a razor blade. Virus stocks were used to inoculate
 domestic cottontail rabbits (Hazelton Research Products, Denver, Pa.); a
 40 .mu.l aliquot of virus was dropped onto the surface of 4 locations on
 the dorsal skin. The 2 left sites on each rabbit received untreated virus
 and the 2 right sites received treated virus. Inactivation of either a
 10.sup.-1 or 10.sup.-2 solution of virus stock was accomplished by
 addition of concentrated SDS solutions which were 40.times. the final
 indicated concentrations. Incubation of SDS and virus was at 37.degree. C.
 for 10 min and virus was immediately used for inoculation of rabbits.
 Virus was not subsequently diluted following inactivation and the
 concentration of SDS present during inactivation and inoculation was
 0.05%. Papillomas were first observed to develop in control sites around 2
 weeks after inoculation. The geometric mean diameter (GMD) of all visible
 lesions was measured and is equal to the cube root of the
 length.times.width.times.height of the lesions as measured in mm by
 calipers.
 Human Papilloma Induction. Stocks of experimentally generated infectious
 HPV 11 were prepared and represented 10% w/v cell free extracts of virus
 in PBS. Undiluted aliquots of virus stocks (39 .mu.l) were mixed with a
 40.times. solution of SDS 1 .mu.l), incubated at 37.degree. C. for 10 min
 and immediately used to infect split thickness grafts of newborn human
 foreskin epithelium. Virus was not subsequently diluted. Control grafts
 were infected with untreated virus stock. Virus adsorption was for 1 hr at
 37.degree. C. The concentration of SDS present during the inactivation
 period and during virus adsorption was 0.05%. Grafts were then
 transplanted beneath the renal capsule of athymic mice. Animals were
 maintained in isolator bubbles with antibiotic supplemented drinking water
 in the animal colony of the Hershey Medical Center. Three months following
 infection, animals were sacrificed, their kidneys were removed, and the
 xenografts were grossly examined. The remaining organs were examined for
 any apparent abnormalities and none were found. Portions of each graft
 were immediately fixed in 10% neutral-buffered formalin and processed by
 standard histologic techniques for staining with hematoxylin and eosin.
 A second set of control grafts was exposed only to identical concentrations
 of SDS and no virus. These grafts were harvested on days 1, 5, 11 and 20
 following transplantation in order to follow the viability and growth of
 the grafts after SDS exposure.
 Results
 Inactivation of the Infectivity of HSV-2 by SDS. In five separate
 experiments, treatment concentrations of SDS as low as 0.0125% to 0.025%
 were effective in eliminating the ability of the virus to induce plaques
 in a monolayer of monkey kidney cells (Table 1). Total HSV-2 inactivation
 was achieved with SDS concentrations between 0.0025% and 0.0125%. These
 effective concentrations are similar to the concentrations of N-9 needed
 for destruction of HSV infectivity (data not shown).
 TABLE 1
 *% SDS during Plaques / Plate
 treatment **% final SDS (5 experiments)
 0 0 57/73/343/145/145
 1 .times. 10.sup.-1 1 .times. 10.sup.-3 0/0/0/0/0
 5 .times. 10.sup.-2 5 .times. 10.sup.-4 0/0/0/0/0
 2.5 .times. 10.sup.-2 2.5 .times. 10.sup.-4 0/0/0/0/0
 1.25 .times. 10.sup.-2 1.25 .times. 10.sup.-4 0/0/0/0/0
 2.5 .times. 10.sup.-3 2.5 .times. 10.sup.-5 28/54/322/145/104
 *Sterile SDS sticks if 40X of the treatment concentration were added to
 virus aliquots to achieve the treatment concentration. After mixing,
 samples were incubated at 37.degree. C. for 10 minutes.
 **Following SDS treatment, virus stocks were diluted 100 fold and 1 ml
 aliquots were immediately adsorbed onto CV-1 cells. Plaques were counted
 after 20-24 hours of infection. Each number represents an average of 2
 plates.
 Inactivation of the infectivity of HIV-1 by SDS and the non-ionic detergent
 C31G. It is established that N-9 can inactivate HIV-1. We compared in
 activation of HIV-1 by a second non-ionic detergent, C31G, and by SDS.
 High titer virus stocks of HIV-1 were incubated with either C31G or SDS
 and then virus was assayed on indicator cells expressing .beta.-gal under
 the control of the HIV-1 LTR. After 48 hours, cells were stained and the
 number of cells expressing increased .beta.-gal counted. Both of these
 detergents were highly effective in the inactivation of HIV-1 (Table 2).
 Total inactivation of HIV-1 was achieved with C31G concentrations as low
 as 0.0125% and with SDS concentrations as low as 0.025%.
 TABLE 2
 *% cells expressing
 LTR-.beta. gal gene
 (duplicate wells) cells counted
 % C31G during
 treatment
 5 .times. 10.sup.-2 (toxic) 0,0 &gt;10.sup.6
 2.5 .times. 10.sup.-2 0,0 &gt;10.sup.6
 1.25 .times. 10.sup.-2 0,0 &gt;10.sup.6
 6.25 .times. 10.sup.-3 19 +/-6.1, 19 +/-6.4 1080,805
 2.5 .times. 10.sup.-3 22 +/-7.4, 29 +/-8.1 1620, 1820
 % SDS
 during treatment
 5 .times. 10.sup.-2 0,0 &gt;10.sup.6
 2.5 .times. 10.sup.-2 0,0 &gt;10.sup.6
 1.25 .times. 10.sup.-2 24 +/-3.3, 24 +/-10 2810,2190
 6.25 .times. 10.sup.-3 10 +/-1.7, 15 +/-2.1 2390,2290
 2.5 .times. 10.sup.-3 9 +/-5.5, 11 +/-3.5 1940,1910
 Mock Infected Cells 0,0 &gt;10.sup.6
 HIV-1 Infected 17+/-4.8, 24 +/-5.4 2680, 1480
 Cells
 * Five random fields of cells were counted in each plate displaying blue
 cells.
 *Duplicate plates were assayed for each sample; individual numbers are the
 standard deviation within 5 fields of one plate.
 Destruction of the Ability of BPV-1 to Induce Morphologically Transformed
 Foci in Monolayers of C127 Mouse Cells. Although SDS could effectively
 reduce HSV-2 infectivity, it remained possible that this destruction was
 mediated by envelope removal. Because papillomaviruses are non-enveloped,
 the possibility remained that SDS would fail to inactivate these viruses.
 We utilized BPV-1 as a prototype PV because of its ability to rapidly
 (within 2 weeks) form multi-layered transformed foci in mouse fibroblasts
 in an in vitro assay. Table 3 describes results of two separate
 experiments in which stocks of BPV-1 were incubated at 37.degree. C. with
 various concentrations of SDS (5% to 5.times.10.sup.-4 %) for either 10 or
 30 minutes, diluted to lower the SDS concentration (to avoid cell
 toxicity) and then used to infect C127 cells. Following incubation of
 control or infected cultures, foci were counted at 14 and 17 days after
 infection. Results indicate that SDS in concentrations as low as 0.05% or
 0.005% can totally inactivate BPV-1 transforming ability after treatment
 of the virus at 37.degree. C. for 10 or 30 minutes, respectively.
 Inactivation of BPV-1 by the lower concentration of 0.005% after 30
 minutes indicated that inactivation is proportional to time as well as to
 detergent concentration. Table 4 lists several other commercially
 available detergents that were tested for possible inactivation of BPV-1.
 None of these detergents inactivated the morphologic transforming
 properties of the BPV-1.
 TABLE 3
 *% SDS foci/plate foci/plate
 during **% final Exp. 1 Day Exp. 2 Day
 treatment SDS 12 14 Day 17
 0 0 266 255 153
 5 5 .times. 10.sup.-3 0 N.D. N.D.
 5 .times. 10.sup.-1 5 .times. 10.sup.-4 0 N.D.
 N.D.
 5 .times. 10.sup.-2 5 .times. 10.sup.-5 0 0 0
 5 .times. 10.sup.-3 5 .times. 10.sup.-6 0 271
 150
 2.5 .times. 10.sup.-3 2.5 .times. 10.sup.-6 N.D. 273
 162
 5 .times. 10.sup.-4 5 .times. 10.sup.-7 N.D. 229
 151
 *Sterile SDS stocks of 40x the treatment concentration were added to virus
 aliquots to achieve the treatment concentration.
 **Following virus treatment, treated virus stocks were further diluted
 1:1000 in order to dilute the detergent
 In Experiment 1, virus and SDS were mixed and incubated at 37.degree. C.
 for 30 minutes.
 In Experiment 2, virus and SDS were mixed and incubated at 37.degree. C.
 for 10 minutes.
 N.D.=not done
 In control plates, without BPV-1, no foci appeared.
 TABLE 4
 Detergents that failed to inactivate BPV-1
 morphologic transformation of C127 cells
 Nonoxynol-9
 C31G
 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-
 propane sulfonate (CHAPSO)
 N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate
 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane
 sulfonate (CHAPS)
 isotridecylpoly (ethylene-glycolether).sub.n
 octanoyl-N-methyl-glucamide (MEGA-8)
 Triton X-100
 Thesit
 All detergents except C31G and N-9 were purchased from Boehringer-Mannheim,
 N-9 was purchased from Rhone-Poulenec Rorer Pharmaceuticals, C31G was
 provided by Biosyn, Inc.
 None of the above reduced foci.
 Positive control SDS @ 1% completely eliminated virus foci.
 All detergents were incubated with virus; 1% final concentration,
 37.degree. C., 10 min.
 Effect of SDS Inactivation of CRPV on Formation of Shope Papillomas in
 Rabbits. To extend the observation of PV inactivation by SDS to an in vivo
 animal model system, we utilized the well established CRPV model system. A
 standard CRPV stock known to form papillomas with 100% efficiency was
 used. The infectious dose 50 (ID.sub.50) for the virus stock corresponds
 to 50 .mu.l of a 10.sup.-3 dilution of the stock virus. In our
 experiments, 40 .mu.l of a 10.sup.-1 dilution and subsequently 40 .mu.l of
 a 10.sup.-2 dilution of the virus stock solution were used. Both of these
 concentrations far exceeded the ID.sub.50. SDS was mixed with virus to a
 final concentration of 0.05% and subsequently incubated at 37.degree. C.
 for 10 min. Immediately following incubation, virus was inoculated by skin
 scarification of the backs of the rabbits. Inoculated sites contained two
 untreated (left; L) and two treated (right; R) virus samples on the same
 rabbit. FIG. 1a demonstrates the average GMD of six lesions inoculated
 with normal (10.sup.-1 dilution) and six lesions inoculated with
 SDS-treated CRPV. GMDs were measured and compared on post inoculation days
 18, 21, 25, 32, 42 and 50. Results indicate that a 10.sup.-1 dilution of
 virus stock was substantially inactivated by a 10 min, 0.05% SDS treatment
 at 37.degree. C. FIG. 1b shows the growth curves over 50 days following
 inoculation for each individual lesion. It should be noted that each of
 the six sites that received SDS treated preparations (.circle-solid.) were
 delayed in the development of papillomas, indicating a substantial
 inactivation of virus. Once papillomas developed, however, the growth rate
 of the lesions appeared similar to the ones that developed from the
 untreated inoculum.
 In a subsequent experiment (FIGS. 2a and 2b), a 10.sup.-2 dilution of CRPV
 virus stock was also incubated at 37.degree. C. for 10 min with either
 0.05% SDS or 0.05% N-9. This dilution of the stock virus not only
 contained less virus but also a lower total protein concentration.
 Following incubation, detergent treated and control virus samples were
 inoculated into five rabbits for the N-9 samples and five rabbits for the
 SDS treated samples. Untreated virus samples were also inoculated onto the
 same rabbits at different sites. This experiment was undertaken for two
 purposes: to observe the inactivation of a smaller amount of CRPV by SDS
 and to directly compare inactivation with SDS to that achieved by the N-9
 treatment. As in the previous experiment, the left inoculation sites (two
 per animal) received untreated virus and the right inoculation sites (two
 per animal) received treated virus. FIG. 2a shows the GMD of ten
 inoculation sites that received SDS treated virus compared to ten
 inoculation sites that received normal virus. GMDs were measured 3, 4, 5,
 and 6 weeks after virus inoculation. In 8 of 10 sites inoculated with SDS
 treated virus, papillomas failed to develop; the remaining 2 sites
 developed very small papillomas beginning to appear 4 weeks after
 inoculation. Although quantitative measurements were not performed, the
 SDS inoculated sites did not exhibit any irritation during the experiment.
 In the 10 sites inoculated with normal CRPV, papillomas developed in 10 of
 10 sites within 2 weeks after inoculation and grew progressively.
 FIG. 2b demonstrates comparative growth of papillomas in 10 sites that
 received normal CRPV compared to 10 sites that received CRPV treated with
 N-9. The GMD of each papilloma was measured 3, 4, 5 and 6 weeks after
 virus inoculation. There was not a difference in the growth of the lesions
 arising after inoculation with these two virus preparations. In addition,
 growth rates of control and experimental papillomas in the N-9 animals did
 not differ from growth rates of control lesions in the SDS treated animals
 (data not shown).
 Effect of SDS Inactivation on the Ability of HPV 11 to Induce Experimental
 Condylomata in Human Foreskin Epithelial Xenografts. Standard stocks of
 HPV 11 were used as undiluted virus. These virus stocks normally induce
 Condylomata in 90-100% of infected xenografts when diluted 1000 fold. In
 this experiment, 39 .mu.l of undiluted HPV 11 stock was mixed with 1 .mu.l
 of SDS to a final concentration of 0.05% SDS and then incubated at
 37.degree. C. for 10 min. Infection was then carried out for 1 hr and the
 grafts subsequently transplanted in vivo. Eight animals (16 kidneys)
 received grafts infected with SDS treated virus and 9 animals (17 kidneys)
 received normal virus. Table 5 shows the results of the harvested grafts.
 In the normal infections, 17 of 17 grafts survived and of these, 14 were
 transformed morphologically upon histologic examination and had typical
 papillomatous appearance. In animals receiving SDS treated virus, 13 out
 of 16 xenografts showed viable tissue at the time of harvest and
 histologic examination of the grafts revealed normal, viable
 differentiating human epithelium. The latter results are compatible with
 our previous observations using uninfected grafts in that normal grafts
 are occasionally resorbed in the mice and do not survive 3 months. We
 concluded that the SDS had effectively prevented virus infection by
 inactivation of the virus.
 TABLE 5
 surviving
 grafts/
 *% SDS during total transplanted
 treatment % final SDS papillomas grafts
 0 0 14 17/17
 0.025 0.025 0 13/16
 *Sterile SDS stocks of 40X the treatment concentration were added to virus
 aliquots to achieve the treatment conaentration.
 Effect of SDS Exposure on the Viability of Human Foreskin Xenografts.
 Because of concern about the potential for SDS to kill human epithelium,
 control experiments were performed in which split thickness grafts of
 neonatal foreskin were exposed to 0.05% SDS alone and then subsequently
 grafted. All conditions in this experiment were identical to those used in
 the HPV 11 infections with treated virus, except that virus was not
 present. SDS-exposed grafts (2 animals at each time) were harvested, fixed
 and sectioned immediately after exposure, and on days 1, 5, 11 and 20
 after treatment. Examination of the tissues demonstrated fully viable
 epithelium on all days and no apparent necrosis associated with detergent
 exposure. The original split thickness grafts were approximately 1
 mm.times.1 mm.times.1 mm in size; in addition they were punctured many
 times with the tip of a needle in order to allow entrance of the HPV 11
 and/or the SDS into the epithelial layers. Although it is possible that
 some epithelial cells may have been damaged or killed during SDS exposure,
 damage was minimal and epithelial growth in the grafts was normal.
 EXAMPLE 2
 Microbicidal Activity of Alkyl Sulfate Derivatives
 The following data show that other members of the alkyl sulfate group,
 namely lithium dodecyl sulfate, lauric acid and the sodium salt of lauric
 acid, have anti-papillomavirus activity in the C127 focus assay using
 bovine papillomavirus. In dose response curves, SDS remains the most
 potent.
 TABLE 6
 COMISON OF SODIUM DODECYL
 SULFATE AND LITHIUM DODECYL SULFATE
 IN THE BPV-1 FOCUS ASSAY
 Treatment # of FOCI
 Negative Control 0,0,0,0
 Positive Control 30,29,32,26
 0.1% SDS Alone 0, 0, 0, 0
 0.1% LDS Alone 0, 0, 0, 0
 0.1% SDS + Virus 0,0,0,0
 0.1% LDS + Virus 7,11,9,10
 In all cases, treatment was for 10 min at 37.degree. C., followed by a
 1:1000 dilution of the virus preparation.
 TABLE 7
 COMISON OF SODIUM DODECYL
 SULFATE WITH LAURIC ACID, THE SODIUM
 SALT OF LAURIC ACID, IN
 THE BPV-1 FOCUS ASSAY
 Treatment # of FOCI
 Negative Control 0,0,0
 Positive Control 50+,50+,36
 0.1% SDS + Virus 0,0,0
 0.1% Lauric Acid + Virus 9,28
 0.1% NA + Lauric Acid + Virus 0,0,0
 In all cases, treatment was for 10 min at 37.degree. C., followed by a
 1:1000 dilution of the virus preparation.
 EXAMPLE 3
 SDS Toxicity and Anti-HSV-2 Activity
 The following data represent an in vivo experiment to test both the
 toxicity and the efficacy of SDS in the protection of mice from vaginal
 infection with live herpes simplex virus (HSV-2).
 GROUP 1 Normal Control
 GROUP 2 Live HSV-2 (Approximately 5.times.10.sup.6 Infectious Units)
 GROUP 3 Live HSV-2 Plus 4% SDS
 GROUP 4 Live HSV-2 Plus 2% SDS
 GROUP 5 Live HSV-2 Plus 1% SDS
 GROUP 6 Live HSV-2 Plus 0.5% SDS
 The experiment used outbred, female, Swiss-Webster mice. Mice were
 anesthetized and then SDS or control solutions (25 .mu.l) was instilled
 into the vagina using a yellow pipette tip. The SDS was not formulated
 into a vaginal cream or foam but merely dissolved into phosphate buffered
 saline. These solutions have low viscosity. Fluids were instilled into
 groups of 10 mice at one time. Following administration of SDS or control
 solutions to the group of 10, then an additional 25.mu.l of virus or
 control fluid was instilled. Mice were allowed to recover from the
 anesthesia and then the mice were checked daily for symptoms, beginning on
 day three and until 12 days after inoculation. Vaginal swabs were also
 performed on the mice on a daily basis, beginning on day three, in order
 to determine shedding of virus. FIGS. 3A-3G show the total symptoms per
 group on days 3 through 12 for each of the following symptoms: swelling,
 vaginal exudate, redness, death, leg paralysis, perianal hair loss and any
 symptom.
 The results clearly show that all concentrations of SDS provided
 significant protection from HSV-2 inoculation of the vagina. In addition a
 dose response was evident for every symptom checked; with 4% SDS providing
 the most protection. The results of determinations of virus shedding are
 not shown but confirm and support these data.
 EXAMPLE 4
 Spermicidal Activity
 The following data represent an in vitro experiment to test the efficacy of
 SDS and other detergents as spermicidal agents. Frozen samples of bull
 semen were obtained, thawed and placed in a test tube. Aliquots were taken
 out and put in a separate test tube where they were mixed with detergent
 (SDS, C31G, N-9 or a mixture of SDS and N-9) to a final percent as listed
 in Tables 8 and 9 below. After mixing, the samples were immediately placed
 on a microscope slide and visually examined for sperm movement The
 experiment was conducted one sample at a time so that the visual
 examination was conducted immediately after addition of detergent to the
 sample. As a result, indications in the table of complete inactivation
 indicate virtually instantaneous inactivation of the sperm. Delayed
 inactivation indicates delay of approximately 10 minutes for those sperm
 cells that did not immediately stop swimming. Occasional swimmers means
 very few, on the order of approximately 1% of the population of sperm
 present in the sample, indicating approximately 99% of the sperm were
 inactivated by the detergent.
 TABLE 8
 Bovine Sperm Motility Following Detergent Addition
 N-9* C31G** SDS*** N-9/SDS
 2% -- -- --
 1% -- -- --
 0.5% -- -- --
 0.025% -- -- --
 0.0125% +++ +/- +/-
 (delayed) (delayed)
 *No Coagulation
 **Major Coagulation
 ***Moderate Coagulation
 When coaguation was present, amount decreased with decreasing
 concentration.
 Key
 +++ Vigorous swimming
 ++/- Many swimmers/some dead
 +/- Occasional swimmer/most dead
 - All dead
 TABLE 9
 Bovine Sperm Motility Following Detergent Addition
 SDS/ SDS/ N-9/
 N-9 C31G SDS N-9 C31G C31G
 2% -- -- +/- +/- +/- +/-
 1% ++/- +/- ++/- ++/- ++/- ++/-
 0.5% +++ +++ ++/- +++ +++
 0.25% +++ +++ ++/- +++ +++
 0.125% +++ +++ ++/- +++ +++
 Key
 +++/- Vigorous swimming
 ++/- Many swimmers/some dead
 +/- Occasional swimmer/most dead
 - All dead
 The invention having now been fully described, it will be apparent to those
 skilled in the art that many variations and modifications can be made
 thereto without departing from the spirit or scope of the pending claims.