AU3903800A - Gel-microemulsion formulations - Google Patents

Description

WO 00/56366 PCT/US00/07419 1 Gel-Microemulsion Formulations Field of the Invention The present invention relates to a pharmaceutical formulation, and more particularly to a gel-microemulsion formulation. In some embodiments. the gel 5 microemulsion formulation has spermicidal activity and can be used as a contraceptive. In other embodiments, the gel-microemulsion can act as a formulation base for other theraputic agents, such as anti-microbial agents to provide an anti-microbial formulation or spermicidal agents to enhance the spermicidal effectiveness of the formulation. In yet other embodiments, the gel 10 microemulsion, having spermicidal activity, can act as a formulation base for the anti-microbial agents to provide a dual function contraceptive/anti-microbial formulation. Background of the Invention At present, all commercially available spermicidal contraceptives have 15 detergent ingredients that disrupt cell membranes. These include the neutral surfactants isononyl-phenyl-polyoxyethylene (9) ether ornonoxynol-9 (N-9), p menthanyl-phenyl-polyoxyethylene (8,8) ether ormenfegol, and isooctyl-phenyl polyoxyethylene (9) ether or octoxynol- 9 (0-9) (Digenis GA, et al., Pharm Dev Technol, 1999;4:421-30; Furuse K, et al., JPharmacobiodyn, 1983;6:359-72.) The 20 detergent-type vaginal spermicide, N-9, available without a prescription, is the most commonly used spermicidal contraceptive in the UK and USA (OTC Panel, Federal Register, 1980;45:82014-49; Chantler E., Brit Fam Plann, 1992:17:118-9.) Worldwide, the cationic surfactant benzalkonium chloride and the anionic detergent sodium docusate (dioctyl sodium sulphosuccinate) are also used as vaginal 25 spermicides (Mendez F, et al., Contraception, 1986;34:353-62.) N-9. sodium oxychlorosene, and benzalkonium chloride, have been used as gels, suppositories, ovules, sponges, or film. N-9 has been in use for more than 30 years in creams, gels, foams and condom lubricants. However, in several large studies for users of N-9. the average 6-month pregnancy rate is 26%. and the first-year pregnancy rates 30 range from 11 to 31%. Thus. N-9 is approximately 75% effective in preventing pregnancy (Trussell J. et al. Stud Fai Plann. 1987;18:237-83: Kulig JW. Ped Clinic North Am, 1989;36:717-30; Raymond E, et al.. Obstet Gynecol, 1999;93:896-903). The spermicidal activities of these surfactants are associated with their structural affinity to the membrane lipids (Schill WB. et al.. Andrologia, 35 1981:13:42-9: Helenious A. et al.. Biocheim Biophys Acta. 1975:415:29-79). STJST!TTJTP VWPPT 1Prr r PA WO 00/56366 PCT/US00/07419 2 Therefore, the major drawback of using N-9 or other currently used surfactants is their detergent-type effect on epithelial cells and normal vaginal flora. N-9 displays antiviral and spermicidal activities only at cytotoxic doses (D'Cruz OJ, et al., Mol Hum Reprod, 1999;5:421-32; D'Cruz OJ, et al., Biol Reprod, 2000;62:37-44). 5 Frequent use of N-9 as a vaginal contraceptive/microbicide has been associated with an increased risk of vaginal or cervical infection, irritation, or ulceration (Niruthisard SR, et al., Sex Transm Dis, 1991;18:176-79; Rekart ML, Defic Syndr, 1992;5:425 27; Roddy RE, et al., IntJSTD & HIV 1993;4:165-70; Weir SS, et al., Genitourin Med, 1995;71:78-81). Detergent-type spermicides alter vaginal bacteria or flora, 10 and lead to an increased risk of opportunistic infections (Hooten TM, et al., JAMA, 1991;265:64-9.; Stafford MK, JAcquir Immune Defic Syndr Hum Retrovirol, 1998; 17:327-31.; Rosenstein IJ, et al. J Infect Dis, 1998;177:1386-90.; Patton DL, et al., Sex Trans Dis, 1996;23:489-93.) Such opportunistic infections are known to enhance the susceptibility of the ectocervical epithelium and the endocervical 15 mucosa to HIV-1 infection (Augenbraun MH, et al., Infect Dis Clin North Am, 1994; 8:439-48.) Chemical irritation that disrupts the vaginal mucosa may actually enhance the risk of vaginal transmission of sexually transmitted diseases (STDs) including HIV-1, by mucosal erosion and local inflammation (Weir SS, et al., Genitourin Med, 1995;71:78-81.;. Kreiss J, JAMA, 1992;268:477-82.). In a 20 study conducted among commercial sex workers in Nairobi, in which some of the women used N-9 containing sponges, a significantly higher rate of genital ulceration and HIV-1 seroconversion was found compared with those not using N-9 (Kreiss J, JAMA, 1992;268:477-82.). Furthermore, recent clinical trials have shown that vaginal contraceptive 25 preparations containing N-9 have no effect on the transmission of HIV/AIDS and other STDs when provided as part of an overall program to prevent heterosexual transmission of HIV/AIDS (Hira SK, et al, Int JSTD AIDS, 1997;8:243-50.; Roddy RE, et al., NEngl J Med, 1998;339:504-10.) Since heterosexual transmission of HIV-1 is the predominant mode of the epidemic spread of HIV, new, effective, and 30 safe vaginal spermicides lacking detergent-type membrane toxicity may offer significant clinical advantage over the currently available detergent-type spermicides. Because vaginal spermicides would likely be used repeatedly over decades, an ideal spermicide should have an established safety record and lack genital 35 epithelial toxicity. Moreover, it should be inexpensive and be produced from WO 00/56366 PCT/US00/07419 3 commonly available resources and should have a broad specificity for solubilizing drugs effective for prevention of sexual transmission of several STDs including HIV-1. Therefore, there is a continuing need for new and better spermicidal 5 formulations. Summary of the Invention The inventors have developed novel gel-microemulsion formulations for use as spermicides that in numerous respects overcomes many of the problems of the 10 commercially available detergent-type spermicides. Embodiments of the novel gel microemulsion spermicide formulations have been show to be very effective as contraceptive agents with reduced levels of toxicity to subjects, and are also useful as formulation bases for anti-microbial agents. Some novel pharmaceutical formulations embodying the gel-microemulsion 15 of the invention contain common pharmaceutical excipients as the active ingredients, and provide for safe in vitro and in vivo spermicidal activity. In some embodiments, it is contemplated that drug solubilizing agents, such as Cremophor EL® and Phospholipon 90G®, may be active ingredients since these agents were spermicidal against highly motile fraction of sperm. Although, the individual 20 components of some such gel-microemulsion formulations alone lacked spermicidal activity in semen, the combined components in such gel-microemulsion formulations containing the pharmacological excipients rapidly inactivated sperm in human semen. The lack of cytotoxicity of individual components of such formulations in human semen and their synergestic spermicidal property in the gel 25 microemulsion formulation shows unique clinical potential to formulate them as the active ingredients for a novel and effective contraceptive, such as a vaginal contraceptive for example. In testing, some embodiments of gel-microemulsion formulations of the invention were significantly more effective as a contraceptive than a commercially-available N-9 gel formulation. 30 Embodiments of the gel-microemulsion of the invention can also be used as an effective formulation base for other agents, for example anti-microbial agents, that can be incorporated into the formulation. Embodiments incorporating anti microbial agents are particularly useful to prevent the transmission of diseases. Additionally, such embodiments are especially useful as a dual function 35 spermicide/anti-microbial formulation, and can be especially useful in inhibiting the OF T"D(''r'VrTT'r," ~ ,, WO 00/56366 PCT/US00/07419 4 transmission of sexually transmitted diseases, for example AIDS, genital herpes, gonorrhea and chlamydia. In yet other embodiments, additional spermicidal agents can be incorporated into the formulation to increase the effectiveness of the gel microemulsion as a spermicide. 5 One aspect of the invention is directed to a pharmaceutical composition adapted for use as a spermicide, the composition comprising a gel-microemulsion comprising an oil-in-water microemulsion and a polymeric hydrogel. Another aspect is a method of using such a composition as a spermicide. Another aspect of the invention is directed to a gel-microemulsion 10 pharmaceutical composition adapted for use as a formulation base for additional theraputic agents. Examples of additional agents include, anti-microbial agents and spermicidal agents. Another aspect is a method of using such a composition as a formulation base for additional theraputic agents. Another aspect is the use of the combined gel-microemulsion formulation base with additional theraputic agents for 15 appropriate theraputic treatment. Another aspect of the invention is directed to a gel-microemulsion pharmaceutical composition that is adapted for use as both a spermicide and formulation base for anti-microbial agents to provide a dual function contraceptive/anti-microbial formulation. Another aspect is a method of using such 20 a composition as a dual function contraceptive/anti-microbial formulation. Brief Description of the Drawings Figure 1 is a ternary phase diagram of one embodiment of a microemulsion system. The non-grid area represents the single phase microemulsion region. The 25 asterisk represents the microemulsion which was used for GM-4 formulation listed in Table 5. Figure 2 shows the effect of individual components of GM-4 on the motility of washed and enriched human sperm. Highly motile fractions of sperm were incubated with increasing concentrations of listed compounds in the assay medium, 30 and the percentage of motile sperm was evaluated by CASA. The plots show mean values of two representative measurements. Cremophor EL and phospholipon 90G were spermicidal at all concentration tested. Captex 300, PEG-200, propylene glycol, seaspan carrageenan, viscarin carrageenan, and sodium benzoate demonstrated little or no inhibition over the range of concentrations tested. CrTTPCrTTTrn crrr-7,' -- - ,, WO 00/56366 PCT/US00/07419 5 Figure 3 shows the effect of individual components of GM-4 on the motility of human sperm in semen. Aliquots of liquefied semen were mixed with an equal volume of assay medium containing the final concentrations of components of GM 4 formulation. At timed intervals, sperm motility was evaluated. The plot shows 5 mean values from two representative experiments. All the components of GM-4 formulation demonstrated little or no inhibition of sperm motility in human semen over the entire range of time course tested. Figure 4 is are light microscopic images of GM-4 and N-9 (4%)-treated rabbit vaginal sections. Representative hematoxylin- and eosin-stained, paraffin 10 embedded sections of the mid vaginal region of a rabbit treated intravaginally with GM-4 formulation (Left panels, A and C) or 4% N-9 in GM-4 (Right panels, B and D) for 10 consecutive days (x200). Higher magnification (x400) shows the intactness of vaginal epithelium (VE) in a GM-4-treated rabbit (C) versus an N-9 treated rabbit (D) which shows epithelial cell layer disruption (arrows) and 15 leukcocyte influx characteristic of inflammation. Figure 5 shows the mean body weights of 10 female B6C3Fl mice with and without intravaginal application of GM-4, 5 days/week for 13 consecutive weeks. Detailed Description of the Invention 20 Terms and Definitions All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the following meanings, unless otherwise indicated: 25 "Microemulsions" are thermodynamically stable, transparent, dispersions of water and oil, stabilized by an interfacial film of surfactant molecules. Microemulsions are characterized by their submicron particle size of 0.1 pm or below. "Lipid" is an inclusive term for fats or fat derived materials. 30 "Surfactant" is any compound that reduces surface tension when dissolved in water or water solutions, or that reduces interfacial tension between two liquids or between a liquid and a solid. An example of one type are emulsifying agents. "Humectant" is a substance having affinity for water with stabilizing action on the water content of a material.

WO 00/56366 PCT/US00/07419 6 As used herein, the terms "analog" or "derivative" are used interchangeably to mean a chemical substance that is related structurally and functionally to another substance. An analog or derivative contains a modified structure from the other substance, and maintains a similar function of the other substance. The analog or 5 derivative need not be, but can be synthesized from the other substance. As used herein, "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, but are not limited to, (a) acid addition salts formed with inorganic acids, for example hydrochloric 10 acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) 15 salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or (c) salts formed with an organic cation formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (d) combinations of (a) and (b) or (c), e.g., a zinc tannate salt; and the like. The preferred acid addition salts are the trifluoroacetate salt and the 20 acetate salt. "Pharmaceutically acceptable carrier" means any material which, when combined with a biologically active compound, allows the compound to retain biological activity, such as the ability to potentiate antibacterial activity of mast cells and macrophages. 25 The term "inhibit" means to reduce by a measurable amount, or to prevent entirely. The term "to treat" means to inhibit or block at least one symptom that characterizes a pathologic condition, in a mammal threatened by, or afflicted with, the condition. 30 "Mammals" means any class of higher vertebrates that nourish their young with milk secreted by mammary glands, e.g., humans, rabbits, mice, monkeys, etc. "N-9" means the virucidal/spermicide, nonoxynol-9. "Organometallic compound" is an organic compound comprised of a metal attached directly to carbon (R-M). rrrTnr.'rY v n... - .. ......

WO 00/56366 PCT/US00/07419 7 "Coordination compound" is a compound formed by the union of a central metal atom or ion with a nonmetal atom, ion or molecule called a ligand or complexing agent. "Ligand" or a "complexing agent" is a molecule, ion or atom that is attached 5 to the central metal atom or ion of a coordination compound. "Monodentate ligand" is a ligand having a single donor atom coordinated to the central metal atom or ion. "Bidentate ligand" is a ligand having two donor atoms coordinated to the same central metal atom or ion. 10 "Chelate" or "chelated compound" a type of coordination compound in which a central metal ion is attached by chelated ligand containing two or more non metal atoms in the same molecule. One or more heterocyclic rings are formed with the central metal atom to form the coordination compounds. "Oxovanadium (IV) complex" is a coordination compound including 15 vanadium as the central metal atom or ion, and the vanadium has an oxidation state of +4 (IV), and is double bonded to oxygen. "Metallocene" is an organometallic coordination compound containing cyclopentadienyl rings attached to a transition metal or transition metal halide. "Vanadocene" is a metallocene including vanadium as the transition metal 20 ion. "Transition metals" is any of a number of elements in which the filling of the outermost shell to eight electrons within a period is interrupted to bring the penultimate shell from 8 to 18 or 32 electrons. Transition metals include elements 21 through 30, 39 through 48, 57 through 80, and from 89 on. 25 "Halo" is Br, Cl, F, or I. "Alkyl" is straight chained or branched chained alkyl, and includes halo substituted alkyl. "Alkoxy" is straight chained or branched chained alkoxy, and includes an O in the alkyl group. 30 "Aryl" refers to monovalent unsaturated aromatic carbocyclic radicals having a single ring, such as cyclopentadieneyl or phenyl, or multiple condensed rings, such as naphthyl or anthryl, which can be optionally substituted by substituents such as halogen, alkyl, arylalkyl, alkoxy, aralkoxy, and the like. "Carboalkoxy" is straight chained or branched chained alkoxy, and includes 35 carbamium carbom.

WO 00/56366 PCT/US00/07419 8 Gel-Microemulsion Formulations One aspect of the invention is directed to a pharmaceutical composition adapted for use as a spermicide. The spermicidal activity of the pharmaceutical 5 composition can be in vitro or in vivo. The spermicidal compositions of the present invention are suitable for use, for example, in mammals. The spermicidal compositions comprise a gel-microemulsion. The gel-microemulsions comprise an oil-in-water microemulsion and a thickening agent, such as polymeric gel thickening agent. 10 The microemulsion generally includes one or more lipids, one or more surfactants, optionally one or more humectants, and water as a diluent. Suitable lipids include those generally know to be useful for creating oil-in water microemulsions. Preferred examples include fatty acid glyceride esters, preferably medium chain C 6 -C 1 2 fatty acid glyceride esters, and the like. Preferred 15 C 6

-C

1 2 fatty acid glyceride esters include medium chain C6-C 12 monoglycerides and triglycerides, with the triglycerides being more preferred. Triglycerides of caprylic/capric acid are particularly suitable for use as the lipid component in the composition. Suitable triglycerides of caprylic/capric acid include Captex 300®, Captex® 355, Captex® 350 and Captex® 200, which are commercially available 20 from Abitec Corp., (Columbus, OH.), with the most preferred being Captex 300®. Mixtures of suitable lipids can be used. Suitable surfactants include those generally know to be useful for creating oil-in-water microemulsions wherein lipids are used as the oil component in the microemulsion, and preferably are well suited to aid in emulsifying the particular 25 lipid being used. Non-ionic surfactants are generally preferred. Examples of suitable surfactants include ethoxylated castor oil, and phospholipids. One suitable ethoxylated castor oil is Cremophor EL® commercially available from BASF Corp., (Mount Olive, NJ,). Preferred phospholipids include purified soy bean phospholipid or lecithins such as phosphatidylcholine. One suitable purified soy bean 30 phospholipid or lecithins is Phospholipon® 90G commercially available from American Lecithin (Oxford, CT.). Other suitable non-ionic surfactants include block copolymers of ethylene oxide and propylene oxide. Suitable commercially available block copolymers of ethylene oxide and propylene oxide include; Pluronic TTDrCr'7 T "rTTT -F - T T. , ,.

WO 00/56366 PCT/US00/07419 9 F-68, Pluronic F-77, Pluronic F-87, and Pluronic F-88, commercially available from BASF Corp., (Mount Olive, NJ.). Mixtures of suitable surfactants can be used. In some preferred embodiments, both ethoxylated caster oil and phospholipids are used as surfactants. 5 The microemulsion also optionally includes one or more humectants. Preferred humectants include propylene glycol such as 1,2-propanediol, and polyethylene glycol (PEG) with an average molecular weight in the range of 100 to 500, preferably in the range of 150 to 300, and more preferably in the range of 190 to 210. Mixtures of suitable humectants can be used. Preferably both propylene 10 glycol and polyethylene glycol are used as humectants. Suitable propylene glycol is commercially available from under the name Propylene Glycol USP from Sigma Chemical Co., (St. Louis, MO). Suitable polyethylene glycol includes Carbowax Polyethylene Glycol 200 commercially available from Union Carbide Corporation (Danbury, CT). 15 Water is used as the diluent, and preferably purified or distilled water is used. The microemulsions alone can be used, for example, as spermicites or in drug delivery systems to enhance the solubility of poorly water soluble substances, such as some anti-microbial compounds, as will be discussed in more detail below. 20 However, to enhance the usefulness of the microemulsions, especially as an effective spermicide and as a base formulation for anti-microbial compounds in certain application, thickening agents are added. Therefore, the gel-microemulsion formulation also includes one or more thickening agents, such as a polymeric hydrogel. Generally, the hydrogel is a 25 hydrophilic natural or synthetic gel-forming polymer, preferably, a natural gel forming polymer. Suitable examples of natural gel-forming polymers include carrageenan, xanthan gum, gum karaya, gum acacia, locust bean gum, guar gum. Mixtures of suitable humectants can be used. Suitable carrageenans include Seaspen® carrageenan and Viscarin® carrageenan commercially available from 30 FMC Corporation (Philadelphia, PA). Suitable xanthan gums include XANTURALTM 75 commercially available from Monsanto Pharmaceutical Ingredients (St. Louis, MO).

WO 00/56366 PCT/US00/07419 10 The formulation can also optionally include one or more additives such as preservatives or antioxidants to help maintain and prolong the useful life of the gel microemulsion. Preservatives and antioxidants that are generally known, and do not detract significantly from the usefulness of the gel-microemulsion for the particular 5 purpose it is being used, can be incorporated into the gel-microemulsion formulation. Particularly suitable preservatives include sodium benzoate, methyl parabens, propyl parabens, sorbic acid, and the like. Sodium benzoate is most preferred, and is commercially available from Cultor Food Science, Inc. (Ardsley, NY). The prevention of the action of microorganisms in the formulation can be 10 brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. If desired, other additives, such as colorants, scents, isotonic agents, for example, sugars, buffers or sodium chloride, can be added to the gel-microemulsion to the extent desired, and to the extent that the usefulness of the gel-microemulsion 15 is not disrupted. The composition is formulated to provide a gel-microemulsion with a submicron particle size, preferably in the range of 30-80 nm. Additionally, the viscosity of the gel-microemulsion is in the range of about 100 to about 1100 centipoise, more preferably from about 150 to about 1000, and more preferably from 20 about 200 to about 1000 centipoise. Those of skill in the art will recognize that the amounts of each of the individual components used to produce a suitable gel-microemulsion are dependent upon the amounts and type of other components used. Therefore, the amounts and types of components are interdependent. 25 Those of skill in the art will also recognize that suitable microemulsions can be identified through systematic mapping of ternary phase diagrams. The ternary phase diagram of the microemulsion components used for the preparation of one embodiment of the invention, GM-4, is shown in Figure 1, and discussed in the Examples below. The non-grid area represents the single phase microemulsion 30 region suitable for use. The concentration of the components can be selected from within this region. The asterisk represents the particular concentration of components of the microemulsion which was used for the GM-4 formulation. Suitable gel-polymer suspensions can then be selected as additives to the WO 00/56366 PCT/US00/07419 11 microemulsion-based system to obtain a gel of desirable viscosity with high thickening capability and compatibility with the microemulsion. It is preferable that the gel-microemulsion be stable at ambient temperature. Representative examples of constituent concentration ranges for base 5 components of some gel-microemulsion formulations embodying the invention can be found in Table 1, wherein the values are given in wt. % of the ingredients in reference to the total weight of the formulation. Table 1 Constituent Ranges Preferred Ranges More Preferred Ranges Lipid 2 to 25 6 to 23 8 to 15 Surfactant 3 to 30 4 to 17 8 to 15 Humectant 2 to 24 3 to 12 5 to 10 Polymer Gel 0.5 to 4 1 to 2 1.2 to 1.8 Additives 0 to 0.5 0.1 to 0.3 0.15 to 0.2 Water Balance Balance Balance 10 In some preferred embodiments, the formulation includes the specific constituent concentrations for base components as found in Table 2, wherein the values are given in wt. % of the ingredients in reference to the formulation weight. 15 Table 2 Ingredients Preferred Ranges More Preferred Ranges Medium Chain Tryglyceride 6 to 23 8 to 15 Ethoxylated Castor Oil 3 to 10 5 to 9 Phospholipid 1.5 to 6 3 to 6 Propylene Glycol 1.5 to 6 3 to 6 PEG-200 1.5 to 6 3 to 6 Natural Polymer Gel 1 to 2 1.2 to 1.8 Preservative 0.1 to 0.2 0.15 to 0.2 Water Balance Balance WO 00/56366 PCT/US00/07419 12 Preparation of Gel-microemulsions: A simple procedure allows for the preparation of a gel-microemulsion at even a one-milliliter scale. The following generally describes such a simple 5 procedure: Combine surfactants, hydrophilic components, and the lipids (preferably medium chain tryglycerides) in an appropriate container. Mix the components using a stir bar with mild heat until a clear and homogeneous microemulsion is formed. Remove the composition from the heat, and wait until it reaches room temperature. Add two parts of a pre-prepared polymer dispersion to each part of microemulsion 10 with continued mixing. The resulting gel-microemulsion is a dispersion with a viscosity in the range of 200-1000 centipoise, and a submicron particle size, preferably in the range of 30-80 nm. Sperimicidal Use of the Gel-microemulsion The contraceptive compositions of the present invention are preferably 15 administered to a site for contacting sperm, in a dosage which is effective to immobilize sperm. Such compositions are intended particularly for use in mammals, but use outside of mammals is contemplated. It is also contemplated that the compositions may be used as sperm immobilization compositions. It is expected that the present invention will be used by humans in most practical applications. 20 Preferably, the amount of spermicide employed will be that amount necessary to achieve the desired spermicidal results. Appropriate amounts can be determined by those skilled in the art. The contraceptive compositions of the present invention may be delivered to the vagina of a mammal by any means known to those skilled in the art. The gel 25 microemulsion can be applied directly. Other typical forms for delivery of the compositions include, for example, intervaginal devices such as sponges, condoms, including female condoms, suppositories, and films. In addition, the compositions of the present invention may be used as personal care lubricants, such as, for example, condom lubricants, and the like. The contraceptive compositions may be located 30 within or on a condom for example. Inter-vaginal devices may also be used to aid in the administration of the composition as described in U.S. Patent 5,069,906. Further details concerning the materials, ingredients, proportions and procedures of such delivery forms are known to those skilled in the art, and can be selected in accordance with techniques well-known in the art.

WO 00/56366 PCT/US00/07419 13 It is also contemplated that the formulation of the invention may be incorporated into a spermicidal article such as a vaginal insert, a condom, or other such device, such that when the article is used, the spermicidal can be delivered to contact sperm. 5 Gel-Microemulsion as a Formulation Base for Other Therapeutic Agents Another aspect of the invention is the use of the above described gel microemulsion formulations as formulation bases for incorporating therapeutically active agents. The base gel-microemulsion formulations generally include the 10 components and concentrations discussed above. The therapeutically active agents can include any generally known therapeutic agent where it would be desirable to administer such an agent with a gel-microemulsion formulation. Some embodiments of the gel-microemulsions of the invention are especially suitable as solubilizing vehicles for poorly water soluble compounds. In some preferred 15 embodiments, gel-microemulsion formulations are used, for example, as formulation bases for anti-microbial agents, spermicidal agents, or dual function anti-microbial/spermicidal agents. Anti-Microbial Gel-Microemulsion Formulations 20 Suitable examples of anti-microbial theraputic agents, include anti-viral agents, anti-bacterial agents, anti-fungal agents, and the like. Such agents can be incorporated into the gel-microemulsion formulations to provide for an anti microbial formulation, or a dual function anti-microbial/spermicidal formulation. Suitable examples of preferred anti-microbial agents include those used for the 25 treatment of sexually transmitted diseases, for example, AIDS (HIV-1, HIV-2, FIV, SIV, etc.) genital herpes, gonorrhea, chlamydia, and the like. Examples of Preferred Anti-Microbial Agents: Preferred examples of anti-microbial agents, such as anti-viral agents, include those disclosed in the following copending patent applications, which are hereby incorporated by reference 30 herein: U.S. Patent Application No. 09/047,609, which is incorporated herein by reference, and corresponding published PCT Application No. PCT/US99/06381 (International Publication Number WO 99/48902), which is incorporated herein by reference; WO 00/56366 PCT/US00/07419 14 U.S. Patent Application No. 09/450,082 which is incorporated herein by reference, and corresponding published PCT Application No. PCT/US99/14774 (International Publication Number WO 00/00501) which is incorporated herein by reference; and 5 U.S. Patent Application No. 09/107,716, which is incorporated herein by reference. Examples of preferred anti-microbial/anti-viral compounds disclosed in copending U.S. Patent Application No. 09/047,609 and corresponding PCT Patent 10 Application No. PCT/US99/06381, include AZT derivatives disclosed therein. Many of the AZT derivatives disclosed therein also have spermicidal activity. Examples of such anti-microbial AZT derivatives include compounds of the formula: 0 CH3 HN R2 0 N R3 0 HO HO__ R) 15 where R, is H, N 3 , halo, CN, COOH or NH, R, is halo (particularly Cl, Br or I, and more particularly Br) and R 3 is alkoxy (particularly C1-3 alkoxy, and more particularly methoxy (-OCH 3 )). The NH 2 group can be functionalized, for example with -CH 3 , -COCH 3 , -Ph, -COPh, and -CHIPh. Pharmaceutically acceptable salt or ester forms also can be used, such as sodium, potassium or ammonium salts. 20 The derivatives of the formula above include substitution on the AZT pentose ring member. The derivatives of this aspect of the present invention have the chemical structure illustrated below: WO 00/56366 PCT/US00/07419 15 O

CH

3 0 N R3 0 R'O RI where R, is H, N3, halo, CN, COOH or NH 2 , R 2 is halo (particularly CI, Br or I, and more particularly Br), R 3 is alkoxy (particularly C1-3 alkoxy, and more particularly methoxy (-OCH 3 )) and R' is a group that facilitates the passage of the compound 5 into a cell. As in the first formula, the NH, group can be converted, for example to

NHCH

3 , NHCOCH 3 , NHPh, NHCOPh, and NHCH 2 Ph. The R' group can be, for example, H, a phosphate, lipid or fatty acid group. Alternatively, sperm-reactive antibodies or cytokines could be used to derivatize these compounds (as well as those of the first formula) at the R' or R, positions for targeted delivery. 10 Pharmaceutically acceptable salt or ester forms, such as the sodium, potassium or ammonium salts, can be used as well. The R' group forms a phosphate group. The H of an -OH member of the phosphate can be replaced with C 1-4 alkyl or aryl substituents (e.g. phenyl-, naphthyl- or anthracinyl-substitution), which optionally may be substituted, and SH 15 or NH, groups can replace the OH of the phosphate, and in each of these cases a H of the NH 2 or SH can be replaced in the same manner as the H of the OH group discussed previously. The aryl phosphate group is surprisingly effective in maintaining excellent anti-HIV activity. A general structure of an exemplary aryl phosphate OR' group is illustrated below: R6

R

5 '015 0 R4 O--P-0 20 R 6

R

5 where R 4 , R 5 , and R 6 are the same or different and are selected from hydrogen, methyl, ethyl, fluoro, chloro, bromo, iodo, dichloro, dibromo, difluoro, WO 00/56366 PCT/US00/07419 16 trifluoromethyl, nitro, cyano, methoxy, trifluromethoxy and ethoxy, particularly hydrogen, fluoro, bromo and methoxy and R" is an amino acid residue that may optionally be substituted and/or esterified, for example an alaninyl group ( NHCH(Me)COOMe). In the case of the alaninyl group, the methyl group attached 5 to the CH group can be substituted, for example with a phenyl group, and the methyl esterification can be replaced with other C 2 or C 3 esterification. Of these compounds, the following compound, hereinafter referred to as WHI-07, is particularly preferred: H3 Br NH 0 MeO Br / O-H - NH N3 Me CO 2 Me 10 Chemical name: (5R,6R)-and (5S,6S)-5-bromo-6-methoxy-5,6-dihydro-AZT 5'-(para- bromophenyl methoxyalaninyl phosphate). Examples of preferred anti-microbial, preferably anti-viral, compounds 15 disclosed in U.S. Patent Application No. 09/450,082, and corresponding published PCT Application No. PCT/US99/14774; and U.S. Patent Application No. 09/107,716, include aryl phosphate derivatives of nucleosides disclosed therein, particularly derivatives of d4T and AZT. Examples of suitable nucleoside derivative disclosed therein include those of the formula: Y Rr--Y-P-R 2 I

N-R

3

R

5 0R4 20 O or a pharmaceutically acceptable salt thereof, in which Y is oxygen or sulfur, preferably oxygen; R, is unsubstituted aryl or aryl substituted with an electron withdrawing group; R, is a nucleoside of one of the following formulae: WO 00/56366 PCT/US00/07419 17 0 O R, 0 O R H Rio R. R,: in which R 6 is purine or pyrimidine, preferably pyrimidine; and R, R, R9, R 1 o, R, and R 1 ,2 are independently hydrogen, hydroxy, halo, azido, -NO, -NR 1 3

R

14 , or 5 N(ORs 5

)R

6 , in which R, 3 , R 14 , R, 5 , and R, are independently hydrogen, acyl, alkyl, or cycloalkyl;

R

3 is hydrogen, acyl, alkyl, or cycloalkyl; R 4 is a side chain of an amino acid; or R 3 and R 4 may be taken together to form the side chain of proline or hydroxyproline; and R s is hydrogen, alkyl, cycloalkyl, or aryl. 10 As used in the definitions of the aryl phosphate derivatives of nucleosides disclosed above, and in U.S. Patent Application No. 09/450,082, the following terms have the following meanings: The term "aryl" includes aromatic hydrocarbyl, such as, for example, phenyl, including fused aromatic rings, such as, for example, naphthyl. Such groups may be 15 unsubstituted or substituted on the aromatic ring by an electron-withdrawing group, such as, for example, halo (bromo, chloro, fluoro, iodo), NO,, or acyl. Preferably, aryl substituted with an electron-withdrawing group is bromophenyl, more preferably 4-bromophenyl. The term "acyl" includes substituents of the formula R,, 17 C(O)-, in which RI 7 20 is hydrogen, alkyl, or cycloalkyl. The term "alkyl" includes a straight or branched saturated aliphatic hydrocarbon chain having from 1 to 6 carbon atoms, such as, for example, methyl, ethyl, propyl, isopropyl (1-methylethyl), butyl, tert-butyl (1,1-dimethylethyl), and the like. Such groups may be unsubstituted or substituted with hydroxy, halo, azido, 25 -NO, -NR, 3

R,

4 , or -N(ORis)R 6 , in which R, 3 , R 14 , R, 5 , and R 16 are as defined above. The term "cycloalkyl" includes a saturated aliphatic hydrocarbon ring having from 3 to 7 carbon atoms, such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Such groups may be unsubstituted or WO 00/56366 PCT/US00/07419 18 substituted with hydroxy, halo, azido, -NO, -NR 3

RI

4 , or -N(ORis)RI 6 , in which R3 ,

RI

4 , R 5 , and R, 6 are as defined above. The term "purine" includes adenine and guanine. The term "pyrimidine" includes uracil, thymine, and cytosine. Preferably, 5 the pyrimidine is thymine. The term "side chain of an amino acid" is the variable group of an amino acid and includes, for example, the side chain of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, hydroxylysine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, 10 tyrosine, valine, and the like. Preferably, the side chain of an amino acid is the side chain of alanine or tryptophan. Generally, compounds substituted with an electron-withdrawing group, such as an ortho-orpara-substituted halogen or NO, provide for more efficient hydrolysis to active inhibitory compounds. Preferred is halogen substitution, and most 15 preferred are para-bromo substitution and para-chloro substitution. Preferred nucleoside derivative disclosed therein include those of the formulas: HHH H3CO2 CH3 H3CO :H,CH N3 20 wherein X is an electron withdrawing group, for example halo or NO 2 , and most preferably, X is bromo or chloro. Gel-Microemulsion Formulations with Additional Spermicidal Agents Suitable examples of spermicidal agents, include any spermicidal agent 25 generally known that is compatible for formulation with the gel-microemulsion. Such agents can be incorporated into the gel-microemulsion formulations to provide for additional spermicidal activity of the formulation. Many of the spermicidal agents discussed below also have anit-microbial activity, such as anti-viral activity, WO 00/56366 PCT/US00/07419 19 and can be incorporated into the formulations as an anti-microbial agent as well as a spermicidal agent. Preferred SpermicidalAgents For Incorporation Into the Gel Microemulsion Formulation: Preferred examples of spermicidal agents include 5 those disclosed in the following copending patent applications: U.S. Patent Application No. 09/008,898, which is incorporated herein by reference, and corresponding published PCT Application No. PCT/US99/01171 (International Publication Number WO 99/36063), which is incorporated herein by reference; 10 U.S. Patent Application No. 09/187,115, which is incorporated herein by reference; U.S. Patent Application No. 09/224,677, which is incorporated herein by reference. 15 Examples of preferred spermicidal compounds disclosed in copending U.S. Patent Application No. 09/008,898, and corresponding published PCT Application No. PCT/UJS99/01171, include the vanadium (IV) compounds disclosed therein. Examples of such vanadium (IV) compounds include organometallic cyclopentadienyl vanadium IV complexes. Preferred such compounds include: 20 vanadocene dichloride, bis (methylcyclopentadienyl) vanadium dichloride, vanadocene dibromide, vanadocene diiodide, vanadocene diazide, vanadocene dicyanide, vanadocene dioxycyanate, vanadocene dithiocyanate, vanadocene diselenocyanate, vanadocene ditriflate, vanadocene monochloro oxycyanate, vanadocene monochloroacetonitrilo tetrachloro ferrate, vanadocene acetylacetonato 25 monotriflate, vanadocene bipyridino ditriflate, vanadocene hexafluoro acetylacetonato monotriflate, vanadocene acethydroxamato monotriflate, and vanadocene N-phenyl benzohydroxamato monotriflate. Particularly preferred compounds include vanadocene diselenocyanate, and vanadocene dichloride. Examples of preferred spermicidal compounds disclosed in copending U.S. 30 Patent Application No. 09/187,115 include oxo-vanadium (IV) compounds disclosed therein. Preferred the oxovanadium (IV) complexes include at least one bidentate ligand. Suitable bidentate ligands include N,N'; N,O; and O,O' bidentate ligands. Examples of suitable bidentate ligands include bipyridyl, bridged bipyridyl, WO 00/56366 PCT/US00/07419 20 and acetophenone. Particularly, preferred oxovanadium compounds are those having the formulas shown and described below. Some suitable oxo-vanadium (IV) compounds include a bidentate ligand wherein the bidentate ligand is a bipyridyl and the oxovanadium IV complex has the 5 general formulae: R 0 N"' X V N X (OH2) n R where R and R, are the same or different and are independently selected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, 10 carboalkoxy (e.g. C 2

-C

6 ) and nitro; X and X 1 are the same or different and are independently selected from: monodentate and bidentate ligands; and n is 0 or 1. Other suitable oxo-vanadium (IV) compounds have a bidentate ligand wherein the bidentate ligand is a bridged bipyridyl and the 15 oxovanadium IV complex has the general formulae: R2 |

X

2 / z V N X N(OH2)nX

R

3 where R 2 and R 3 are the same or different and are selected from H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C 2

-C

6 ) and nitro; 20 X 2 and X 3 , are the same or different and are selected from monodentate and bidentate ligands; Z is selected from O, CH, CH,-CH,, and CH=CH; and n is 0 or 1.

WO 00/56366 PCT/US00/07419 21 Other suitable oxo-vanadium (IV) compounds have a bidentate ligand wherein the bidentate ligand is a bridged bipyridyl, and the bridged bipyridyl is phenanthroline, and the oxovanadium IV complex has the general formulae: 5

R

4 0 N

X

4 V

R

5 / N x5 (OH2)n

R

6 where R 4 , R and R 6 are the same or different and are independently selected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, 10 carboalkoxy (e.g. C 2

-C

6 ) and nitro; X 4 and X 5 are the same or different and independently selected from: monodentate and bidentate ligands; and n is 0 or 1. Other suitable oxo-vanadium (IV) compounds have a bidentate ligand wherein the bidentate ligand is an O,O' bidentate ligand, the 15 oxovanadium IV complex has the general formulae: R O 0 R OV / o \Y

R

9 (OH2) n where R 7 and R 9 are the same or different and are independently selected 20 from: H, lower alkyl, lower alkoxy, and halogenated alkyl; R 8 is selected from H, lower alkyl, halo, lower alkoxy, and halogenated alkyl; Y and Y' are the same or different and independently selected from the group consisting of: monodentate and bidentate ligands; and n is 0 or 1.

WO 00/56366 PCT/US00/07419 22 Preferred monodentate ligands for the oxovanadium complex include HO, halides and carboxylates. Preferred bidentate ligands include N, N' bidentate ligands, N, O bidentate ligands, and O, O' bidentate ligands. Examples of suitable N, N' bidentate ligands include diamines and other such known suitable N, N' 5 bidentate ligands. Examples of diamines include bipyridal, derivatives of bipyridal, bridged bipyridal, such as phenanthroline, derivatives of phenanthroline, and other such compounds. Examples of suitable N, O bidentate ligands include amino acids and Schiff base type groups. Examples of suitable O, O' bidentate ligands include dicarboxylate, 2 -hydroxyacetophenone, acetylacetone type and catechol type groups. 10 Particularly useful oxo-vanadium (IV) complexes are the following: (diaqua)(2,2'-bipyridyl)oxovanadium(IV) sulfate; (aqua)bis(2,2'-bipyridyl)oxovanadium(IV) sulfate; (diaqua)(4,4'-dimethyl-2,2'-bipyridyl)oxovanadium(IV) sulfate; (aqua)bis(4,4'-dimethyl-2,2'-bipyridyl)oxovanadium(IV) sulfate; 15 (diaqua)(1,10-phenanthroline)oxovanadium(IV) sulfate; (aqua)bis(1,10-phenanthroline)oxovanadium(IV) sulfate; (diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate; (aqua)bis(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate; (diaqua)(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate; 20 (aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate; and bis(5'-bromo-2'-hydroxyacetophenone) oxovanadium(IV). Examples of preferred spermicidal compounds disclosed in copending U.S. Patent Application No. 09/224,677 include phenethyl-5-bromopyridylthiourea 25 (PBT) and dihydroxalkoxybenzylopyrimidine (DABO) derivatives disclosed therein. Examples of such PBT derivatives include those having the following chemical formula, or a pharmaceutically acceptable salt thereof: WO 00/56366 PCT/US00/07419 23 S R, RN N R H Br where R, R 1 , R 2 , R 3 , and R 4 are independently hydrogen, F, Cl, Br, or I, and where at least one of R, R 1 , R 2 , R 3 , and R4 is F, Cl, Br, or I. Preferably, one of R, R 1 , R 2 , R 3 , 5 and R 4 in structure above is F or Cl. Some, but not all of the suitable halogen substituted PBT derivatives of the invention are listed below: N-[2-(2-fluorophenethyl)]-N'-[ 2 -(5-bromopyridyl)]-thiourea N-[2-(2-chlorophenethyl)]-N'-[ 2 -(5-bromopyridyl)]-thiourea N-[2-(3-fluorophenethyl)]-N'-[2-(5 -bromopyridyl)] -thiourea 10 N-[2-(3-chlorophenethyl)]-N'-

[

2 -(5-bromopyridyl)]-thiourea N-[2-(4-fluorophenethyl)]-N'-[ 2 -(5-bromopyridyl)]-thiourea N-[2-(4-chlorophenethyl)]-N'-[ 2 -(5-bromopyridyl)]-thiourea. One of the more preferred PBT derivatives is N-[2-(2-fluorophenethyl)] 15 N'-[ 2 -(5-bromopyridyl)]-thiourea (F-PBT) which has the chemical structure shown below: S HN N H N Br F-PBT PBT derivatives can be synthesized as described in Vig et al., BIOORG. MED. 20 CHEM., 6:1789-1797 (1998). In brief, 2 -amino-5-bromopyridine is condensed with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl derivative.

WO 00/56366 PCT/US00/07419 24 Further reaction with appropriately halogen-substituted phenylethylamine gives the target halogenated PBT derivatives. Examples of suitable DABO derivatives include those of the formula, or a pharmaceutically acceptable salt thereof: 5 O R , R2

HN

3 *5 S N R-- Y R2 where R 1 and R 2 are alike or different, and are hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl. 10 Preferably, one or more of R 1 and R 2 is an alkyl having 1 to 3 carbonatones, (C 1 C 3 ), such as methyl (Me), ethyl (Et), or isopropyl (i-Pr). Preferably, R, is alkyl, alkenyl, ROH, or RNH 2 . R 2 is preferably halo, alkyl, or C 1

-C

3 alkoxy; Y is S or O, and is preferably S. R 3 is alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R is alkyl, and is preferably CI-C 3 alkyl. 15 Preferred DABO derivatives include compounds having the chemical structure shown below, or a pharmaceutically acceptable salt thereof: O R, R, IHN S N

R

2 Me-S where R 1 is Me, Et, or i-Pr and R 2 is H or Me. 20 Some, but not all, of the suitable DABO derivative compounds include compounds (a) through (d) listed below, or a pharmaceutically acceptable salt thereof: WO 00/56366 PCT/US00/07419 25 (a) 5-methyl-2-[(methylthiorethyl)thio]-6-benzyl-pyrimidin-4-1H one, (b) 5-ethyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1

H

one, 5 (c) 5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4 1 H-one, and (d). 5-isopropyl-2-[(methylthiomethyl)thio]-6-(3,5-dimethylbenzyl) pyrimidin-4-1 H-one. One of the more preferred DABO derivatives is the compound 5-isopropyl 10 2 -[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-ones (S-DABO), and pharmaceutically acceptable salts thereof, which is exemplified by the chemical structure shown below: O Me Me HN S N S-Me S-DABO 15 DABO derivatives can be prepared as described in described in Vig et al., BIOORG MED CHEM LETTERS, 8:1461-1466 (1998). The general synthesis scheme for the preparation of DABO derivatives (a) through (d) listed above is as follows: WO 00/56366 PCT/US00/07419 26 R ' N R OEt c / R, CN a, b R, P CH R2 R 1 R 1 H la-d 2a-d 0 R1 S R, me-) 3a-d 1-3

R

1

R

2 aH Me b H Et c H i-Pr d Me i-Pr Reagents and conditions a) RFCHBrCOOEt/ZfTHF, b) HCI(aq), c) (H 2 NECS/Na/EtOH, d) DMF, K2CO 3 , Chloromethyl rnethyl sulfide, 15h. Briefly, ethyl- 2 -alkyl-4-(phenyl)-3-oxobutyrates la-d were obtained from commercially available phenyl acetonitrile. The 3-ketoesters were condensed with 5 thiourea in the presence of sodium ethoxide to furnish the corresponding thiouracils 2a-d. Compounds (1 a-d and 2 a-d) were produced by a methods previously described (Danel, K. et al., Acta Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J Med. Chem., 1997, 40, 1447-1454; Danel, K. et al., J Med. Chem., 1998, 41, 191-198). 10 Subsequent reaction of thiouracil with methylchloromethyl sulfide in N,N-dimethylformamide (DMF) in the presence of potassium carbonate afforded compounds 3a-d in moderate yields. A mixture of thiouracil compound 2 (1 mmol), WO 00/56366 PCT/US00/07419 27 methylchloromethyl sulfide (1 mmol), and potassium carbonate (1 mmol) in anhydrous DMF (5ml) was stirred overnight at room temperature. After treatment with water (50 ml), the solution was extracted with ethyl acetate (3 x 50 ml). The combined extracts were washed with saturated NaCl (2 x 50 ml), dried (MgSO 4 ), 5 filtered and concentrated in vacuo to give the crude products 3a-d which were purified by column chromatography (hexane : ethyl acetate eluent). Other examples of therapeutic agents includes vanadium (IV) complexes containing a substituted or un-substituted catacholate ligand. Examples of such 10 compounds include complexes having the following structural formula, or pharmaceutically acceptable salts thereof: R1 R5, nj R2 V O R6' '0 R (OH2)n R4 15 wherein R 1 , R 2 , R 3 and R 4 are the same or different and are independently selected from H, halo, OH 2 , O 3

SCF

3 , N 3 , CN, OCN, SCN, SeCN, NO 2 , C]-C 4 alkyl, Cj-C 4 alkoxy, and aryl; and n is 0 or 1; and R 5 and R 6 are the same or different and are either monodentate ligands or R s and R 6 together comprise a bidentate ligand. Suitable monodentate ligands include, for example, aryl, halo, H 2 0, O 3

SCF

3 , 20 N 3 , COOH, CN, OCN, SCN, SeCN, NO 2 , C 1

-C

4 alkyl, C 1

-C

4 alkoxy. Preferred monodentate ligands comprises one or more unsubstituted or substituted aromatic ring. More preferred monodentate ligands comprise substituted or un-substituted cyclopentadienyl ligands. Suitable bidentate ligands include, for example, N,N'; N,O; and 0,0' 25 bidentate ligands. Examples of suitable N, N' bidentate ligands include diamines and other such known suitable N, N' bidentate ligands. Examples of diamines include vrr, - - - - - -- T7 - - WO 00/56366 PCT/US00/07419 28 bipyridal, derivatives of bipyridal, bridged bipyridal, such as phenanthroline, derivatives of phenanthroline, and other such compounds. Examples of suitable N, O bidentate ligands include amino acids and Schiff base type groups. Examples of suitable O, O' bidentate ligands include dicarboxylate, 2-hydroxyacetophenone, 5 acetylacetone type and catechol type groups. Preferred bidentate ligands comprise one or more aromatic ring. Preferred examples of suitable bidentate ligands comprising aromatic rings include substituted or un-substituted bipyridyl, bridged bipyridyl, and acetophenone ligands. One example of a bridged bipyridyl includes phenanthroline. 10 Some preferred vanadium (IV) catacholate complexes include "bent sandwich" vanadocene monocatacholate complexes having the following structure formula, or pharmaceutically acceptable salts thereof: R1 CP,,v.-0 R2 Cp~ p V Cp "O

R

3 R4 15 wherein Cp is unsubstituted cyclopentadienyl, or cyclopentadieneyl substituted with one or more substituents selected from substituted or unsubstituted aryl, C 1

-C

4 alkyl, C 1

-C

4 alkoxy, halo, OH2, O 3

SCF

3 , N 3 , CN, OCN, SCN, SeCN,

NO

2 . Preferably, Cp is unsubstituted cyclopentadienyl. 20 R 1 , R 2 , R 3 and R 4 are the same or different and are independently selected from H, halo, OH2, O 3

SCF

3 , N 3 , CN, OCN, SCN, SeCN, NO 2 , C 1

-C

4 alkyl, and Ci

C

4 alkoxy Preferably, electron donating groups, for example electron donating alkyl WO 00/56366 PCT/US00/07419 29 groups, are present as substituents of the catacholate ring in positions R', R 2 , R 3 and/or R 4 Particularly preferred such compounds include vanadocene catacholate, vanadocene mono-tertbutyl catacholate, and vanadocene 1, 3-diisopropyl 5 catacholate. Another example of vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is a bipyridyl has the general formula shown below, or pharmaceutically acceptable salts thereof: R7 RI N On R2 IV 0 N R3 (OH2)n RR4 10 where R 7 and R 8 are the same or different and are independently selected from: H, aryl, C 1 i-C 4 alkyl, halo, C 1

-C

4 alkoxy, carboalkoxy (e.g.

C

2

-C

6 ), cyano, and nitro; n is 0 or 1; R 1 , R 2 , R 3 and R 4 are the same or different and are independently selected from H, halo, OH 2 , O 3

SCF

3 , N 3 , CN, 15 OCN, SCN, SeCN, NO 2 , CI-C 4 alkyl, and Ci-C 4 alkoxy. Another example of vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is a bridged bipyridyl has the general formula shown below, or pharmaceutically acceptable salts thereof: 20 WO 00/56366 PCT/US00/07419 30 R9 R1 N On R2 z V N R3 (OH2)n RR4 where R 9 and R 0 are the same or different and are selected from H, aryl, C 1 C 4 alkyl, halogen, CI-C 4 alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. 5 C 2

-C

6 ) and nitro; Z is selected from O, CH 2 , CH 2

-CH

2 , and CH=CH; n is 0 or 1; and halo, OH 2 , O 3

SCF

3 , N 3 , CN, OCN, SCN, SeCN, NO 2 , CI-C 4 alkyl, and C 1

-C

4 alkoxy. Another example of vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is a 10 bridged bipyridyl, and the bridged bipyridyl is phenanthroline, has the general formula shown below, or pharmaceutically acceptable salts thereof: R IR R1 N On R2 RIV N R3 N (OH2)n 4 RR 15 where R", R 12 and R 1 3 are the same or different and are independently selected from: H, aryl, C 1

-C

4 alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C 2

-C

6 ) and nitro; n is 0 or 1; and R1, R 2 , R 3 and R 4 are the same or different and are independently selected from H, halo, WO 00/56366 PCT/US00/07419 31

OH

2 , O 3

SCF

3 , N 3 , CN, OCN, SCN, SeCN. NO2, C 1

-C

4 alkyl, and C 1

-C

4 alkoxy. Another example of vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is an O,O' 5 bidentate ligand, and the complex has the general formula VI, is shown below, or pharmaceutically acceptable salts thereof:

R

4 0 R1 I~ (OH2))n R4 10 where R 14 and R 16 are the same or different and are independently selected from: H, aryl, C 1

-C

4 alkyl, and Ci-C 4 alkoxy, and halogenated alkyl; R 15 is selected from H, C 1

-C

4 alkyl, halo, C 1

-C

4 alkoxy, and halogenated alkyl; n is 0 or 1; and R',

R

2 , R 3 and R 4 are the same or different and are independently selected from H, halo,

OH

2 , O 3

SCF

3 , N 3 , CN, OCN, SCN, SeCN, NO 2 , Cl-C 4 alkyl, and C 1

-C

4 alkoxy. 15 Formulation of the gel-microemulsions formulations including additional therapeutic agents Gel-microemulsions formulations including additional therapeutic agents, for example the anti-microbial agents or spermicidal agents discussed above, are 20 formulated and prepared in substantially the same way as the primary gel microemulsions discussed above, with the only difference being the addition of the additional therapeutic agent. The amount of additional ingredient added is dependent upon the desired effective amount of the ingredient in the final gel microemulsion formulation. It is still desirable to provide a dispersion with a 25 viscosity in the range of 200-1000 centipoise, and a submicron particle size, preferably in the range of 30-80 nm. "'TrDvJr( T'rr rr' '-,r v-,,, WO 00/56366 PCT/US00/07419 32 Representative examples of constituent concentration ranges for base components of some gel-microemulaion formulations embodying the invention can be found in Table 3, wherein the values are given in wt. % of the ingredients in reference to the total weight of the formulation. 5 Table 3 Constituent Ranges Preferred Ranges More Preferred Ranges Therapeutic up to 10 up to 5 up to 2 Ingredient Lipid 2 to 25 6 to 23 8 to 15 Surfactant 3 to 30 4 to 17 8 to 15 Humectant 2 to 24 3 to 12 5 to 10 Polymer Gel 0.5 to 4 1 to 2 1.2 to 1.8 Additives (e.g. 0 to 0.5 0.1 to 0.3 0.15 to 0.2 Preservatives) Water Balance Balance Balance In some preferred embodiments, the formulation includes the specific constituent concentrations for base components as found in Table 4, wherein the 10 values are given in wt. % of the ingredients in reference to the formulation weight. Table 4 Ingredients Preferred Ranges More Preferred Ranges Anti-microbial agent or up to 2 1 to 2 Spermicidal agent, or mixtures thereof Medium Chain Tryglyceride 6 to 23 8 to 15 Ethoxylated Castor Oil 3 to 11 5 to 9 Phospholipid 1.5 to 6 3 to 6 Propylene Glycol 1.5 to 6 3 to 6 PEG-200 1.5 to 6 3 to 6 Natural Hydrogels 1 to 2 1.2 to 1.8 Preservative 0.1 to 0.2 0.15 to 0.2 Water Balance Balance v r TP ("r'7T 7'7r r "....-- .

WO 00/56366 PCT/US00/07419 33 The following generally describes a simple formulation procedure for producing formulations with additional therapeutic agents, such as an anti-microbial agent or a spermicidal agent: Combine surfactants, hydrophilic components, and the 5 lipids (preferably medium chain tryglycerides) in an appropriate container. Mix the components using a stir bar with mild heat until a clear and homogeneous microemulsion is formed. Add the appropriate amount of therapeutic agents with continued stiffing for approximately 10 minutesto assure complete solubilization of the drug. Remove the composition from the heat, and wait until it reaches room 10 temperature. Add two parts of a pre-prepared polymer dispersion to each part of microemulsion with continued mixing. The resulting gel-microemulsion is a dispersion with a viscosity in the range of 200-1000 centipoise, and a submicron particle size, preferably in the range of 30-80 nm. 15 Use Theraputic Agent Containing Gel-Microemulsion Formulations When used as a spermicide, or a dual function spermicide/anti-microbial composition, the gel-microemulsion formulations resulting from the addition of additional theraputic agents are contemplated for used in generally the same manner 20 as the gel-microemulsion spermicide use discussed above. However, it is contemplated that formulations including other theraputic agents, for example anti viral agents, can be used in non-spermicidal applications. In such applications, the formulation is preferably administered to a site appropriate for the theraputic activity desired in a dosage which is effective to 25 effectuate the desired theraputic effect. For example, in anti-microbial applications, the formulation is preferably administered to a site appropriate for desired anti microbial activity in a dosage which is effective to effectuate the desired anti microbial effect. Appropriate amounts can be determined by those skilled in the art. Such theraputic compositions are intended particularly for use in mammals, but use 30 outside of mammals is contemplated. It is expected that the formulations will be used by humans in most practical applications. The invention may be further clarified by reference to the following Examples, which serve to exemplify some of the preferred embodiments, and not to limit the invention. tYTTD 'rVT"rT~ll I'VTl'lr, r, -. rr - - WO 00/56366 PCT/US00/07419 34 EXAMPLES Example 1: Synthesis of Gel-Microemulsion Formulations 5 Materials: Captex 300 was obtained from ABITEC Corp., Janeswille, WI. Cremophor EL was from BASF Corp., Mount Olive, NJ. Phospholipon 90G was purchased from American Lecithin Co., Danbury, CT. PEG-200 was from Union Carbide Corp., Danbury, CT while propylene glycol was obtained from Spectrum Quality Products Inc., New Brunswick, NJ. Seaspan and viscarin 10 carrageenan were obtained from FMC Corp., Newark, DE. N-9 (IGEPAL CO-630) was a generous gift from Rhone Poulenc, Cranbury, NJ. Gel-Microemulsion Formulation: A lipophilic sub-micron (30-80 nm) particle size microemulsion was developed using commonly used pharmaceutical excipients through systemic mapping of ternary phase diagrams (Eccleston GM, In: 15 Swarbrick J, Boylan JC, eds. Encyclopedia of Pharmaceutical Technology, New York:Marcel Dekker, 1992:375-421.; Ritschel WA, Meth Find Exp Clin Pharmacol, 1993; 13: 205-20.). Several microemulsion compositions were screened for particle size, stability, and responses to in vitro spermicidal activity. The ingredients tested included: medium chain triglycerides, purified soya 20 phospholipid, Pluronic F-68, ethoxylated castor oil, propylene glycol, and polyethylene glycol. The ingredients selected included, drug solubilizers and stabilizers (Captex 300, Cremophor EL, phospholipon 90 G, propylene glycol, and PEG 200) and a preservative (sodium benzoate). Various polymeric gels were screened to produce a gel with desirable viscosity. Polymer suspensions of seaspan 25 and viscarin carrageenan were selected as additives to the microemulsion-based system to obtain a gel with desirable viscosity with high thickening capability and compatibility with vaginal mucosa. A submicron (30-80 nm) particle size microemulsion-based system containing the pharmacological excipients, Captex 300, Cremophor EL, 30 phospholipon 90G, propylene glycol, PEG 200, and sodium benzoate, with high solubilizing capacity for lipophilic drugs was identified through systematic mapping of ternary phase diagrams, and lipophilic drug solubilization studies. The ternary phase diagram of the microemulsion components used for the preparation of GM-4 is shown in Figure 1. The non-grid area represents the single phase microemulsion 35 region. The asterisk represents the microemulsion which was used for GM-4 CrTTCTT'rTT'r" orx"7rr, /"yy.

WO 00/56366 PCT/US00/07419 35 formulation listed in Table 5. Polymer suspensions of seaspan and viscarin carrageenan were selected as additives to the microemulsion-based system to obtain a gel of desirable viscosity with high thickening capability and compatibility with the microemulsion. These polymers did not cause precipitation or alter the 5 microemulsion particle size. The GM-4 was found to be very stable at ambient temperature. Particle size determination was made using Nicomp Model 380 laser diode source (Particle Sizing Systems, Santa Barbara, CA). Viscosity measurements were made using the Brookfield digital viscometer (Model DV-II+; Brookfield 10 Engineering Laboratories, Spoughton, MA). Table 5: Components of GM-4 Formulation Excipient Type Final concentration (%, by wt) Captex 300 Lipid 10.8 Cremophor EL Surfactant 7.6 Phospholipon 90G Phospholipid 5.1 Propylene Glycol Humectant 4.2 PEG-200 Humectant 4.2 Seaspan carrageenan Natural polymer 0.9 Viscarin carrageenan Natural polymer 0.5 Sodium benzoate Preservative 0.2 Water Diluent 66.5 15 EXAMPLE 2: Screening of Pharmaceutical Excipients of GM-4 for Spermicidal Activity Against Human Sperm Methods and Materials Computer-Assisted Spermicidal Assay: To evaluate the spermicidal 20 activity of the pharmaceutical excipients used in the GM-4 formulation, a highly ('lTYD('rYg'rr'r' frrr'.-., . ., - v -.

WO 00/56366 PCT/US00/07419 36 motile fraction of pooled donor sperm (n = 9) was prepared by discontinuous (90 45%) gradient centrifugation using Enhance-S-Plus cell isolation medium (Conception technologies, San Diego, CA) and the "swim-up" method as previously described (D'Cruz OJ, et al., Biol Reprod 1995;53:118-30.; D'Cruz OJ, et al., Biol 5 Reprod, 1998; 59:503-15.). All donor specimens were obtained after informed consent and in compliance with the guidelines of the Parker Hughes Institute Institutional Review Board. Highly motile fraction of sperm (_ 10x10 6 /ml) were suspended in 1 ml of Biggers, Whitten, and Whittingam's medium (BWW) containing 25 mM HEPES (Irvine Scientific, Santa Ana, CA) and 0.3% BSA 10 (fraction V; Sigma Chemical Co., St. Louis, MO) in the presence and absence of serial 2-fold dilutions of test substance. The ingredients evaluated were Captex 300 (1.35% - 10.8%), Cremophor EL (0.95% - 7.6%), phospholipon 90G (0.637% 5.1%), propylene glycol (0.52% - 4.2%), PEG 200 (0.52% - 4.2%), seaspan carrageenan (0.11% - 0.9%), viscarin carrageenan (0.06% - 0.5%), and sodium 15 benzoate (0.025% - 0.2%). After 3 h incubation at 37 0 C, the sperm head centroid derived sperm motility parameters were determined using a Hamilton Thorne Research (Danvers, MA) Integrated Visual Optical System (IVOS), version 10 instrument, as previously described (D'Cruz OJ, et al., Biol Reprod, 1998;58:1515 26.; D'Cruz OJ, et al., Mol Hum Reprod, 1998;4:683-93.; D'Cruz OJ, et al., Biol 20 Reprod, 1999;60:435-44.; D'Cruz OJ, et al. Biol Reprod, 1999;60:1419-28.). The attributes of sperm kinematic parameters evaluated included numbers of motile (MOT) and progressively (PRG) motile sperm; curvilinear velocity (VCL); average path velocity (VAP); straight-line velocity (VSL); beat-cross frequency (BCF); and the amplitude of lateral head displacement (ALH) and the derivatives, straightness 25 (STR) and linearity (LIN). Data from each individual cell track were recorded and analyzed. For each aliquot sampled, >200 sperm were analyzed. The percentage motilities were compared with those of sham-treated control suspensions of motile sperm. The spermicidal activity of the test compound was expressed as EC50 (the final concentration of the compound in the medium that decreased the proportion of 30 motile sperm by 50%). Results The effects of individual components of GM-4 on the motility of washed and enriched motile fraction of sperm evaluated by CASA are summarized in Figure 2. At the final concentrations used for GM-4 formulation, Captex 300, PEG 200, 35 seaspan carrageenan, viscarin carrageenan, and sodium benzoate, demonstrated little WO 00/56366 PCT/US00/07419 37 or no inhibitory effects on human sperm motility. Further, sperm motion kinematics using CASA confirmed that these excipients did not significantly alter the sperm motion parameters, such as the progressive velocity, straightness of the swimming pattern, linearity of the sperm tracks, beat-cross frequency, and the amplitude of 5 lateral sperm head displacement. In contrast, Cremophor EL and phospholipon 90G were spermicidal over the entire range of concentrations tested whereas propylene glycol was partially spermicidal at the highest concentration tested (EC50 = >4.2%). The concentration-dependent spermicidal activity by Cremophor EL and phospholipon 90G was associated with a parallel decline in sperm kinematics, 10 particularly with respect to track speed (VCL), path velocity (VAP), and straight line velocity (VSL). Example 3: Spermicidal Activity of Pharmaceutical Excipients of GM-4 and of the GM-4 Formulation in Human Semen 15 Methods and Materials The effect of duration of incubation on spermicidal activity in the presence of each of the eight pharmaceutical excipients was tested by mixing an aliquot of semen with equal volume of test compounds in BWW-0.3% BSA to yield the final 20 concentrations contained in GM-4. At timed intervals of 15, 30, 45 and 60 min, 5 pl samples were transferred to two 2 0-pm Microcell (Conception Technologies) chambers, and sperm motility was assessed by CASA. Sperm motility in samples too viscous for CASA analysis (seaspan carrageenan, viscarin carrageenan, Cremophor EL and phospholipon 90G) were determined by phase contrast 25 microscopy, and the number of motile sperm per treatment were enumerated for a total of 200 sperm. The time course test was performed in 3 separate trials, with semen obtained from three different donors. Modified Sander-Cramer Assay: The spermicidal activity of GM-4 formulation, as produced in Example 1, was tested by a modified Sander-Cramer 30 assay (Sander FV, et al., Hum Fertil, 1941;6:134-7.; D'Cruz OJ, et al., Contraception, 1999;59:319-31.). Briefly, aliquots (0.1 ml) of freshly liquefied semen were rapidly mixed with an equal volume of freshly prepared GM-4 formulation. A 5-pl sample was transferred to a 20 pm Microcell chamber (Conception Technologies) and examined immediately under a phase contrast 35 microscope (Olympus BX-20; Olympus Corporation, Lake Success, NY) attached to a CCD camera (Hitachi Deneshi Ltd., Tokyo, Japan) and a videomonitor. The time £YTTD('LrqT vr.TT,-.T -- V .. ... .

WO 00/56366 PCT/US00/07419 38 required for sperm immobilization was recorded in seconds. This test was performed in six separate trials, with semen obtained from six different donors. 5 Results: The results of spermicidal activity of individual components of GM-4 tested in human semen rather than using washed motile fraction of sperm are shown in Figure 3. A time course study of sperm motility impairment by each of the individual components of GM-4 formulation revealed that none of the eight 10 components tested including Captex 300, phospholipon 90G, Cremophor EL, propylene glycol, PEG 200, viscarin carrageenan, seaspan carrageenan and sodium benzoate was spermicidal in human semen (tl/2 = > 60 min). By contrast, the submicron particle size GM-4 formulation completely immobilized sperm in human semen in less than 2 min (1.2 ± 0.3 min). Thus, the 15 combination of these pharmaceutical excipients as a gel-microemulsion formulation was a potent spermicide in semen. EXAMPLE 5 Preparation and Characterization of GM-4 Formulation Containing 2% WHI-07. 20 WHI-07 is a phenyl phosphate derivative of bromo-methoxy zidovudine (WHI-07) with potent anti-HIV and spermicidal activities. WHI-07 is a lipophilic zidovudine (AZT) derivative which has extremely low solubility in water. WHI-07 has the following chemical structure:

H

3 Br NH 0 MeO A Br HO- -- O

N

3 25 M CO 2 Me Chemical name: (5R,6R)-and (5S, 6 S)-5-bromo-6-methoxy-5,6-dihydro AZT-5'-(para- bromophenyl methoxyalaninyl phosphate). Molecular weight: 698 30 WHI-07 was synthesized using the following synthetic scheme.

WO 00/56366 PCT/US00/07419 39 Synthetic Scheme for WHI-07 0 E 3 N/EbO Br / OH + C--P-CI EN Br/ O-pCi I I CI CI Me

NH

2 CHMeCQMe AZT/THF > B O-P-N CO 2 Me Et 3

N/CC

2 C I H N-Methylimidazole O O O H3Y ,H Br 2 /MeOH O Me NH B O-NO N B O-POO

N

3 I N, M C2Me M CO 2 3e Chemical Characterization: 5 Melting Point: 59-60 0 C; Rf: 0.56 ( 10%MeOH/ 90% CHC13 ); UV (MeOH) 209, 218, 221, & 261 nm; IR (Neat): 3218, 3093, 2925, 2850, 2105, 1712, 1484, 1378, 1241, 1153, 1010, 929 cm - 1 H NMR (CDCl3) d 8.66 (1H, br, 3-NH), 7.43 (2H, d, J = 9.0Hz, Aryl H), 7.14 (2H, d, J = 9.0 Hz, Aryl H), 6.01 (0.68H, t, J = 6.3 Hz, -CH at C-1'), 5.37 (0.32H, 10 m, -CH at C-1i'), 4.87 (0.68H, s, -CH at C-6), 4.61 ( 0.33H, s, -CH at C-6 ), 4.35 3.96 (6H, m, -CH at C-3', 4', 5' and Ala-NH, a-CH), 3.74 (3H, s, -COOCH3), 3.44 (3H, s, -OCH3 at C-6), 2.56-2.30 (2H, m, -CH2 at C-2'), 1.93 (3H, s, -CH3 at C-5), 1.38 (3H, m, a-CH3 of Ala).; 13C NMR (CDCl3) d 173.6, 166.7, 150.1, 148.9, 132.7, 132.5, 121.9, 121.7, 118.1, 87.7, 85.1, 81.7, 81.5, 81.4, 65.7, 65.6, 15 60.0, 57.9, 57.8, 53.6, 52.7, 50.3, 50.2, 36.9, 36.7, 22.8, 22.7, 21.2, 21.1.; 3 1 P NMR (CDCl3) d 2.70, 2.60, 2.54, 2.32.; MS (CI, m/e) 700.6 (M , 8 1 Br+ 8 1 Br), 698.6 (M', 8 1 Br+ 79 Br), 696.6 (M +, 79 Br+ 79 Br), 588.8 (M + - Br-OCH3, 8 1 Br), 586.9 (M - Br-OCH3, 79Br).; HPLC: 39.06, 40.28, 45.33, & 49.25 min (Column: LiChrospher 100 RP-18e (5 Itm); Flow rate: 1 mL/min; Solvent: H20 (0.1% TEA + 20 0.1% TFA) : CH3CN = 62:38). c~C~j r 'rr 'rr r'T- ,-, WO 00/56366 PCT/US00/07419 40 Appearance: White solid at room temperature; Solubility: < 0.003% solubility in water, soluble in oil (medium chain triglyceride, 4.1%), and quite soluble in some hydrophilic cosolvents such as polyethylene glycol 300 (13.1%). It is also soluble in several organic solvents including chloroform, ethanol, methanol, and DMSO.; 5 Octanol/Water partition coefficient: Log KD = 2.05 (see below) Partition coefficient of WHI-07: Four samples of WHI-07, 51.8 mg, 64.6 mg, 56.5 mg, and 74.5 mg were weighed and dissolved in 5 ml of octanol in four test tubes. After the drug was completely dissolved in octanol, 5 ml of water was added 10 to each octanol solution. The mixtures were handshaken vigorously for 10 min, and afterward let to stand overnight until a complete phase separation occurred. Samples of water and octanol were taken from each mixture, and directly injected into the HPLC for analysis. Peak areas of WHI-07 in the water layer and in corresponding octanol layer are shown in Table 6. The peak area ratios represent the 15 partition coefficient. Table 6 Sample Octanol layer Water layer Peak area ratio Peak area Peak area Octanol/water (mAU*s) (mAU*s) 1 5324.3 46.74 113.9 2 6159.2 55.32 111.3 3 5712.4 49.32 115.8 4 7347.4 67.25 109.2 The average octanol/water partition coefficient is: Log KD = 2.05 20 The suitable microemulsion compositions were identified by first constructing a series of ternary phase diagrams. The solubilizations of WHI-07 in several microemulsions selected from within the single phase microemulsion region in the phase diagrams were determined to identify microemulsions with high solubilizations of WHI-07. 25 Solubility of WHI-07 The solubility of WHI-07 in Captex 300 and polyethylene glycol 300 was carried out using UV-Vis spectrophotometer at 272 nm.

WO 00/56366 PCT/US00/07419 41 Calibration curve: Two stock solutions of WHI-07 were prepared in ethanol at concentrations of 4.2 mg/ml and 3.85 mg/ml respectively. These stock solutions were diluted in ethanol to prepare the standard solutions with concentrations and A272 shown in Table 7. The plot of the calibration curve is 5 shown in Curve 1. Table 7 Calibration sample Conc. in mg/ml A272 1 0.06 0.07 2 0.12 0.128 3 0.24 0.240 4 0.48 0.467 5 0.96 0.927 6 1.93 1.802 7 0.11 0.084 8 0.21 0.182 9 0.42 0.414 10 0.53 0.479 11 0.7 0.652 12 1.05 0.969 13 2.10 1.886 WO 00/56366 PCT/US00/07419 42 Curve 1 2 WHI-07 Conc mg/ml = 0.0134 + 0.912 x Correlation coefficient R^2 = 0.999 0 0 3 0 1 2 3 Abs (272 nrm) Sample preparation and assay 5 Appropriate amounts of WHI-07 and Captex 300 or PEG300 were weighed and placed in glass containers. Each sample was stirred with a stir bar at ambient temperature for 2 hours. Each sample was then filtered through 0.45 pm filter, and diluted with ethanol for UV-Vis measurement. The results are shown in Table 8. 10 Table 8 WHI-07 Solubility (mg/ml) Captex 300 40.6 +/-4.1 (n = 3) PEG300 131.5 +/- 4.9 (n = 3) Polymer suspensions were added to the microemulsions to increase their viscosity. Two types of polymers, xanthan gum and Carrageenan, were found to be particularly suitable but other types of polymers can also be used to produce gel 15 microemulsions. In general, a total polymer concentration of about between 0.5% and 3% is needed to provide a gel-microemulsion with adequate viscosity. Based on these results, we postulated that WHI-07 can be delivered using the GM-4 formulation. To this end, 18.7 mg of WHI-07 was first dissolved in 305 WO 00/56366 PCT/US00/07419 43 mg of a microemulsion consisting 32.4% of Captex 300 (medium chain triglyceride), 4.3% of purified water, and 63.3% of surfactant-cosolvent mixture containing 36% of Cremophor EL, 24% of Phospholipon 90G (purified soya lecithin), 20% of polyethylene glycol 200 and 20% of propylene glycol all by weight. The mixture 5 was heated to 700C. The drug was completely dissolved in the mixture after about 5 minutes of mixing with a stir bar. The drug solution was then removed from the heat. The resulting composition was a clear microemulsion. This WHI-07-containing microemulsion concentrate was diluted with water to bring the intensity to between 300 and 500 KHZ for optimal particle size 10 measurement. The particle diameter of the microemulsion in the absence of polymers was determined by laser light scattering using Nicomp 380 Submicron Particle Sizer (Particle Sizing Systems, Inc., Santa Barbara, CA). The size distribution and mean particle diameter value became stabilized quickly after a few minutes of run time. A average (mean±SD) particle diameter of 18.5+8.1 nm was 15 determined by Nicomp 380 photon correlation light scattering particle sizer. WHI-07 concentration analysis in WHI-07 Formulations Routine analysis of WHI-07 were performed by dissolving the samples in ethanol or acetonitrile and analyzed with HPLC after appropriate dilution. WHI-07 20 gel formulations, however, are not totally soluble in either ethanol or acetonitrile because of the polymers in the formulation. The following WHI-07 analytical methods were developed for the gel formulations. The accuracy and precision of the methods meet pharmaceutical requirements. 25 WHI-0 7 calibration curve 101.3 mg of WHI-07 (96% purity) was dissolved in a 10 ml glass vial with 1.5 ml of a microemulsion with the following composition: 36% Cremophor EL, 24% Phospholipon 90G, 20% PEG 200, and 20% propylene glycol. The mixture was stirred for 10 min at 500C until the drug was completely dissolved and the 30 mixture was clear. The vial was removed from the hot plate and 3.5 ml of the polymer suspension (1.3% Seaspen Carrageenan, 0.7% Xantural, and 0.3% sodium benzoate in DI water) was added to the vial. The vial was hand shaken for 1 min and vortexed for 1 min or until the mixture was homogeneous. c'YTDr~TrrrTrrF yrrr' -- ~r ,- .- WO 00/56366 PCT/US00/07419 44 The control (drug-free) gel microemulsion was prepared by mixing 1.2 ml of the microemulsion with 2.8 ml of the polymer suspension. The calibration curve was prepared as following: Five standard solutions with varying WHI-07 contents were prepared as 5 shown in Table 9. Each vial was hand shaken and the polymers in the gel was precipitated out. The mixture was then centrifuged at room temperature for 10 min at 2000 rpm. The vials were carefully removed from the centrifuge and 0.5 ml of the supernatant was pipetted out from each vial for HPLC analysis. 10 The condition for HPLC analysis (WHI-07) was as followed: Column: RP18e (5pm) Lot # L228433 Eluent: Acetonitrile / (0.1% TFA and 0.1% TEF) = 45 / 55 Eluent flowrate: 1 ml/min Sample injection volume: 20 pl 15 Method run time: 30 min Under these conditions, the retention times for the three isomeric peaks of WHI-07 were 20.5 min, 23.2 min, and 24.9 min. The area ratio of these peaks were 31.9 : 5.7 : 3.3. The peak with retention time of 20.5 min was used to construct the 20 calibration curve by plotting WHI-07 concentration vs. peak area. The calibration curve was linear in the WHI-07 concentration range of 0 to 1.3 mg/ml with a correlation coefficient of 1.000 (Curve 2). Table 9 25 Vial # WHI-07 gel Control gel ACN added WHI-07 concentration Peak(20 min) added (ml) added (ml) (ml) (mg/ml) (mAU*s) (20.26 mg/ml) 1 0 0.50 4.0 0 0 2 0.05 0.45 4.0 0.225 1121.2 30 3 0.10 0.40 4.0 0.450 2348.4 4 0.20 0.30 4.0 0.900 4682.6 5 0.30 0.20 4.0 1.351 6998.1 CTTIC'TI'rTT'r7' C]7 ELrr /nrr - "I WO 00/56366 PCT/US00/07419 45 Curve 2: Calibration curve for WHI-07 gel formulation 2 WHI-07 Conc mg/mi = 0.0022 + 1.924 x Peak area / Correlation coefficient R^2 = o 1 0 2000 4000 6000 8000 HPLC Peak areas Precision of the analytical method 5 To test the precision of the method, ten WHI-07 gel samples were prepared as following, and HPLC analysis was performed. 101.3 mg of WHI-07 (99.4% purity) was dissolved in a 10 ml glass vial with 1.5 of the microemulsion with composition of 36% Cremophor EL, 24% Phospholipon 90G, 20% PEG 200 and 20% propylene glycol. The mixture was stirred for 10 min at 50 0 C until the drug 10 was dissolved and the mixture was clear. The vial was removed from the hot plate and 3.5 ml of the polymer suspension (1.3% Seaspen Carrageenan, 0.7% Xantrual and 0.3% sodium benzoate in DI water) was added to the vial. The vial was hand shaken for 1 min and vortexed for 1 min or until the mixture was homogeneous. The control gel was prepared by mixing 1.2 ml of the microemulsion with 15 2.8 ml of the polymer suspension. To each of the 10 vials, 0.2 ml of the WHI-07 gel, 0.3 ml of the control gel and 4 ml of acetonitrile were added. The vials were then handshaken for 1 min and vortexed for 1 min. The mixture was then centrifuged at room temperature for 10 min at 2000 rpm. The vials were carefully removed from the centrifuge and 0.5 ml of the supernatant was pipetted out from 20 each vial for HPLC analysis. The data was analyzed to determine the precision of the method. The results are shown in Table 10. The relative standard deviation of WO 00/56366 PCT/US00/07419 46 the method was 0.61%. These data indicate that the developed method has a good precision for the analysis of WHI-07 content in the gel formulation. Table 10 5 Vial # Peak area(mAU*s) deviation relative standard deviation (RSD) 1 2312.8 18.04 2 2285.2 -7.56 3 2272.0 -22.76 10 4 2279.4 -15.36 5 2296.0 1.24 6 2275.6 -19.16 7 2299.4 4.64 8 2291.2 -3.56 15 9 2332.8 38.04 10 2303.2 8.44 Average 2294.76 14.08 0.61% 20 Procedures for WHI-07 formulation sample preparation: 240 mg of WHI-07 was weighed in a 10 ml glass shell vial. 4.0 ml of the microemulsion was transferred to the vial with a 1.0 ml Drummond pipette. A 12 X 4 mm magnetic stir bar was placed in the vial and the vial was placed on the Coming stir/hot plate for mixing. The dials for both stir and heat were set at #3. The mixture 25 was heated for 5 min until the drug crystals were disappeared and the mixture was clear. WHI-07 concentration in this microemulsion was 60 mg/ml. 2% WHI-07 gel was prepared by mixing 2 ml of the concentrated WHI-07 microemulsion with 4 ml of the polymer suspension in a 10 ml glass shell vial. 1% WHI-07 gel was prepared by mixing 1 ml of the concentrated WHI-07 30 microemulsion with 1 ml of the microemulsion and 4 ml of the polymer suspension. 0.5% WHI-07 gel was prepared by mixing 0.5 ml of the concentrated WHI-07 microemulsion with 1.5 ml of the microemulsion and 4 ml of the polymer suspension.

WO 00/56366 PCT/US00/07419 47 The control gel was prepared by mixing 2 ml of the microemulsion and 4 ml of the polymer suspension. WHI-07formulation concentration analysis: Two samples, 100 pl each, were taken from each preparation for concentration analysis. The samples were 5 placed in 5 ml glass shell vials. 4.2 ml of acetonitrile was added to each of the sample vials. The vials were hand shaken for 10 seconds and were placed in a shaker at 200 rpm for 30 min. The vials were hand shaken again for 10 seconds and span for 10 min at 2000 rpm in the Beckman GS-6 Centrifuge. The vials were carefully removed from the centrifuge and 1 ml each of the supernatant was pipette 10 out for HPLC analysis. The results were shown in Table 11. All of the samples meet pharmaceutical requirement (RSD less than 5.0%). Table 11 Vial # Sample ID WHI-07 Average RSD 15 concentration 1 0.5% WHI-07 0.52% 2 0.5% WHI-07 0.49% 0.5% 0 3 1.0% WHI-07 1.02% 4 1.0% WHI-07 0.92% 0.97% 3.0% 20 5 2.0% WHI-07 1.88% 6 2.0% WHI-07 1.99% 1.94 3.0% 25 We next examined the shelf life/stability of the WHI-07. A gel microemulsion with WHI-07 concentration of 0.5% and 2.0% were prepared and were analyzed for WHI-07 concentrations at day 1, day 4 and day 8. The result is shown in the following Table 12: WO 00/56366 PCT/US00/07419 48 Table 12 Time (day) 0.5% WHI-07 2.0% WHI-07 gel gel microemulsion microemulsion 1 0.5 2.0 4 0.49 2.0 8 0.51 1.97 Percent change at day 2% 1.5% 8 The result indicates that WHI-07 was stable in the gel microemulsion 5 formulations were stable at room temperature in the 8 day observations. Formation of gel-microemulsion: This drug-containing microemulsion was then mixed with 600 mg of a polymer suspension containing 1.3% of Seaspan Carrageenan, 1.3% of Viscarin Carrageenan, and 0.3% of sodium benzoate by weight with gentle mixing. The resulting gel microemulsion had a pH of 7.2 and 10 was a translucent gel with the following composition: % (by weight) WHI-07 2.0 Captex 300 10.7 15 Phospholipon 90G 5.0 Cremophor EL 7.5 Propylene glycol 4.2 PEG 200 4.2 SeaSpan Carrageenan 0.9 20 Viscarin Carrageenan 0.9 Sodium benzoate 0.2 Water 64.4 The viscosity of the GM-4 formulation with and without WHI-07 was 25 determined using a Brookfield DV-E Viscometer with spindle #3 (speed: 10 rpm). Drug-free GM-4 formulation had a viscosity of 301.9 centripoises. The GM-4 formulation containing 2% WHI-07 had a viscosity of 64.7.

WO 00/56366 PCT/US00/07419 49 Although a viscosity of 64.7 appeared to be sufficient for the utility of the 2% WHI-07 containing GM-4 formulation to prevent transvaginal or transrectal transmission of FIV, the viscosity could easily be increased by using a different polymer suspension. For example, a thicker formulation was prepared as follows: 5 This drug-containing microemulsion in Example was mixed with 600 mg of a polymer suspension containing 1.3% of Seaspan Carrageenan, 0.7% of Xantural, and 0.3% of sodium benzoate by weight with gentle mixing. The resulting gel microemulsion was a translucent gel-like liquid with the following composition: 10 % (by weight) WHI-07 2.0 Captex 300 10.7 Phospholipon 90G 5.0 Cremophor EL 7.5 15 Propylene glycol 4.2 PEG 200 4.2 SeaSpan Carrageenan 0.9 Xantural 0.5 Sodium benzoate 0.2 20 Water 64.8 The viscosity of this formulation was 380 centripoises. This formulation was also found to have good colloidal stability when stored at room temperature for several months. 25 EXAMPLE 4: Preclinical Studies: Methods and Materials Rabbits: Fifty nine female and 12 male, sexually mature (> 6 months old; > 30 7 lbs), specific-pathogen-free, New Zealand White rabbits were obtained from Charles River Laboratories (Wilmington, DE). For each fertility trial, 24 does and 12 bucks were used. All rabbits were uniquely identified with metal ear tags. Tap water and rabbit food pellets (Teklad LM-485; Harlan Teklad) were available ad libitum. The does and bucks were maintained in separate rooms that were kept at 22 35 + 2oC with relative humidity of 50 + 20% and a 12-h fluorescence light cycle. The WO 00/56366 PCT/US00/07419 50 rabbits were isolated for a minimum of 4 weeks before the fertility trials. All procedures were approved by the Parker Hughes Institute Animal Use and Care Committee. All animal husbandry operations were conducted under current USDA Guidelines. 5 Mice: Twenty, female B6C3F 1 mice of approximately 6 weeks of age were obtained from Charles River Laboratories (Wilmington, DE) and were uniquely identified with metal ear tags and ear notches. Tap water and laboratory diet (Teklad LM-485; Harlan Teklad) were available ad libitum. The animals were maintained in a room that was kept at 22 ± 2 0 C with relative humidity of 50 ± 10% and a 12-h 10 fluorescence light cycle. All animal husbandry operations were conducted under NIH 1996 Guidelines. In Vivo Contraceptive Efficacy in the Rabbit Model: For each fertility trial, 24 does and 12 bucks were used. For each contraceptive test, the does were divided into 3 subgroups of 8; 1) control does; 2) GM-4 group and; 3) N-9 group. 15 Semen was obtained from bucks (n = 12) of proven fertility via a prewarmed (45 0 C) artificial vagina immediately before use. Sperm count and motility was assessed to ensure that the males were ejaculating good quality semen. Prior to artificial insemination, semen samples without the contamination of urine or gel were pooled and 0.5 ml (>30 x 107 sperm/ml) aliquots were transferred to 1 ml tuberculin 20 syringes. Two ml of a GM-4 formulation or a commercial 2% N-9 formulation (Gynol II; Ortho Pharmaceutical Corp., Raritan, NJ) was applied intravaginally by means of a 3 ml disposable plastic syringe. The doe was held in a supine position during the application of 2 ml of the test agent followed by the application of semen dose (0.5 ml) which was deposited within 1-2 min by inserting approximately 8 cm 25 of the syringe into the vagina for the delivery of the test agent. At the time of artificial insemination, ovulation was induced by an intravenous injection of 100 IU of human chorionic gonadotropin (Sigma Chemical Co., St. Louis, MO) into the marginal ear vein. After ovulation and artificial insemination, the does were allowed to complete their pregnancy (31 ± 2 days). Pregnant does were transferred to cages 30 containing nest boxes (16 x 12 x 6 in). The litter size, weight, fetal length, and the condition of each offspring at birth were recorded. The in vivo spermicidal effect of GM-4 formulation versus 2% N-9 formulation was assessed based on the level of pregnancy reduction achieved in comparison to controls and the consistency of this response. The vaginal delivery/artificial insemination and pregnancy cycle was 35 repeated a second time.

WO 00/56366 PCT/US00/07419 51 Rabbit Vaginal Irritation Test: For the vaginal irritation study, eleven female rabbits were treated intravaginally with 1 ml of GM-4 (seven rabbits) or 1 ml of GM-4-containing 4% N-9 (four rabbits), for 10 consecutive days. Animals were sacrificed on day 11 and the reproductive tract was examined grossly and 5 microscopically after completion of the study (Eckstein P, et al., J Reprod Fertil, 1969;20:85-93.). The vaginal tissues were rapidly removed and parts of the caudal, mid, and distal regions of each vagina were fixed in 10% buffered formalin. Tissues were embedded in paraffin, sectioned at 4-6 gm and stained with hematoxylin and eosin and examined under x200 and x400 magnification using a Leica light 10 microscope (Milton Keynes, Buckinghamshire, UK) interfaced with an image analysis system. The images were captured using the ImagePro Plus program (Media Cybernetics, Silver Spring, MD) in conjunction with a 3CCD camera (DAGE-MTI Inc., Michigan City, KS), and images were transferred to Adobe Photoshop 5.5 software (Adobe Systems Inc., San Jose, CA) for observation and 15 analysis. Each of the three regions of vagina were examined for epithelial ulceration, edema, leukocyte infiltration, and vascular congestion. The scores were assigned based on the scoring system of Eckstein et al., (Eckstein P, et al., JReprod Fertil, 1969;20:85-93.) which was as follows: Individual score: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = intense irritation; Total score: <8 acceptable, 20 9-10 marginal, and 11 unacceptable. Results were expressed as the mean + SD values. Thirteen-Week Toxicity Study in Mice: Twenty, female B6C3F1 mice were allocated to two groups. The test group of 10 mice received 50 pl of the GM-4 formulation intravaginally for 5 days per week for 13 consecutive weeks. Ten mice 25 without intravaginal treatment served as the control group. The GM-4 formulation was prepared weekly and the intravaginal treatment was performed inside a microisolator. All animals were individually observed daily for signs of toxic effects. Body weights were obtained before exposure (day 0), weekly during exposure, and preceding sacrifice. At the end of the study, animals were sacrificed 30 for pathologic and histopathologic examinations and determination of blood chemistry. Hematological analyses were performed from blood obtained from 5 control and 5 test mice. Hematology Parameters: Complete blood counts and differentials were obtained using an Abbot CELL-DYN 3200 multiparameter, automated hematology 35 analyzer (Abbot Laboratories, Abbot Park, IL) which was standardized for mouse WO 00/56366 PCT/US00/07419 52 blood. This instrument uses flow cytometric techniques to provide the hemograms for anticoagulated whole blood samples: red blood cell count (RBC; 10 6 /l), total and differential leukocyte count (lymphocytes [LYM], neutrophils [NEU], monocytes [MONO], eosinophils [EOS], and basophils [BASO] as 10 3 /ptl or %), 5 hemoglobin concentration (HGB; g/dl), hematocrit (HCT; %), mean corpuscular volume (MCV; fl), mean cell hemoglobin content (MCH; pg), mean cell hemoglobin concentration (MCHC; g/dl), red cell distribution width (RDW; %), platelet count (PLT; 10 3 / t1), and mean platelet volume (MPV; fl). Clinical Chemistry Profiles: Biochemical analyses were performed using a 10 Beckman SYNCHRON CX5CE random access analyzer (Beckman Coulter Inc., Fullerton, CA). After 13 weeks of intravaginal application of GM-4, blood was obtained from GM-treated and control mice in lithium heparin tubes and the clarified plasma was used for the determination of serum/plasma levels of total protein (TP), albumin/globulin (ALB/G), blood urea nitrogen (BUN), creatinine 15 (CRE), total cholesterol (CHO), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), amylase (AMY), total bilirubin (TBIL), glucose (GLU), and calcium (Ca), phosphorous (P), sodium (Na), potassium (K), and chloride (Cl) using reagents and methods provided by the manufacturer. Necropsy and Histopathology: Mice were killed after 13 weeks of 20 intravaginal exposure of GM-4 for complete necropsy evaluations. The thymus, lungs, heart, liver, pancreas, spleen, kidneys, reproductive organs (ovaries, uteri and vaginal tissue) and brain from each of 10 mice were weighed at necropsy. Organ weights were recorded as absolute weights and as a percentage of body weight. The above mentioned organs and the bone and bone marrow, large and small intestine, 25 skeletal muscle, skin, spinal cord, and urinary bladder were fixed in 10% buffered formalin solution, trimmed, embedded in paraffin, sectioned at 4-6 Itm, and stained with hematoxylin and eosin. Complete histopathological examination of all tissues was performed on mice from the control and GM-4 group. StatisticalAnalysis: Group means and standard deviations were calculated 30 from initial and terminal body weights, organ weights, hematology and clinical chemistry parameters. Statistical significance of the differences between the treated group mean versus the control group was analyzed by a one-way analysis of variance, followed by Dunnett's multiple comparison test using GraphPad Prism software (San Diego, CA). The significance of differences in fertility between the WO 00/56366 PCT/US00/07419 53 groups was analyzed by Fisher's exact test. Differences were considered statistically significant ifp < 0.05. Cat Studies: FeT-J cell line is a feline T-cell line chronically infected with the FIV strain Bangston (FIVBang). Cats were challenged with FIV by inoculating 5 7x1 0 6 infected FeT-J cells mixed in 0.2 mL of infected culture fluid in the vagina or in the rectum 1 min after insertion of 0.4 mL of the WHI-07 gel formulation. The FIV load in the peripheral blood mononuclear cells (PBMC), lymph node (LN) cells, and bone marrow (BM) cells was measured by quantitative virus isolation and PCR analysis, as previously described (Rey MA et al., Biochem Biophys. Res. Commun. 10 1984, 121:126-33; Diehl LJ et al., J Virol 1995, 69:2328-32; Greene WK et al., Arch Virol 1993, 133:51-62; Okada S et al., AIDS Res Hum Retroviruses 1994, 10:1739-46; Tellier MC et al., Vet Microbiol 1997, 57:1-11). The cells from treated and untreated cats ( 5x10 6 cells/culture) were cocultured for 3 weeks with 5x10 6

T

cell enriched PBMCs from specific pathogen-free (SPF) cats in a total volume of 5 15 ml in a 25 cm 2 flask. Culture supernatants were harvested and cells were resuspended in fresh culture media every 3 days. Viral production was determined by measuring the levels of RT activities in the culture supernatants and examining the cells for proviral DNA by FIV gag-specific and env-specific PCR at the termination of cultures (Diehl LJ et al., J Virol 1995, 69:2328-32; Greene WK et 20 al., Arch Virol 1993, 133:51-62; Okada S et al., AIDS Res Hum Retroviruses 1994, 10:1739-46). Serum samples were also examined by standard Western blot analysis for the presence of FIV core antigen p25. Cats were considered FIV positive if one of the following criteria was met during the 18-week observation period: (1) Serum samples from two different bleeding dates were positive by Western blot analysis; 25 (2) A single Western blot result and a single virus isolation test from a different bleeding date were positive, with or without a positive PCR test; (3) PBMC, lymph node or bone marrow cells from two different bleeding dates were positive by virus isolation test; or (4) mononuclear cells from two different bleeding dates were positive by PCR with the same tissue source. 30 Results In Vivo Contraceptive Activity of GM-4 versus N-9 Formulation in the Rabbit Model: Because of the rapid spermicidal activity of GM-4 formulation, we performed in vivo contraceptive efficacy studies of GM-4 formulation in the 35 standard rabbit model. Gynol II, a commercial contraceptive containing 2% N-9, C~TTDC-rT'7rr77 C'T TF' , , - - - - WO 00/56366 PCT/US00/07419 54 was tested in the same way for comparison. Ovulated NZW rabbits were given intravaginal application of GM-4 formulation or N-9 formulation immediately prior (< 2 min) to artificial insemination with fresh pooled semen and the females were allowed to complete their pregnancy. The efficacy of GM-4 formulation versus 5 Gynol II for preventing pregnancy in the rabbit model are summarized in Table 13. In the control group, 15 out of 16 (93.7%) rabbits artificially inseminated became pregnant and delivered a total of 123 newborn rabbits. By contrast, none of the 16 rabbits given GM-4 formulation prior to artificial insemination became pregnant (p <0.0001, Fisher's exact test). Whereas rabbits given Gynol II, 5 out of 16 (31.2%) 10 rabbits became pregnant (p = 0.0006) and delivered a total of 34 newborn rabbits. Thus, the GM-4 formulation was far more effective than Gynol II as a vaginal spermicidal contraceptive (100% vs 6 8

.

7 %,p < 0.05, Fisher's exact test). Table 13 15 Fertility of female rabbits after artificial insemination/ovulation induction with and without intravaginal application of GM-4 formulation or Gynol II containing 2% N-9 reatment No. of does No. of does fertile (% Litter size inseminated None 16 15 (93.7) 123 GM-4 16 0(0) 0 Gynol II (2%-N-9) 16 5 (31.2) ' 34 Aliquots (0.5 ml) of fresh, pooled semen obtained from fertile bucks (n = 12) 20 were used to artificially inseminate the does within 1-2 min following intravaginal application of 2 ml of GM-4 or N-9 formulation. Does were induced to ovulate by an intravenous injection of 100 IU ofhCG and allowed to complete term pregnancy. Significantly different from control by Fisher's exact test, (p < 0.0001) 25 Significantly different from control by Fisher's exact test, (p = 0.0006) Lack of Vaginal Irritation from GM-4 in the Rabbit Model: Histological evaluation of three different regions of the vaginal tissue after daily intravaginal application of GM-4 for 10 consecutive days showed lack of significant vaginal 30 irritation in all seven rabbits examined (mean individual scores 0-1; total score 2, C rTDC11T'r1rYTTT C Tlr~r ,,T - -r~ - " , WO 00/56366 PCT/US00/07419 55 range 0-3) [Table 14]. In contrast, all rabbits treated with 4% N-9 had epithelial ulceration, edema, leukocyte influx, and vascular congestion characteristic of inflammation (mean individual scores 1-3; total score 9, range 7 to 11) as quantitated by histological scoring according to the method of Eckstein et al 5 (Eckstein P, et al., JReprod Fertil, 1969;20:85-93.). Figure 4 shows the representative vaginal section from a GM-4-treated rabbit which showed intact vaginal epithelium when compared with the vaginal section of a N-9-treated rabbit which revealed disruption of the epithelial lining, and an inflammatory response with influx of leukocytes, consistent with previously published observations in rats 10 (Tryphonas L, et al., Toxicol Lett, 1984;20:289-95.). Table 14 Scoring of histological changes in the rabbit vaginal tissue after 10 days of intravaginal application of GM-4 formulation with and without 4% N-9 15 GM-4 GM-4 + 4% N-9 (n = 7) (n = 4) Epithelial ulceration 0 3 ± 2 *t Lamina propria thickness 1 ± 1 2 ± 1 Leukocyte Infiltration 1 ± 1 3 ± 2 Vascular congestion 0 1 ± 1 Total score 2 ± 1 t 9 ± 2 Seven rabbits were administered intravaginally with 1 ml of GM-4 and 4 rabbits S were exposed to GM-4 containing 4% N-9. Mean ± SD values representing the caudal, middle, and distal sections of vagina 20 from each rabbit. ('FTD r T'7'7TT'rr' C'TTr'T"T' ,r,77T T- ', WO 00/56366 PCT/US00/07419 56 Semiquantitative scoring based on Eckstein et al [35]. Individual score: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = intense. Total score: <8 acceptable, 9-10 marginal, and I l unacceptable. 5 Lack of Systemic Toxicity from GM-4 in Mice: Female B6C3F I mice (n = 10) were treated with intravaginal application of GM-4, 5 days per week, for 13 consecutive weeks. Mortality did not occur and there were no clinical signs attributed to intravaginal exposure of GM-4 throughout these studies. All animals were clinically healthy at the end of the study. Mean body weight gain and final 10 mean body weight of control mice (27.4 ± 1.6 g; n = 10) and test mice (28.0 ± 3.0 g; n = 10) exposed to GM-4 formulation were similar (Figure 5). Complete blood counts of mice revealed no biologically significant differences between GM-4 -treated and control mice. The values of hematologic parameters for red cell, leukocyte, lymphocyte, platelet counts, and hemoglobin were 15 within normal limits (Table 15). Analysis of blood chemistry parameters for female mice revealed no significant treatment-related differences between GM-4-treated and control groups (Table 16). The kidney function (BUN and CRE), liver function (TBIL, AST, ALT, ALB/G, GLU, and TG), pancreas function (AMY and GLU), and nutritional status (TP), were not affected adversely by repeated intravaginal exposure 20 to GM-4 formulation. Table 15 Hematological Findings for B6C3F1 Mice Given GM-4 Intravaginally for 13 Weeks Parameter Control GM-4 A0/p1 * * RBC (x 104/l) 949 ± 62 905 ± 61 WBC (x 104/tl) 2.6 1.2 2.0 ± 0.7 LYM (%) 76.3 ± 17.7 85.4 ± 5.6 NEU(%) 3.0 1.2 2.5 ± 0.8 (OTTDVrr'rT'T'- ',vIr -',",,, I - - , WO 00/56366 PCT/US00/07419 57 Others (%) 20.6 ± 17.9 19.8 ± 14.9 HGB (g/dl) 14.3 ± 0.8 13.5 ± 0.8 HCT (%) 54.3 ± 3.5 51.4 ± 3.0 MCV (fl) 57.2 ± 1.6 56.4 ± 0.8 MCH (pg) 15.1 ± 0.2 15.0 ± 0.1 MCHC (g/dl) 26.4 ± 0.9 26.4 ± 0.4 RDW 9.2 ± 3.5 8.8 ± 1.9 PLT (x 10 4 /Il) 358 ± 226 413 ± 327 MPV (fl) 7.4 ± 2.7 6.2 ± 0.7 .Mean ± SD for groups of 5 mice. Others = MONO, EOS, AND BASO. RBC, red blood cells; WBC, white blood cells; LYM, lymphocytes; 5 NEU, neutrophils; MONO, monocytes; EOS, eosinophils; BASO, basophils; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; RDW, red cell distribution width; PLT, platelets; MPV, mean platelet volume. 10 Table 16 Blood Chemistry Profiles for B6C3F1 Mice Given GM-4 Intravaginally for 13 Weeks Parameter Control GM-4 TP g/dl 4.8 0.1 4.8 ± 0.1 ALB/G g/dl 3.6 0.2 3.8 ± 0.3 WO 00/56366 PCT/US00/07419 58 BUN mg/dl 24 ± 2 26 ± 4 CRE mg/dl 0.25 ± 0.05 0.27 ± 0.04 CHO mg/dl 109 ± 6 104 ± 6 TG mg/dl 174 ± 38 166 ± 33 AST IU/1 210 ± 138 315 ± 206 ALT IU/I 125 ± 87 103 ± 89 AMY IU/1 1783 ± 134 1667 ± 377 TBIL mg/dl 0.16 ± 0.05 0.18 ± 0.06 GLU mg/dl 325 ± 23 310 ± 50 Ca mg/dl 9.2 ± 0.2 8.6 ± 0.7 P mg/dl 6.9 ± 1.4 6.4 ± 1.2 Na mg/dl 152 ± 2 150 3 K mg/dl 3.9 ± 0.4 5.2 1.4 Cl mg/dl 109 ± 1 110 ± 2 Mean ± SD for groups of 8 mice. Significantly different from control group (p < 0.05). 5 13-Week Necropsy/Organ Weights and Histopathology: Table 17 summarizes the terminal body weight, terminal absolute and relative organ weights observed at the conclusion of the 13-week study. No statistically significant WO 00/56366 PCT/US00/07419 59 differences were observed between the absolute and relative organ weights of test versus control mice. Microscopic examination of bone and bone marrow, brain, gut, heart, kidney, liver, lung, ovaries, pancreas, skeletal muscle, skin, spinal cord, spleen, urinary bladder, uterus, and vaginal specimens taken from the study animals 5 did not reveal any treatment-related lesions (data not shown). No histopathological lesions were observed in the ovarian, uterine and vaginal tissues of GM-4-treated mice which suggests lack of toxicity to repeated intravaginal exposure of the lipophilic and spermicidal GM-4 formulation. 10 Table 17: Absolute and Relative Organ Weights of B6C3F1 Mice Given GM-4 Intravaginally for 13 Weeks Control GM-4 Organ Absolute Relative Absolute Relative (g) (g%) (g) (g%) Terminal 27.4 1.6 28.0 3.0* Body weight Thymus 0.09 ± 0.01 0.32 + 0.03 0.10 ± 0.03 0.35 + 0.10 Lung 0.28 ± 0.02 1.02 ± 0.07 0.29 ± 0.03 1.03 ± 0.10 Heart 0.14 ± 0.01 0.51 ± 0.03 0.13 ± 0.01 0.46 ± 0.03 Liver 1.62 ± 0.13 5.91 ± 0.47 1.73 ± 0.22 6.17 ± 0.78 Pancreas 0.15 ± 0.02 0.54 ± 0.07 0.14 ± 0.03 0.49 ± 0.10 WO 00/56366 PCT/US00/07419 60 Spleen 0.12 ± 0.02 0.43 ± 0.07 0.10 ± 0.01 0.35 ± 0.03 Rep Org 0.17 ± 0.02 0.62 ± 0.07 0.17 ± 0.04 0.60 + 0.14 Kidney 0.18 ± 0.01 0.65 ± 0.03 0.17 ± 0.01 0.60 ± 0.03 Brain 0.55 ± 0.02 2.00 + 0.07 0.53 ± 0.04 1.89 ± 0.14 Mean ± SD for groups of 10 mice. Reproductive organs (ovaries, uteri, and vagina). 5 WHI-07/GM-4 Gel Formulation Prevents Vaginal and Rectal FIV Transmission in Cats. A total of 10 cats, including 5 control cats and 5 cats treated with WHI 07(2%)/GM-4 vaginal gel formulation were challenged with an intravaginal inoculum of 7x1 06 FIVBang-infected FeT-J cells mixed in 0.2 mL of infected 10 culture fluid. As shown in Table 18, WHI-07/GM-4 formulation provided 60% protection against FIV. Similarly, WHI-07/GM-4 was also able to prevent the transrectal transmission of FIV (Table 19).

WO 00/56366 PCT/US00/07419 61 Table 18 WHI-07(2%)/GM-4 Gel Formulation Prevents Vaginal Transmission of FIV in Cats 5 Cat No. Treatment WB Virus Isolation PCR Overall Protection Serum PBMC PBMC Conclusion Rate 1 None 3X 3X 3X + 10 2 None 5X 4X 2X + 3 None 1X 2X 2X + Not applicable 4 None 5X 5X 6X + 5 None 4X 2X 2X + 6 WHI-07/GM-4 0 0 0 15 7 WHI-07/GM-4 0 0 0 8 WHI-07/GM-4 5X 5X 4X + 60% 9 WH1I-07/GM-4 0 0 0 10 WHI-07/GM-4 5X 6X 5X + 20 The FIV status of cats was examined at 0, 3, 6, 9, 12, 15, and 18 weeks post intravaginal FIV challenge. The number of different assessment times with positive test results is indicated for each assay. WB: Western blot; PCR: polymerase chain reaction; PBMC: peripheral blood mononuclear cells.

WO 00/56366 PCT/US00/07419 62 Table 19 WHI-07(2%)/GM-4 Gel Formulation Prevents Rectal Transmission of FIV in Cats 5 Cat No. Treatment WB Virus Isolation PCR Overall Infection Serum PBMC PBMC Conclusion Rate 1 None 6X 6X 6X + 10 2 None 0 0 0 - 67% 3 None 2X 3X 4X + 4 WHI-07/GM-4 0 0 0 5 WHI-07/GM-4 0 0 0 - 0% 6 WHI--07/GM-4 0 0 0 15 The FIV status of cats was examined at 0, 3, 6, 9, 12, 15, and 18 weeks post intrarectal FIV challenge. The number of different assessment times with positive test results is indicated for each assay. WB: Western blot; PCR: polymerase chain reaction; PBMC: peripheral blood mononuclear cells. 20 Discussion: The first objective of our studies was to determine the in vivo contraceptive efficacy of spermicidal GM-4. Since the rabbit provides a standard animal model for testing vaginal agents for antifertility activity (Castle PE, et ai., Biol Reprod, 1997;56:153-9.; Castle PE, et al., Contraception, 1998;58:51-60.), we tested the 25 ability of intravaginally applied GM-4 to prevent pregnancy in ovulated rabbits. We confirmed that vaginal delivery of GM-4 formulation prior to artificial insemination can prevent pregnancy in the rabbit. Our in vivo contraceptive efficacy studies included term pregnancy as well as the analysis of normalcy of the resulting pregnancies. The GM-4 formulation showed remarkable contraceptive activity in 30 the rigorous rabbit model. In two separate fertility trials, a 100% contraceptive effect was obtained despite the fact that the rabbit ejaculate used contained >1000 fold larger inseminating doses than in humans (Castle PE, et al., Biol Reprod, 1997;56:153-9.). To our knowledge, these experiments are the first to demonstrate the in vivo contraceptive efficacy of a GM formulation prepared from commonly 35 used pharmaceutical excipients. The 100% contraceptive efficacy obtained with GM-4 is most likely due to rapid spreadability of GM-4 across the vaginal mucosa WO 00/56366 PCT/US00/07419 63 as well as to its rapid spermicidal activity. In contrast, the contraceptive effect of N 9 has been shown to be highly dependent on the time interval between delivering the agent to the vagina and coitus or artificial insemination. It takes several minutes for N-9 gel to distribute in the rabbit vagina (Castle PE, et al., Contraception, 5 1998;58:51-60.). Thus, gels may be slower to mix with vaginal secretions than the GM-4 formulation. Therefore, a large excess of N-9 (400-fold greater dose) is required to achieve in vivo contraceptive activity (Castle PE, et al., Contraception, 1998;58:51-60.). In fact, in over-the-counter formulations, N-9 is being used at concentrations of 2 to 6% in creams and gels, 12% in foams and as high as 18% in 10 condom lubricants. The partial (68.7%) contraceptive effect of a commercial 2% N 9 gel observed in our study when compared with 100% efficacy of GM-4 is in agreement with the high contraceptive failure rates reported for N-9 (Trussell J, et al. Stud Fam Plann, 1987;18:237-83.; Kulig JW, Ped Clinic North Am, 1989;36:717-30.; Raymond E, et al., Obstet Gynecol, 1999;93:896-903.). Our 15 studies suggest that this is most likely due to incomplete mixing of semen with N-9 gel or inadequate distribution of the agent throughout the vagina. The second objective of these studies was to determine the toxic effects, if any, resulting from repeated intravaginal application of spermicidal GM-4. Because of the potent in vitro and in vivo spermicidal activity of GM-4 formulation, it was 20 necessary to evaluate the toxicity to vaginal mucosa particularly in the rabbit vaginal irritation test. In the rabbit vaginal tolerance test, the GM-4 formulation lacked mucosal toxicity in contrast to 4 %-N-9-containing GM-4 formulation after daily application for 10 days. Our results clearly demonstrated that the GM-4 is not damaging to vaginal mucosa of the rabbit despite the fact that it was a potent 25 spermicidal agent when added to human or rabbit semen. The spermicidal components used for GM-4 formulation are non-toxic solubilizers for lipophilic drugs used in the preparation of a variety of topical, oral, and injectable medications. Cremophor EL (polyethoxylated castor oil), phospholipon 90G (purified soya lecithin), PEG 200, propylene glycol and Captex 30 300 (medium chain triglyceride), are widely used parenteral vehicles as non-toxic solubilizers for lipophilic drugs and vitamins (Castle PE, et al., Contraception, 1998;58:51-60.; Lundberg BB, JPharm Pharmacol, 1997;49:16-21.) Cremophor EL when used up to 10% w/v did not cause any apparent membrane damage to cell monolayers and did not cause lysis of human leukemic cells (Woodcock DM, et al., 35 Cancer Res, 1990;50:4199-203.; Nerurkar MM, et al., Pharmaceutical Research, T T DCT'rlT* Vq T 'r" T, r r'- ,- - r WO 00/56366 PCT/US00/07419 64 1996;13:528-34.). These components by themselves were not spermicidal in human semen. Therefore, unlike the currently used non-ionic and cationic detergent spermicides, the submicron particle-based GM-4 formulation is not likely to cause harmful side effects following repetitive intravaginal application. 5 In short-term toxicity studies, intravaginal administration of the lipophilic and spermicidal GM-4, to female B6C3F1 mice for 13 weeks displayed no adverse effects on survival, growth, hematological, clinical chemistries, absolute or relative organ weights and histopathology. The kidney, liver, and pancreas function as well as the nutritional status were not affected adversely by GM-4 formulation exposure. 10 Based on the preclinical results reported here, we are hopeful that repetitive intravaginal application of GM-4 will have no significant adverse systemic side effects in clinical settings. Experiments to formally test the safety of the intravaginally applied GM-4 on the long-term health and reproductive performance of test animal species are currently in progress. 15 Conclusion Within the above examples we described the in vitro and in vivo spermicidal activity and safety of a novel pharmaceutical formulation, in the form of a gel microemulsion GM, which contains common pharmaceutical excipients as the active 20 ingredients. In some embodiments of the Examples, drug solubilizing agents, for example Cremophor EL and Phospholipon 90G, are contemplated as the active ingredients since these agents were spermicidal against highly motile fraction of sperm. Although, the individual components of GM-4 formulation alone lacked spermicidal activity in semen, the GM-4 formulation containing all eight 25 pharmacological excipients rapidly inactivated sperm in human semen. The lack of cytotoxicity of individual components of GM-4 in human semen and their synergestic spermicidal property in a GM formulation shows uniqe clinical potential to formulate them as the active ingredients for a novel and effective vaginal contraceptive. 30 The microemulsion-based lipophilic and vaginal spermicide, GM-4, appears to offer several benefits for vaginal delivery including increased absorption, improved contraceptive efficacy, and decreased toxicity. Under the described conditions of its intended use, a 13-week intravaginal application of GM-4 formulation in B6C3F 1 mice did not result in systemic toxicity and no other specific 35 target organs were identified. Therefore, the spermicidal GM-4 formulation shows (try rrr rn r ' r r" flr-r T" r " - WO 00/56366 PCT/US00/07419 65 unique clinical potential to become a clinically useful vaginal contraceptive for preventing the sexual transmission of STDs while preventing unwanted pregnancies. As a potent contraceptive agent which is inexpensive and devoid of mucosal toxicity, the lipophilic GM-4 formulation meets the criteria for a vaginal spermicide 5 and warrant further evaluation in vivo in humans. In addition, this non-toxic lipophilic gel-microemulsion formulation may also be useful for intravaginal application of anti-microbial agents to prevent the sexual transmission of diseases such as AIDS, genital herpes, gonorrhea and chlamydia. All publications, patents, and patent documents described herein are incorporated by reference as if fully set forth. The invention described herein may be modified to include alternative embodiments. All such obvious alternatives are within the spirit and scope of the invention, as claimed below.

Claims (50)

1. A pharmaceutical composition adapted for use as a spermicide, the composition comprising a gel-microemulsion comprising a oil-in-water microemulsion and a polymeric hydrogel.

2. The composition of claim 1, wherein the oil-in-water microemulsion comprises a medium chain C 6 -C 1 2 fatty acid glyceride ester, one or more pharmaceutically acceptable surfactants, one or more pharmaceutically acceptable humectants, and water as a diluent.

3. The composition of claim 2, wherein the surfactant comprises ethoxylated castor oil and phospholipid.

4. The composition of claim 2, wherein the fatty acid glyceride ester comprises a triglyceride.

5. The composition of claim 2, wherein the humectant comprises propylene glycol and PEG-200.

6. A composition according to claim 1, wherein the hydroge! is a hydrophilic natural or synthetic gel-forming polymer.

7. A composition according to claim 6, wherein the gel-forming polymer is a natural gum.

8. A composition according to claim 7, wherein the natural gum is selected from the group consisting of xanthan gum, carrageenan, gum karaya, gum acacia, locust bean gum, guar gum, and a mixture thereof.

9. The composition of claim 1, wherein the composition comprises: in the range of 6 to 23 % by weight lipid; in the range of 4 to 17% by weight surfactant; in the range of 3 to 12% by weight humectant; WO 00/56366 PCT/US00/07419 67 in the range of 1 to 2% by weight polymer gel; and the balance being water.

10. The composition of claim 9, wherein; the lipid comprises a medium chain tryglyceride; the surfactant comprises ethoxylated castor oil and phospholipid; the humectant comprises propylene glycol and PEG-200; and the polymer gel comprises a natural polymer gel.

11. The composition of claim 9, further including in the range of 0.1 to 0.2% by weight a preservative.

12. The composition of claim 1, wherein the oil-in-water microemulsion comprises a medium chain C 6 -C 12 fatty acid glyceride ester, a phospholipid, and water as a diluent.

13. The composition of claim 1, wherein the oil-in-water microemulsion comprises a medium chain C 6 -C 1 2 fatty acid glyceride ester, ethoxylated castor oil, and water as a diluent.

14. The composition of claim 1, wherein the oil-in-water microemulsion comprises a medium chain C 6 -C 1 2 fatty acid glyceride ester, ethoxylated castor oil, phospholipid, and water as a diluent

15. The composition of claim 1, in the form of a vaginal cream.

16. The composition of claim 1, further including an additional therapeutically active agent wherein the gel-microemulsion acts as a formulation base for the additional therapeutic agent.

17. The composition of claim 9, further including up to 10% by weight a therapeutically active agent. WO 00/56366 PCT/US00/07419 68

18. The composition of claim 10, further including in the range of 1 and 2% by weight a therapeutically active agent.

19. The composition of claim 16, wherein the additional therapeutic agent comprises an anti-microbial agent.

20. The composition of claim 16, wherein the additional therapeutic agent comprises an AZT derivative.

21. A composition according to claim 20, wherein the AZT derivative is a compound of formula: 0 CH 3 HN ,! R2 0 N R3 0 RO R 1 wherein R 1 is H, N 3 , halo, CN, COOH or NH,; R, is halo; R 3 is alkoxy; and R'is a group that facilitates the passage of the compound into a cell, or a pharmaceutically acceptable salt or ester thereof.

22. The composition of claim 21, wherein said AZT derivative is 5-bromo-6 methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxyalaninyl phosphate.

23. The composition of claim 16, wherein the theraputic agent comprises a nucleoside derivative of the following formula, or a pharmaceutically acceptable salt thereof: WO 00/56366 PCT/USOO/07419 69 Y RF-Y-P-R 2 I N-R3 R 5 0 R4 O or a pharmaceutically acceptable salt thereof, in which Y is oxygen or sulfur; R, is unsubstituted aryl or aryl substituted with an electron-withdrawing group; R, is a nucleoside of one of the following formulae: SO O R 0 H R, RR in which R 6 is purine or pyrimidine; and RT, R 8 , R 9 , RI 0 , RI, and R 12 are independently hydrogen, hydroxy, halo, azido, -NO2 R 13 RI 4 , or -N(ORs)R 6 , in which R 13 , R 14 , Rt 5 , and R 1 6 are independently hydrogen, acyl, alkyl, or cycloalkyl; R 3 is hydrogen, acyl, alkyl, or cycloalkyl; R 4 is a side chain of an amino acid; or R 3 and R 4 may be taken together to form the side chain of proline or hydroxyproline; and R 5 is hydrogen, alkyl, cycloalkyl, or aryl.

24. The composition of claim 23, wherein the nucleoside derivative is selected from those of the formulas: H H X -_F X j0 OJ HN H 3 C0 2 CH H Co~H 3 N wherein X is an electron withdrawing group.

25. The composition of claim 24, wherein X is bromo or chloro. WO 00/56366 PCT/US00/07419 70

26. The composition of claim 16, wherein the therapeutic agent is a spermicidal agent.

27. The composition of claim 16, wherein the therapeutic agent comprises a vanadium (IV) complex, or a pharmaceutically acceptable salt thereof.

28. The composition of claim 27, wherein the vanadium (IV) complex is an organometallic cyclopentadienyl vanadium (IV) complex, or a pharmaceutically acceptable salt thereof.

29. The composition of claim 28, wherein the organometallic cyclopentadienyl vanadium IV complexes is selected from the following: vanadocene dichloride, bis (methylcyclopentadienyl) vanadium dichloride, vanadocene dibromide, vanadocene diiodide, vanadocene diazide, vanadocene dicyanide, vanadocene dioxycyanate, vanadocene dithiocyanate, vanadocene diselenocyanate, vanadocene ditriflate, vanadocene monochloro oxycyanate, vanadocene monochloroacetonitrilo tetrachloro ferrate, vanadocene acetylacetonato monotriflate, vanadocene bipyridino ditriflate, vanadocene hexafluoro acetylacetonato monotriflate, vanadocene acethydroxamato monotriflate, and vanadocene N-phenyl benzohydroxamato monotriflate.

30. The composition of claim 27, wherein the vanadium (IV) complex comprises an oxovanadium (IV) complex.

31. The composition of claim 30, wherein the oxovanadium (IV) complex includes at least one bidentate ligand selected from N,N'; N,O; and O,O' bidentate ligands.

32. The composition of claim 27, wherein the vanadium (IV) complex comprises a substituted or un-substituted catacholate ligand.

33. The composition of claim 32, wherein the vanadium (IV) complex comprises complexes having the structural formula: WO 00/56366 PCT/US00/07419 71 R1 R 5 n R2 R 6 -1O 0 (OH2)n R R4 wherein R', R 2 , R 3 and R 4 are the same or different and are independently selected from H, halo, OH 2 , O 3 SCF 3 , N 3 , CN, OCN, SCN, SeCN, NO 2 , Cl-C 4 alkyl, Ci-C 4 alkoxy, and aryl; and n is 0 or 1; and R 5 and R 6 are the same or different and are either monodentate ligands or R 5 and R 6 together comprise a bidentate ligand.

34. The composition of claim 33, wherein the vanadium (IV) complex comprises complexes having the structural formula: R1 CP1-v "0 R2 Cp~ p CpN "-R * R 3 R4 wherein Cp is unsubstituted cyclopentadienyl, or cyclopentadieneyl substituted with one or more substituents selected from substituted or unsubstituted aryl, Ci-C 4 alkyl, CI-C 4 alkoxy, halo, OH 2 , O 3 SCF 3 , N 3 , CN, OCN, SCN, SeCN, NO 2 ; R', R 2 , R 3 and R 4 are the same or different and are independently selected from H, halo, OH 2 , O 3 SCF 3 , N 3 , CN, OCN, SCN, SeCN, NO 2 , C 1 -C 4 alkyl, and Cj C 4 alkoxy. WO 00/56366 PCT/US00/07419 72

35. A composition according to claim 16. wherein the therapeutic agent comprises a phenethyl-5-bromopyridylthiourea or a dihydroxalkoxybenzylopyrimidine derivative.

36. The composition of claim 35, wherein the therapeutic agent comprises a phenethyl-5-bromopyridylthiourea derivative of the following chemical formula, or a pharmaceutically acceptable salt thereof: R R R-, S HN NR3 H Br where R, RI, R 2 , R 3 , and R 4 are independently hydrogen, F, Cl, Br, or I, and where at least one of R, RI, R 2 , R 3 , and R 4 is F, Cl, Br, or I.

37. The composition of claim 36, wherein the phenethyl-5 bromopyridylthiourea derivative is selected from: N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)] -thiourea; N-[2-(2-chlorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea; N-[2-(3-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea; N-[2-(3-chlorophenethyl)]-N'-[ 2 -(5-bromopyridyl)]-thiourea; N-[2-(4-fluorophenethyl)]-N'-[ 2 -(5-bromopyridyl)]-thiourea; N-[2-(4-chlorophenethyl)]-N'-[2-(5-bromopyridyl)] -thiourea; and mixtures thereof.

38. The composition of claim 35, wherein the therapeutic agent comprises a dihydroxalkoxybenzylopyrimidine derivative of the following chemical formula, or a pharmaceutically acceptable salt thereof: WO 00/56366 PCT/US00/07419 73 O HN 3 5R S N 0 R, Y R, where R 1 and R 2 are alike or different, and are hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl; Y is S or O; and R 3 is alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R is alkyl.

39. The composition of claim 38, wherein the dihydroxalkoxybenzylopyrimidine derivative is selected from: 5-methyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1 H-one, 5-ethyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1 H-one, 5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1 H-one. 5-isopropyl-2-[(methylthiomethyl)thio]-6-(3,5-dimethylbenzyl) pyrimidin-4-1 H-one; and mixtures thereof.

40. The composition of claim 1, wherein the gel-microemulsion has a particle size from about 30-80 nanometers.

41. The composition of claim 1, wherein the gel-microemulsion has a viscosity in the range of about 200 to about 1000 centipoises.

42. A pharmaceutical composition adapted for the topical delivery of a therapeutic agent, the composition comprising a gel-microemulsion comprising an oil-in-water microemulsion and a polymeric hydrogel; and an effective amount of a theraputic, wherein the gel-microemulsion acts as a formulation base for delivery of the therapeutic agent.

43. The composition of claim 42, wherein the gel-microemulsion comprises: in the range of 6 to 23 % by weight lipid; WO 00/56366 PCT/US00/07419 74 in the range of 4 to 17% by weight surfactant; in the range of 3 to 12% by weight humectant; in the range of 1 to 2% by weight polymer gel; and the balance being water.

44. The composition of claim 43, wherein; the lipid comprises a medium chain tryglyceride; the surfactant comprises ethoxylated castor oil and phospholipid; the humectant comprises propylene glycol and PEG-200; and the polymer gel comprises a natural polymer gel.

45. The composition of claim 43, further including in the range of 0.1 to 0.2% by weight a preservative.

46. The composition of claim 43, wherein the oil-in-water microemulsion comprises a medium chain C 6 -C 1 2 fatty acid glyceride ester, a phospholipid, and water as a diluent.

47. The composition of claim 43, wherein the oil-in-water microemulsion comprises a medium chain C 6 -C 1 2 fatty acid glyceride ester, ethoxylated castor oil, and water as a diluent.

48. The composition of claim 43, wherein the oil-in-water microemulsion comprises a medium chain C 6 -C 1 2 fatty acid glyceride ester, ethoxylated castor oil, phospholipid, and water as a diluent

49. A method of delivering a therapeutically active agent to a subject, the method comprising: forming a pharmaceutical gel-microemulsion comprising an oil-in-water microemulsion, a polymeric hydrogel, and an effective amount of the therapeutically active agent; administering the gel-microemulsion to the subject. WO 00/56366 PCT/US00/07419 75

50. The method of claim 49, wherein the therapeutically active agent is an anti viral agent. cTrDrcTr'rT2rL" "rr ' 7 /1rT - - 11,