WO2000056366A1 - Gel-microemulsion formulations - Google Patents

Gel-microemulsion formulations

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Publication number
WO2000056366A1
WO2000056366A1 PCT/US2000/007419 US0007419W WO0056366A1 WO 2000056366 A1 WO2000056366 A1 WO 2000056366A1 US 0007419 W US0007419 W US 0007419W WO 0056366 A1 WO0056366 A1 WO 0056366A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
microemulsion
gel
ofthe
vanadocene
Prior art date
Application number
PCT/US2000/007419
Other languages
French (fr)
Inventor
Seang Yiv
Mingshu Li
Osmond D'cruz
Fatih M. Uckun
Original Assignee
Parker Hughes Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Parker Hughes Institute filed Critical Parker Hughes Institute
Priority to AU39038/00A priority Critical patent/AU3903800A/en
Priority to EP00918179A priority patent/EP1163009A1/en
Priority to JP2000606270A priority patent/JP2002540080A/en
Priority to CA002367266A priority patent/CA2367266A1/en
Publication of WO2000056366A1 publication Critical patent/WO2000056366A1/en
Priority to US09/957,434 priority patent/US7064114B2/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16—Masculine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the present invention relates to a pharmaceutical formulation, and more particularly to a gel-microemulsion formulation.
  • the gel- microemulsion formulation has spermicidal activity and can be used as a contraceptive.
  • 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.
  • the gel- microemulsion. having spermicidal activity can act as a formulation base for the anti-microbial agents to provide a dual function contraceptive/anti-microbial formulation.
  • spermicidal contraceptives have detergent ingredients that disrupt cell membranes. These include the neutral surfactants isononyl-phenyl-polyoxyethylene (9) ether or nonoxynol- 9 (N-9),p- menthanyl-phenyl-polyoxyethylene (8.8) ether o ⁇ menfegol. and isooctyl-phenyl- polyoxyethylene (9) ether or octoxynol- 9 (O-9) (Digenis GA, et al., Pharm Dev Technol, 1999;4:421-30; Furuse K, et al, J Pharmacobiodyn.
  • neutral surfactants isononyl-phenyl-polyoxyethylene (9) ether or nonoxynol- 9 (N-9),p- menthanyl-phenyl-polyoxyethylene (8.8) ether o ⁇ menfegol.
  • vaginal spermicide The 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: 1 18-9.) Worldwide, the cationic surfactant benzalkonium chloride and the anionic detergent sodium docusate (dioctyl sodium sulphosuccinate) are also used as vaginal spermicides (Mendez F. et al., Contraception. 1986:34:353-62.) N-9. sodium oxychlorosene.
  • 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 range from 1 1 to 31 %o. Thus. N-9 is approximately 75% effective in preventing pregnancy (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).
  • N-9 displays antiviral and spermicidal activities only at cytotoxic doses (D'Cruz OJ, et al, Mol
  • vaginal contraceptive 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 J STD AIDS, 1997;8:243-50.; Roddy RE, et al., N Engl J Med, 1998;339:504-10.) Since heterosexual transmission of HIN-1 is the predominant mode ofthe epidemic spread of HIN, new, effective, and safe vaginal spermicides lacking detergent-type membrane toxicity may offer significant clinical advantage over the currently available detergent-type spermicides.
  • vaginal spermicides would likely be used repeatedly over decades, an ideal spermicide should have an established safety record and lack genital epithelial toxicity. Moreover, it should be inexpensive and be produced from commonly available resources and should have a broad specificity for solubilizing drugs effective for prevention of sexual transmission of several STDs including
  • the inventors have developed novel gel-microemulsion formulations for use as spermicides that in numerous respects overcomes many ofthe problems ofthe commercially available detergent-type spermicides.
  • Embodiments ofthe 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 ofthe invention contain common pharmaceutical excipients as the active ingredients, and provide for safe in vitro and in vivo spermicidal activity.
  • 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.
  • Cremophor EL® and Phospholipon 90G® may be active ingredients since these agents were spermicidal against highly motile fraction of sperm.
  • gel-microemulsion formulations ofthe invention show unique clinical potential to formulate them as the active ingredients for a novel and effective contraceptive, such as a vaginal contraceptive for example.
  • some embodiments of gel-microemulsion formulations ofthe invention were significantly more effective as a contraceptive than a commercially-available ⁇ -9 gel formulation.
  • Embodiments ofthe gel-microemulsion ofthe 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 antimicrobial agents are particularly useful to prevent the transmission of diseases.
  • Such embodiments are especially useful as a dual function spermicide/anti-microbial formulation, and can be especially useful in inhibiting the transmission of sexually transmitted diseases, for example AIDS, genital herpes, gonorrhea and chlamydia.
  • additional spermicidal agents can be incorporated into the formulation to increase the effectiveness ofthe gel- microemulsion as a spermicide.
  • One aspect ofthe 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 ofthe invention is directed to a gel-microemulsion pharmaceutical composition adapted for use as a formulation base for additional theraputic agents.
  • 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 ofthe combined gel-microemulsion formulation base with additional theraputic agents for 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 a composition as a dual function contraceptive/anti-microbial formulation.
  • 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 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, 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.
  • 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-
  • Figure 4 is are light microscopic images of GM-4 and N-9 (4%)-treated rabbit vaginal sections. Representative hematoxylin- and eosin-stained, paraffin- 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 ( ⁇ 200). Higher magnification ( ⁇ 400) 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 leukcocyte influx characteristic of inflammation.
  • VE vaginal epithelium
  • Figure 5 shows the mean body weights of 10 female B6C3F ⁇ mice with and without intravaginal application of GM-4, 5 days/week for 13 consecutive weeks.
  • 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 ⁇ m or below.
  • Lipid is an inclusive term for fats or fat derived materials.
  • “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.
  • Human is a substance having affinity for water with stabilizing action on the water content of a material.
  • 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 ofthe other substance. The analog or derivative need not be, but can be synthesized from the other substance.
  • salts refers to a salt that retains the desired biological activity ofthe 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 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) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper,
  • “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.
  • the term “inhibit” means to reduce by a measurable amount, or to prevent entirely.
  • 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.
  • 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.
  • Organic compound is an organic compound comprised of a metal attached directly to carbon (R-M).
  • 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 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.
  • “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 (IN) complex is a coordination compound including vanadium as the central metal atom or ion, and the vanadium has an oxidation state of +4 (IN), and is double bonded to oxygen.
  • Metallocene is an organometallic coordination compound containing cyclopentadienyl rings attached to a transition metal or transition metal halide.
  • Neadocene is a metallocene including vanadium as the transition metal ion.
  • Transition metals is any of a number of elements in which the filling ofthe 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. "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.
  • 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 carbamium carbom.
  • One aspect ofthe invention is directed to a pharmaceutical composition adapted for use as a spermicide.
  • the spermicidal activity ofthe pharmaceutical composition can be in vitro or in vivo.
  • the spermicidal compositions ofthe 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.
  • 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 ⁇ 2 fatty acid glyceride esters, and the like.
  • Preferred C 6 -C ⁇ 2 fatty acid glyceride esters include medium chain -Cn 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 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 lipid being used.
  • Non-ionic surfactants are generally preferred.
  • suitable surfactants include ethoxylated castor oil, and phospholipids.
  • 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.
  • 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 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.
  • both ethoxylated caster oil and phospholipids are used as surfactants.
  • 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
  • humectants can be used.
  • propylene 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). Water is used as the diluent, and preferably purified or distilled water is used.
  • 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.
  • thickening agents are added to enhance the usefulness ofthe microemulsions, especially as an effective spermicide and as a base formulation for anti-microbial compounds in certain application.
  • the gel-microemulsion formulation also includes one or more thickening agents, such as a polymeric hydrogel.
  • the hydrogel is a hydrophilic natural or synthetic gel-forming polymer, preferably, a natural gel- forming polymer.
  • 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 FMC Corporation (Philadelphia, PA).
  • Suitable xanthan gums include
  • the formulation can also optionally include one or more additives such as preservatives or antioxidants to help maintain and prolong the useful life ofthe gel- microemulsion.
  • preservatives and antioxidants that are generally known, and do not detract significantly from the usefulness ofthe gel-microemulsion for the particular 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 ofthe action of microorganisms in the formulation can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • 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 ofthe gel-microemulsion 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 ofthe 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 about 200 to about 1000 centipoise.
  • CM-4 ternary phase diagram ofthe microemulsion components used for the preparation of one embodiment ofthe invention, GM-4, is shown in Figure 1, and discussed in the Examples below.
  • the non-grid area represents the single phase microemulsion region suitable for use.
  • the concentration ofthe components can be selected from within this region.
  • the asterisk represents the particular concentration of components ofthe microemulsion which was used for the GM-4 formulation.
  • Suitable gel-polymer suspensions can then be selected as additives to the 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.
  • the formulation includes the specific constituent concentrations for base components as found in Table 2, wherein the values are given in wt. % ofthe ingredients in reference to the formulation weight.
  • a simple procedure allows for the preparation of a gel-microemulsion at even a one-milliliter scale.
  • 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.
  • compositions ofthe present invention are preferably 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.
  • 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 ofthe present invention may be delivered to the vagina of a mammal by any means known to those skilled in the art.
  • the gel- microemulsion can be applied directly.
  • Other typical forms for delivery ofthe compositions include, for example, intervaginal devices such as sponges, condoms, including female condoms, suppositories, and films.
  • 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 within or on a condom for example. Inter-vaginal devices may also be used to aid in the administration ofthe composition as described in U.S. Patent 5,069,906.
  • the formulation of the invention may be inco ⁇ orated 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.
  • Gel-Microemulsion as a Formulation Base for Other Therapeutic Agents Another aspect ofthe invention is the use ofthe above described gel- microemulsion formulations as formulation bases for incorporating therapeutically active agents.
  • the base gel-microemulsion formulations generally include the 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 ofthe gel-microemulsions ofthe invention are especially suitable as solubilizing vehicles for poorly water soluble compounds.
  • 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 include anti-viral agents, anti-bacterial agents, anti-fungal agents, and the like. Such agents can be inco ⁇ orated 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 treatment of sexually transmitted diseases, for example, AIDS (HIV-1, HIV-2, FIV, SIV, etc.) genital he ⁇ es, gonorrhea, chlamydia, and the like.
  • Preferred Anti-Microbial Agents include those disclosed in the following copending patent applications, which are hereby inco ⁇ orated by reference herein:
  • Examples of preferred anti-microbial/anti-viral compounds disclosed in copending U.S. Patent Application No. 09/047,609 and corresponding PCT Patent Application No. PCT/US99/06381 include AZT derivatives disclosed therein. Many ofthe AZT derivatives disclosed therein also have spermicidal activity. Examples of such anti-microbial AZT derivatives include compounds ofthe formula:
  • R is H, N 3 , halo, CN, COOH or NH 2 , R 2 is halo (particularly Cl, Br or
  • R 3 is alkoxy (particularly Cl-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 -CH,Ph.
  • Pharmaceutically acceptable salt or ester forms also can be used, such as sodium, potassium or ammonium salts.
  • the derivatives ofthe formula above include substitution on the AZT pentose ring member.
  • the derivatives of this aspect ofthe present invention have the chemical structure illustrated below:
  • R is H, N 3 , halo, CN, COOH or NH 2
  • R 2 is halo (particularly Cl, Br or I, and more particularly Br)
  • R 3 is alkoxy (particularly Cl-3 alkoxy, and more particularly methoxy (-OCH 3 ))
  • R' is a group that facilitates the passage ofthe compound into a cell.
  • the NH 2 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.
  • sperm-reactive antibodies or cytokines could be used to derivatize these compounds (as well as those ofthe first formula) at the R' or R, positions for targeted delivery.
  • 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 ofthe phosphate can be replaced with Cl-4 alkyl or aryl substituents (e.g. phenyl-, naphthyl- or anthracinyl-substitution), which optionally may be substituted, and SH or NH 2 groups can replace the OH ofthe phosphate, and in each of these cases a H ofthe NH 2 or SH can be replaced in the same manner as the H ofthe OH group discussed previously.
  • the aryl phosphate group is su ⁇ risingly effective in maintaining excellent anti-HIV activity.
  • a general structure of an exemplary aryl phosphate OR' group is illustrated below:
  • 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, trifiuoromethyl, 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 (-
  • the methyl group attached 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.
  • WHI-07 is particularly preferred:
  • Examples of preferred anti-microbial, preferably anti-viral, compounds 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 is oxygen or sulfur, preferably oxygen;
  • R is unsubstituted aryl or aryl substituted with an electron- withdrawing group;
  • R 2 is a nucleoside of one ofthe following formulae:
  • R 6 is purine or pyrimidine, preferably pyrimidine; and R 7 , R 8 , R,, R l0 , R,,, and R 12 are independently hydrogen, hydroxy, halo, azido, -NO 2 , -NR I3 R ⁇ 4 , or - N(OR 15 )R 16 , in which R l3 , R M , R 15 , and R l6 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
  • R 5 is hydrogen, alkyl, cycloalkyl, or aryl.
  • aryl includes aromatic hydrocarbyl, such as, for example, phenyl, including fused aromatic rings, such as, for example, naphthyl.
  • groups may be unsubstituted or substituted on the aromatic ring by an electron-withdrawing group, such as, for example, halo (bromo, chloro, fluoro, iodo), NO,, or acyl.
  • aryl substituted with an electron-withdrawing group is bromophenyl. More preferably 4-bromophenyl.
  • acyl includes substituents ofthe formula R 17 C(O)-, in which R I7 is hydrogen, alkyl, or cycloalkyl.
  • 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, -NO,, -NR ]3 R 14 , or -N(OR, 5 )R 16 , in which R, 3 , R 14 , R 15 , and R, 6 are as defined above.
  • 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 substituted with hydroxy, halo, azido, -NO,, -NR, 3 R 14 , or -N(OR 15 )R, 6 , in which R 13 ,
  • R ]4 , R I5 , and R, 6 are as defined above.
  • purine includes adenine and guanine.
  • pyrimidine includes uracil, thymine, and cytosine.
  • pyrimidine is thymine.
  • 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, tyrosine, valine, and the like.
  • the side chain of an amino acid is the side chain of alanine or tryptophan.
  • compounds substituted with an electron-withdrawing group such as an ort/zo-or/w ⁇ -substituted halogen or NO, provide for more efficient hydrolysis to active inhibitory compounds.
  • an electron-withdrawing group such as an ort/zo-or/w ⁇ -substituted halogen or NO.
  • Preferred is halogen substitution, and most preferred are para-bromo substitution and para-chloro substitution.
  • Preferred nucleoside derivative disclosed therein include those ofthe formulas:
  • X is an electron withdrawing group, for example halo or NO, and most preferably, X is bromo or chloro.
  • spermicidal agents include any spermicidal agent generally known that is compatible for formulation with the gel-microemulsion.
  • Such agents can be inco ⁇ orated into the gel-microemulsion formulations to provide for additional spermicidal activity ofthe formulation.
  • Many ofthe spermicidal agents discussed below also have anit-microbial activity, such as anti-viral activity, and can be inco ⁇ orated into the formulations as an anti-microbial agent as well as a spermicidal agent.
  • Preferredspermicidal Agents for Incorporation Into the Gel- Microemulsion Formulation Preferred examples of spermicidal agents include those disclosed in the following copending patent applications:
  • Patent Application No. 09/008,898, and corresponding published PCT Application No. PCT US99/01171 include the vanadium (IV) compounds disclosed therein.
  • vanadium (IV) compounds include organometallic cyclopentadienyl vanadium IV complexes.
  • Preferred such compounds include: 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 benzohydroxama
  • Examples of preferred spermicidal compounds disclosed in copending U.S. 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.
  • suitable bidentate ligands include bipyridyl, bridged bipyridyl, and acetophenone.
  • preferred oxovanadium compounds are those having the formulas shown and described below.
  • oxo-vanadium (IV) compounds include a bidentate ligand wherein the bidentate ligand is a bipyridyl and the oxovanadium IV complex has the general formulae:
  • R and Ri are the same or different and are independently selected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, 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.
  • oxo-vanadium (IV) compounds have a bidentate ligand wherein the bidentate ligand is a bridged bipyridyl and the oxovanadium IV complex has the general formulae:
  • 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;
  • X 2 and X 3 are the same or different and are selected from monodentate and bidentate ligands;
  • 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:
  • R 4 , R 5 and R 6 are the same or different and are independently selected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, 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.
  • oxo-vanadium (IV) compounds have a bidentate ligand wherein the bidentate ligand is an O,O' bidentate ligand.
  • the oxovanadium IV complex has the general formulae:
  • R 7 ; and R 9 are the same or different and are independently selected 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 1 are the same or different and independently selected from the group consisting of: monodentate and bidentate ligands: and n is 0 or 1.
  • Preferred monodentate ligands for the oxovanadium complex include H,O, 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' bidentate ligands.
  • diamines include bipyridal, derivatives of bipyridal, bridged bipyridal, such as phenanthroline, derivatives of phenanthroline, and other such compounds.
  • suitable N, O bidentate ligands include amino acids and Schiff base type groups.
  • suitable O, O' bidentate ligands include dicarboxylate, 2-hydroxyacetophenone, acetylacetone type and catechol type groups.
  • Particularly useful oxo-vanadium (IV) complexes are the following:
  • Examples of preferred spermicidal compounds disclosed in copending U.S. Patent Application No. 09/224,677 include phenethyl-5-bromopyridylthiourea (PBT) and dihydroxalkoxybenzylopyrimidine (DABO) derivatives disclosed therein.
  • PBT phenethyl-5-bromopyridylthiourea
  • DABO dihydroxalkoxybenzylopyrimidine
  • PBT derivatives include those having the following chemical formula, or a pharmaceutically acceptable salt thereof:
  • R, Ri, R 2 , R 3 , and I * are independently hydrogen, F, Cl, Br, or I, and where at least one of R, Ri, R 2 , R 3 , and j is F, Cl, Br, or I.
  • R, Ri, R , R . and t in structure above is F or Cl.
  • PBT N-[2-(2-fluorophenethyl)]- N'-[2-(5-bromopyridyl)]-thiourea (F-PBT) which has the chemical structure shown below:
  • F-PBT PBT derivatives can be synthesized as described in Vig et al.. BlOORG. MED. CHEM.. 6:1789-1797 (1998).
  • 2-amino-5-bromopyridine is condensed with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl derivative.
  • Further reaction with appropriately halogen-substituted phenylethylamine gives the target halogenated PBT derivatives.
  • Suitable DABO derivatives include those ofthe formula, or a pharmaceutically acceptable salt thereof:
  • Ri 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.
  • one or more of Ri and R 2 is an alkyl having 1 to 3 carbonatones, ( - C 3 ), such as methyl (Me), ethyl (Et), or isopropyl ( -Pr).
  • Ri is alkyl, alkenyl, ROH, or RNH 2 .
  • R 2 is preferably halo, alkyl, or -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 -C 3 alkyl.
  • Preferred DABO derivatives include compounds having the chemical structure shown below, or a pharmaceutically acceptable salt thereof:
  • DABO derivative compounds include compounds (a) through (d) listed below, or a pharmaceutically acceptable salt thereof: (a) 5-methyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4- 1 H- one,
  • DABO derivatives can be prepared as described in descrioed 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:
  • Reagents and conditions a) R CHBrCOOEt/Zn/THF, b) HCI(aq), c) (H 2 N ⁇ CS Na EtOH, d) DMF, KCQi, Chioromethyl methyl sulfide, 15h.
  • ethyl-2-alkyl-4-(phenyl)-3-oxobutyrates la-d were obtained from commercially available phenyl acetonitrile.
  • the ⁇ -ketoesters were condensed with 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., Ada 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).
  • therapeutic agents includes vanadium (IV) complexes containing a substituted or un-substituted catacholate ligand.
  • IV vanadium
  • examples of such compounds include complexes having the following structural formula, or pharmaceutically acceptable salts thereof:
  • R , R , R and R are the same or different and are independently selected from H, halo, OH 2 , O 3 SCF , N 3 , CN, OCN, SCN, SeCN, NO 2 , Cj-C 4 alkyl, C
  • Suitable monodentate ligands include, for example, aryl, halo, H 2 O, O SCF 3 , N 3 , COOH, CN, OCN, SCN. SeCN, NO 2 , C,-C 4 alkyl, -C 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 O,O' bidentate ligands.
  • suitable N, N' bidentate ligands include diamines and other such known suitable N, N' bidentate ligands.
  • diamines include bipyridal, derivatives of bipyridal. bridged bipyridal. such as phenanthroline, derivatives of phenanthroline, and other such compounds.
  • suitable N, O bidentate ligands include amino acids and Schiff base type groups.
  • suitable O. O' bidentate ligands include dicarboxylate, 2-hydroxyacetophenone. 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.
  • Some preferred vanadium (IV) catacholate complexes include ''bent sandwich" vanadocene monocatacholate complexes having the following structure formula, or pharmaceutically acceptable salts thereof:
  • Cp is unsubstituted cyclopentadienyl, or cyclopentadieneyl substituted with one or more substituents selected from substituted or unsubstituted aryl, - alkyl, d-C 4 alkoxy, halo, OH 2 , O 3 SCF 3 , N 3 , CN, OCN, SCN, SeCN, NO 2 .
  • Cp is unsubstituted cyclopentadienyl.
  • R , R , R 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, and C,- C alkoxy
  • electron donating groups for example electron donating alkyl groups, are present as substituents of the catacholate ring in positions R , R , R and/or R 4 .
  • Particularly preferred such compounds include vanadocene catacholate, vanadocene mono-tertbutyl catacholate, and vanadocene 1 , 3-diisopropyl catacholate.
  • 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:
  • R 7 and R 8 are the same or different and are independently selected from: H, aryl, -C 4 alkyl, halo, C ⁇ C 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 , N 3 , CN, OCN, SCN, SeCN, NO 2 , C,-C 4 alkyl, and C,-C 4 alkoxy.
  • 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:
  • R 9 and R 10 are the same or different and are selected from H, aryl, Cj- C 4 alkyl, halogen, C[-C 4 alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C 2 -C 6 ) and nitro;
  • n is 0 or 1; and halo, OH 2 , O 3 SCF 3 , N 3 , CN, OCN, SCN, SeCN, NO 2 , -C, alkyl, and Cj-C alkoxy.
  • vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is a bridged bipyridyl, and the bridged bipyridyl is phenanthroline, has the general formula shown below, or pharmaceutically acceptable salts thereof:
  • R 11 , R 12 and R 13 are the same or different and are independently selected from: H, aryl, C ⁇ C 4 alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C 2 -C 6 ) and nitro; n is 0 or 1; and R 1 , R 2 , R 3 and R are the same or different and are independently selected from H, halo, OH 2 , 0 3 SCF 3 , N 3 , CN. OCR SCN, SeCN. NO 2 , C,-C 4 alkyl, and C,-C 4 alkoxy.
  • vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is an O,O' bidentate ligand, and the complex has the general formula VI, is shown below, or pharmaceutically acceptable salts thereof:
  • R 14 and R 16 are the same or different and are independently selected from: H, aryl, -C alkyl, and C]-C alkoxy, and halogenated alkyl;
  • R 15 is selected from H, C ⁇ C 4 alkyl, halo, C ⁇ C 4 alkoxy, and halogenated alkyl;
  • n is 0 or 1; and
  • 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 , Cj-C 4 alkyl, and -C 4 alkoxy.
  • Gel-microemulsions formulations including additional therapeutic agents are formulated and prepared in substantially the same way as the primary gel- microemulsions discussed above, with the only difference being the addition ofthe additional therapeutic agent.
  • the amount of additional ingredient added is dependent upon the desired effective amount ofthe ingredient in the final gel- microemulsion formulation. It is still desirable to provide 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.
  • 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. % ofthe ingredients in reference to the total weight ofthe formulation.
  • the formulation includes the specific constituent concentrations for base components as found in Table 4, wherein the values are given in wt. % ofthe ingredients in reference to the formulation weight.
  • additional therapeutic agents such as an anti-microbial agent or a spermicidal agent
  • the gel-microemulsion formulations resulting from the addition of additional theraputic agents are contemplated for used in generally the same manner as the gel-microemulsion spermicide use discussed above.
  • formulations including other theraputic agents, for example antiviral agents can be used in non-spermicidal applications.
  • the formulation is preferably administered to a site appropriate for the theraputic activity desired in a dosage which is effective to effectuate the desired theraputic effect.
  • the formulation is preferably administered to a site appropriate for desired antimicrobial 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 outside of mammals is contemplated. It is expected that the formulations will be used by humans in most practical applications.
  • Captex 300 was obtained from ABITEC Co ⁇ ., Janeswille,
  • Cremophor EL was from BASF Co ⁇ ., Mount Olive, NJ.
  • Phospholipon 90G was purchased from American Lecithin Co., Danbury, CT.
  • PEG-200 was from Union Carbide Co ⁇ ., Danbury, CT while propylene glycol was obtained from Spectrum Quality Products Inc., New Brunswick, NJ. Seaspan and viscarin carrageenan were obtained from FMC Co ⁇ ., 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: 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.
  • Various polymeric gels were screened to produce a gel with desirable viscosity. Polymer suspensions of seaspan 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, 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 ofthe microemulsion components used for the preparation of GM-4 is shown in Figure 1.
  • the non-grid area represents the single phase microemulsion region.
  • the asterisk represents the microemulsion which was used for GM-4 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 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
  • 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 benzoate (0.025%> - 0.2%).
  • 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 Reprod, 1999;60:435-44.; D'Cruz OJ, et al. Biol Reprod, 1999;60:1419-28.).
  • IVOS Integrated Visual Optical System
  • 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 (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 ofthe test compound was expressed as EC50 (the final concentration ofthe compound in the medium that decreased the proportion of motile sperm by 50%). Results
  • the concentration-dependent spermicidal activity by Cremophor EL and phospholipon 90G was associated with a parallel decline in sperm kinematics, 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
  • viscarin carrageenan, Cremophor EL and phospholipon 90G were determined by phase contrast 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 assay (Sander FV, et al., Hum Fertil, 1941;6:134-7.; D'Cruz OJ. et al.,
  • the submicron particle size GM-4 formulation completely immobilized sperm in human semen in less than 2 min (1.2 ⁇ 0.3 min).
  • the combination of these pharmaceutical excipients as a gel-microemulsion formulation was a potent spermicide in semen.
  • 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:
  • WHI-07 was synthesized using the following synthetic scheme. Synthetic Scheme for WHI-07
  • 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 to each octanol solution. The mixtures were handshaken vigorously for 10 min, and afterward let to stand overnight until a complete phase separation occurred.
  • 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.
  • 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- microemulsions.
  • a total polymer concentration of about between 0.5% and 3% is needed to provide a gel-microemulsion with adequate viscosity.
  • WHI-07 can be delivered using the GM-4 formulation.
  • 18.7 mg of WHI-07 was first dissolved in 305 mg of a microemulsion consisting 32.4% of Captex 300 (medium chain triglyceride),
  • This WHI-07-containing microemulsion concentrate was diluted with water to bring the intensity to between 300 and 500 KHZ for optimal particle size measurement.
  • the particle diameter ofthe 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 determined by Nicomp 380 photon correlation light scattering particle sizer.
  • WHI-07 gel 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 gel formulations, however, are not totally soluble in either ethanol or acetonitrile because ofthe polymers in the formulation. The following WHI-07 analytical methods were developed for the gel formulations. The accuracy and precision ofthe methods meet pharmaceutical requirements.
  • 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 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).
  • 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 ofthe 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 C until the drug was dissolved and the mixture was clear. The vial was removed from the hot plate and 3.5 ml ofthe 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 ofthe microemulsion with 2.8 ml ofthe polymer suspension. To each of the 10 vials, 0.2 ml ofthe WHI-07 gel, 0.3 ml ofthe 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 ⁇ m. The vials were carefully removed from the centrifuge and 0.5 ml ofthe supernatant was pipetted out from 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 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.
  • WHI-07 240 mg was weighed in a 10 ml glass shell vial. 4.0 ml ofthe 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 Corning stir/hot plate for mixing. The dials for both stir and heat were set at #3. The mixture 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 ofthe concentrated WHI-07 microemulsion with 4 ml ofthe polymer suspension in a 10 ml glass shell vial.
  • 1% WHI-07 gel was prepared by mixing 1 ml ofthe concentrated WHI-07 microemulsion with 1 ml ofthe microemulsion and 4 ml ofthe polymer suspension.
  • 0.5% WHI-07 gel was prepared by mixing 0.5 ml ofthe concentrated WHI-07 microemulsion with 1.5 ml ofthe microemulsion and 4 ml ofthe polymer suspension.
  • the control gel was prepared by mixing 2 ml ofthe microemulsion and 4 ml ofthe polymer suspension.
  • WHI-07 formulation concentration analysis Two samples, 100 ⁇ l each, were taken from each preparation for concentration analysis. The samples were placed in 5 ml glass shell vials. 4.2 ml of acetonitrile was added to each ofthe sample vials. The vials were hand shaken for 10 seconds and were placed in a shaker at 200 ⁇ m for 30 min. The vials were hand shaken again for 10 seconds and span for 10 min at 2000 ⁇ m in the Beckman GS-6 Centrifuge. The vials were carefully removed from the centrifuge and 1 ml each ofthe supernatant was pipette out for HPLC analysis. The results were shown in Table 11. All ofthe samples meet pharmaceutical requirement (RSD less than 5.0%).
  • 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 was a translucent gel with the following composition:
  • the viscosity of the GM-4 formulation with and without WHI-07 was determined using a Brookfield DV-E Viscometer with spindle #3 (speed: 10 ⁇ m). Drug-free GM-4 formulation had a viscosity of 301.9 centripoises. The GM-4 formulation containins 2% WHI-07 had a viscosity of 64.7. Although a viscosity of 64.7 appeared to be sufficient for the utility ofthe
  • a thicker formulation was prepared as follows: 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
  • the resulting gel microemulsion was a translucent gel-like liquid with the following composition:
  • 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.
  • Rabbits Fifty nine female and 12 male, sexually mature (> 6 months old; > 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 ⁇ 2°C with relative humidity of 50 ⁇ 20% and a 12-h fluorescence light cycle. The 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
  • mice Twenty, female B6C3F1 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°C with relative humidity of 50 ⁇ 10% and a 12-h fluorescence light cycle. All animal husbandry operations were conducted under NIH 1996 Guidelines.
  • 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 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.
  • vaginal tissues were rapidly removed and parts ofthe caudal, mid, and distal regions of each vagina were fixed in 10%> buffered formalin. Tissues were embedded in paraffin, sectioned at 4-6 ⁇ m and stained with hematoxylin and eosin and examined under x200 and ⁇ 400 magnification using a Leica light 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 analysis.
  • ImagePro Plus program Media Cybernetics, Silver Spring, MD
  • 3CCD camera DAGE-MTI Inc., Michigan City, KS
  • images were transferred to Adobe Photoshop 5.5 software (Adobe Systems Inc., San Jose, CA) for observation and analysis.
  • Each ofthe three regions of vagina were examined for epithelial ulceration, edema, leukocyte infiltration, and vascular congestion.
  • mice Thirteen-Week Toxicity Study in Mice: Twenty, female B6C3F1 mice were allocated to two groups. The test group of 10 mice received 50 ⁇ l ofthe GM-4 formulation intravaginally for 5 days per week for 13 consecutive weeks. Ten mice 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 ofthe study, animals were sacrificed 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 analyzer (Abbot Laboratories. Abbot Park, IL) which was standardized for mouse blood. This instrument uses flow cytometric techniques to provide the hemograms for anticoagulated whole blood samples: red blood cell count (RBC; 10 / ⁇ l), total and differential leukocyte count (lymphocytes [LYM], neutrophils [NEU], monocytes [MONO], eosinophils [EOS], and basophils [BASO] as 10 / ⁇ l or %), hemoglobin concentration (HGB; g/dl), hematocrit (HCT; %>), mean co ⁇ uscular volume (MCV; fl), mean cell hemoglobin content (MCH; pg), mean cell hemoglobin concentration (MCHC; g/dl), red cell distribution width (RDW; %), platelet count
  • RBC red blood cell count
  • LYM total and differential
  • PKT 10 3 / ⁇ l
  • MPV mean platelet volume
  • mice were killed after 13 weeks of 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, skeletal muscle, skin, spinal cord, and urinary bladder were fixed in 10%> buffered formalin solution, trimmed, embedded in paraffin, sectioned at 4-6 ⁇ m, and stained with hematoxylin and eosin. Complete histopathological examination of all tissues was performed on mice from the control and GM-4 group.
  • FeT-J cell line is a feline T-cell line chronically infected with the FIV strain Bangston (FlV ⁇ ang)- Cats were challenged with FIV by inoculating 7x10 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 ofthe WHI-07 gel formulation.
  • PBMC peripheral blood mononuclear cells
  • LN lymph node
  • BM bone marrow
  • Cats were considered FIV positive if one ofthe following criteria was met during the 18-week observation period: (1) Serum samples from two different bleeding dates were positive by Western blot analysis; (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.
  • Gynol II for preventing pregnancy in the rabbit model are summarized in Table 13.
  • the GM-4 formulation was far more effective than Gynol II as a vaginal spermicidal contraceptive (100% vs 68.7%, ? ⁇ 0.05, Fisher's exact test).
  • 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 ofthe epithelial lining, and an inflammatory response with influx of leukocytes, consistent with previously published observations in rats (Tryphonas L, et al., Toxicol Lett, 1984;20:289-95.).
  • mice 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 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).
  • RBC red blood cells
  • WBC white blood cells
  • LYM lymphocytes
  • NEU neutrophils
  • MONO monocytes
  • EOS eosinophils
  • BASO basophils
  • HGB hemoglobin concentration
  • HCT hematocrit
  • MCV mean co ⁇ uscular volume
  • MCH mean cell hemoglobin
  • MCHC mean cell hemoglobin concentration
  • RDW red cell distribution width
  • PLT platelets
  • MPV mean platelet volume.
  • Table 17 summarizes the terminal body weight, terminal absolute and relative organ weights observed at the conclusion ofthe 13-week study. No statistically significant 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 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 ofthe lipophilic and spermicidal GM-4 formulation.
  • Thymus 0.09 ⁇ 0.01 * 0.32 ⁇ 0.03 0.10 ⁇ 0.03* 0.35 ⁇ 0.10
  • 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 7x10 FIVBang-infected FeT-J cells mixed in 0.2 mL of infected culture fluid.
  • WHI-07/GM-4 formulation provided 60% protection against FIV.
  • WHI-07/GM-4 was also able to prevent the transrectal transmission of FIV (Table 19).
  • 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.
  • 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 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 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 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 as well as to its rapid spermicidal activity. In contrast, the contraceptive effect of N-
  • N-9 is being used at concentrations of 2 to 6% in creams and gels, 12% in foams and as high as 18% in 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.).
  • the second objective of these studies was to determine the toxic effects, if any, resulting from repeated intravaginal application of spermicidal GM-4. Because ofthe potent in vitro and in vivo spermicidal activity of GM-4 formulation, it was 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 ofthe rabbit despite the fact that it was a potent 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 300 (medium chain triglyceride)
  • Cremophor EL polyethoxylated castor oil
  • phospholipon 90G purified soya lecithin
  • 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., Cancer Res, 1990;50:4199-203.; Nerurkar MM, et al.
  • the submicron particle-based GM-4 formulation is not likely to cause harmful side effects following repetitive intravaginal application.
  • intravaginal administration ofthe 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.
  • a novel pharmaceutical formulation in the form of a gel- microemulsion GM, which contains common pharmaceutical excipients as the active ingredients.
  • 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.
  • GM-4 formulation alone lacked spermicidal activity in semen, the GM-4 formulation containing all eight pharmacological excipients rapidly inactivated sperm in human semen.
  • GM-4 microemulsion-based lipophilic and vaginal spermicide
  • GM-4 The microemulsion-based lipophilic and vaginal spermicide, GM-4, appears to offer several benefits for vaginal delivery including increased abso ⁇ tion, improved contraceptive efficacy, and decreased toxicity.
  • a 13-week intravaginal application of GM-4 formulation in B6C3F1 mice did not result in systemic toxicity and no other specific target organs were identified. Therefore, the spermicidal GM-4 formulation shows unique clinical potential to become a clinically useful vaginal contraceptive for preventing the sexual transmission of STDs while preventing unwanted pregnancies.
  • the lipophilic GM-4 formulation meets the criteria for a vaginal spermicide and warrant further evaluation in vivo in humans.
  • 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 he ⁇ es, gonorrhea and chlamydia.

Abstract

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. The gel-microemulsion can be used in a spermicidal method. Also, a gel-microemulsion pharmaceutical composition adapted for use as a formulation base for additional therapeutic agents. Examples of additional agents include anti-microbial agents and spermicidal agents. Such gel-microemulsions with additional therapeutic agents can be used in methods for appropriate therapeutic treatment. Also, 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. Method of using such a composition as a dual function contraceptive/anti-microbial formulation are also included.

Description

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- 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- 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 detergent ingredients that disrupt cell membranes. These include the neutral surfactants isononyl-phenyl-polyoxyethylene (9) ether or nonoxynol- 9 (N-9),p- menthanyl-phenyl-polyoxyethylene (8.8) ether oτmenfegol. and isooctyl-phenyl- polyoxyethylene (9) ether or octoxynol- 9 (O-9) (Digenis GA, et al., Pharm Dev Technol, 1999;4:421-30; Furuse K, et al, J Pharmacobiodyn. 1983;6:359-72.) The 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: 1 18-9.) Worldwide, the cationic surfactant benzalkonium chloride and the anionic detergent sodium docusate (dioctyl sodium sulphosuccinate) are also used as vaginal 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 range from 1 1 to 31 %o. Thus. N-9 is approximately 75% effective in preventing pregnancy (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).
The spermicidal activities of these surfactants are associated with their structural affinity to the membrane lipids CSchill WB. et al. Andrologia. 1981 : 13:42-9: Helenious A. et al. Biochem Biophys Ada. 1975:415:29-79). 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). 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, and lead to an increased risk of opportunistic infections (Hooten TM, et al., JAMA, 1991;265:64-9.; Stafford MK, J Acquir Immune Defic Syndr Hum Retrovirol, 1998;17:327-31.; Rosenstein IJ, et al. J Infect Dis, 1998;177:1386-90.; Patton DL, et al, Se Trans Dis, 1996;23:489-93.) Such opportunistic infections are known to enhance the susceptibility ofthe ectocervical epithelium and the endocervical mucosa to HIN-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 study conducted among commercial sex workers in Nairobi, in which some ofthe 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 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 J STD AIDS, 1997;8:243-50.; Roddy RE, et al., N Engl J Med, 1998;339:504-10.) Since heterosexual transmission of HIN-1 is the predominant mode ofthe epidemic spread of HIN, new, effective, and 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 epithelial toxicity. Moreover, it should be inexpensive and be produced from commonly available resources and should have a broad specificity for solubilizing drugs effective for prevention of sexual transmission of several STDs including
HIN-1.
Therefore, there is a continuing need for new and better spermicidal formulations.
Summary of the Invention
The inventors have developed novel gel-microemulsion formulations for use as spermicides that in numerous respects overcomes many ofthe problems ofthe commercially available detergent-type spermicides. Embodiments ofthe 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 ofthe 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 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- 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 ofthe invention were significantly more effective as a contraceptive than a commercially-available Ν-9 gel formulation. Embodiments ofthe gel-microemulsion ofthe 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 antimicrobial agents are particularly useful to prevent the transmission of diseases. Additionally, such embodiments are especially useful as a dual function spermicide/anti-microbial formulation, and can be especially useful in inhibiting the 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 ofthe gel- microemulsion as a spermicide. One aspect ofthe 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 ofthe invention is directed to a gel-microemulsion 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 ofthe combined gel-microemulsion formulation base with additional theraputic agents for 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 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 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, 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. 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 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- 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 (χ200). Higher magnification (χ400) 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 leukcocyte influx characteristic of inflammation.
Figure 5 shows the mean body weights of 10 female B6C3Fιmice with and without intravaginal application of GM-4, 5 days/week for 13 consecutive weeks.
Detailed Description of the Invention 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: "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 μm or below.
"Lipid" is an inclusive term for fats or fat derived materials. "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. 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 ofthe other substance. The analog or 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 ofthe 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 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) 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 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. 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. "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). "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 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. "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 (IN) complex" is a coordination compound including vanadium as the central metal atom or ion, and the vanadium has an oxidation state of +4 (IN), and is double bonded to oxygen.
"Metallocene" is an organometallic coordination compound containing cyclopentadienyl rings attached to a transition metal or transition metal halide.
"Nanadocene" is a metallocene including vanadium as the transition metal ion.
"Transition metals" is any of a number of elements in which the filling ofthe 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. "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. "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 carbamium carbom. Gel-Microemulsion Formulations
One aspect ofthe invention is directed to a pharmaceutical composition adapted for use as a spermicide. The spermicidal activity ofthe pharmaceutical composition can be in vitro or in vivo. The spermicidal compositions ofthe 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. 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 C6-Cι2 fatty acid glyceride esters, and the like. Preferred C6-Cι2 fatty acid glyceride esters include medium chain -Cn 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 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 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 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 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. 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 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). 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. However, to enhance the usefulness ofthe 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 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 FMC Corporation (Philadelphia, PA). Suitable xanthan gums include
XANTURAL 75 commercially available from Monsanto Pharmaceutical Ingredients (St. Louis, MO). The formulation can also optionally include one or more additives such as preservatives or antioxidants to help maintain and prolong the useful life ofthe gel- microemulsion. Preservatives and antioxidants that are generally known, and do not detract significantly from the usefulness ofthe gel-microemulsion for the particular 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 ofthe action of microorganisms in the formulation can be 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 ofthe gel-microemulsion 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 ofthe 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 about 200 to about 1000 centipoise.
Those of skill in the art will recognize that the amounts of each ofthe 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. 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 ofthe microemulsion components used for the preparation of one embodiment ofthe invention, GM-4, is shown in Figure 1, and discussed in the Examples below. The non-grid area represents the single phase microemulsion region suitable for use. The concentration ofthe components can be selected from within this region. The asterisk represents the particular concentration of components ofthe microemulsion which was used for the GM-4 formulation. Suitable gel-polymer suspensions can then be selected as additives to the 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 components of some gel-microemulsion formulations embodying the invention can be found in Table 1, wherein the values are given in wt. % ofthe ingredients in reference to the total weight ofthe formulation.
Table 1
Figure imgf000013_0001
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. % ofthe ingredients in reference to the formulation weight.
Table 2
Figure imgf000013_0002
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 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 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 ofthe Gel-microemulsion
The contraceptive compositions ofthe present invention are preferably 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. 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 ofthe present invention may be delivered to the vagina of a mammal by any means known to those skilled in the art. The gel- microemulsion can be applied directly. Other typical forms for delivery ofthe 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 within or on a condom for example. Inter-vaginal devices may also be used to aid in the administration ofthe 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. It is also contemplated that the formulation of the invention may be incoφorated 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.
Gel-Microemulsion as a Formulation Base for Other Therapeutic Agents Another aspect ofthe invention is the use ofthe above described gel- microemulsion formulations as formulation bases for incorporating therapeutically active agents. The base gel-microemulsion formulations generally include the 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 ofthe gel-microemulsions ofthe invention are especially suitable as solubilizing vehicles for poorly water soluble compounds. In some preferred 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 Suitable examples of anti-microbial theraputic agents, include anti-viral agents, anti-bacterial agents, anti-fungal agents, and the like. Such agents can be incoφorated 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 treatment of sexually transmitted diseases, for example, AIDS (HIV-1, HIV-2, FIV, SIV, etc.) genital heφes, 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 incoφorated by reference herein:
U.S. Patent Application No. 09/047,609, which is incoφorated herein by reference, and corresponding published PCT Application No. PCT/US99/06381 (International Publication Number WO 99/48902), which is incoφorated herein by reference; U.S. Patent Application No. 09/450,082 which is incoφorated herein by reference, and corresponding published PCT Application No. PCT/US99/14774
(International Publication Number WO 00/00501) which is incoφorated herein by reference; and U.S. Patent Application No. 09/107,716, which is incoφorated 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 Application No. PCT/US99/06381 , include AZT derivatives disclosed therein. Many ofthe AZT derivatives disclosed therein also have spermicidal activity. Examples of such anti-microbial AZT derivatives include compounds ofthe formula:
Figure imgf000016_0001
where R, is H, N3, halo, CN, COOH or NH2, R2 is halo (particularly Cl, Br or
I, and more particularly Br) and R3 is alkoxy (particularly Cl-3 alkoxy, and more particularly methoxy (-OCH3)). The NH2 group can be functionalized, for example with -CH3, -COCH3, -Ph, -COPh, and -CH,Ph. Pharmaceutically acceptable salt or ester forms also can be used, such as sodium, potassium or ammonium salts. The derivatives ofthe formula above include substitution on the AZT pentose ring member. The derivatives of this aspect ofthe present invention have the chemical structure illustrated below:
Figure imgf000017_0001
where R, is H, N3, halo, CN, COOH or NH2, R2 is halo (particularly Cl, Br or I, and more particularly Br), R3 is alkoxy (particularly Cl-3 alkoxy, and more particularly methoxy (-OCH3)) and R' is a group that facilitates the passage ofthe compound into a cell. As in the first formula, the NH2 group can be converted, for example to NHCH3, NHCOCH3, NHPh, NHCOPh, and NHCH2Ph. 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 ofthe first formula) at the R' or R, positions for targeted delivery. 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 ofthe phosphate can be replaced with Cl-4 alkyl or aryl substituents (e.g. phenyl-, naphthyl- or anthracinyl-substitution), which optionally may be substituted, and SH or NH2 groups can replace the OH ofthe phosphate, and in each of these cases a H ofthe NH2 or SH can be replaced in the same manner as the H ofthe OH group discussed previously. The aryl phosphate group is suφrisingly effective in maintaining excellent anti-HIV activity. A general structure of an exemplary aryl phosphate OR' group is illustrated below:
Figure imgf000017_0002
where R4, R5, and R6are the same or different and are selected from hydrogen, methyl, ethyl, fluoro, chloro. bromo, iodo, dichloro, dibromo, difluoro, trifiuoromethyl, 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 ofthe alaninyl group, the methyl group attached to the CH group can be substituted, for example with a phenyl group, and the methyl esterification can be replaced with other C2 or C3 esterification.
Of these compounds, the following compound, hereinafter referred to as
WHI-07, is particularly preferred:
Figure imgf000018_0001
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 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:
Figure imgf000018_0002
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; R2 is a nucleoside of one ofthe following formulae:
Figure imgf000019_0001
in which R6 is purine or pyrimidine, preferably pyrimidine; and R7, R8, R,, Rl0, R,,, and R12 are independently hydrogen, hydroxy, halo, azido, -NO2, -NRI3Rι4, or - N(OR15)R16, in which Rl3, RM, R15, and Rl6 are independently hydrogen, acyl, alkyl, or cycloalkyl;
R3 is hydrogen, acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino acid; or R3 and R4 may be taken together to form the side chain of proline or hydroxyproline; and R5 is hydrogen, alkyl, cycloalkyl, or aryl. As used in the definitions ofthe 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 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 ofthe formula R17C(O)-, in which RI7 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, -NO,, -NR]3R14, or -N(OR,5)R16, in which R,3, R14, R15, and R,6 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 substituted with hydroxy, halo, azido, -NO,, -NR,3R14, or -N(OR15)R,6, in which R13,
R]4, RI5, and R,6 are as defined above.
The term "purine" includes adenine and guanine.
The term "pyrimidine" includes uracil, thymine, and cytosine. Preferably, 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, 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 ort/zo-or/wα-substituted halogen or NO, provide for more efficient hydrolysis to active inhibitory compounds. Preferred is halogen substitution, and most preferred are para-bromo substitution and para-chloro substitution.
Preferred nucleoside derivative disclosed therein include those ofthe formulas:
Figure imgf000020_0001
wherein X is an electron withdrawing group, for example halo or NO,, and most preferably, X is bromo or chloro.
Gel— Microemulsion Formulations with Additional Spermicidal Agents
Suitable examples of spermicidal agents, include any spermicidal agent generally known that is compatible for formulation with the gel-microemulsion.
Such agents can be incoφorated into the gel-microemulsion formulations to provide for additional spermicidal activity ofthe formulation. Many ofthe spermicidal agents discussed below also have anit-microbial activity, such as anti-viral activity, and can be incoφorated into the formulations as an anti-microbial agent as well as a spermicidal agent.
Preferred Spermicidal Agents For Incorporation Into the Gel- Microemulsion Formulation: Preferred examples of spermicidal agents include those disclosed in the following copending patent applications:
U.S. Patent Application No. 09/008.898, which is incoφorated herein by reference, and corresponding published PCT Application No. PCT/US99/01171 (International Publication Number WO 99/36063), which is incoφorated herein by reference; U.S. Patent Application No. 09/187,115, which is incoφorated herein by reference;
U.S. Patent Application No. 09/224,677, which is incoφorated herein by reference.
Examples of preferred spermicidal compounds disclosed in copending U.S.
Patent Application No. 09/008,898, and corresponding published PCT Application No. PCT US99/01171, include the vanadium (IV) compounds disclosed therein. Examples of such vanadium (IV) compounds include organometallic cyclopentadienyl vanadium IV complexes. Preferred such compounds include: 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. Particularly preferred compounds include vanadocene diselenocyanate, and vanadocene dichloride.
Examples of preferred spermicidal compounds disclosed in copending U.S. 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, 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 general formulae:
Figure imgf000022_0001
where R and Ri are the same or different and are independently selected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C2-C6) and nitro; X and X1 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 oxovanadium IV complex has the general formulae:
Figure imgf000022_0002
where R2 and R3 are the same or different and are selected from H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C2-C6) and nitro; X2 and X3, 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. 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:
Figure imgf000023_0001
where R4, R5 and R6 are the same or different and are independently selected from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C2-C6) and nitro; X4 and X5 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 oxovanadium IV complex has the general formulae:
Figure imgf000023_0002
where R7 ; and R9 are the same or different and are independently selected from: H, lower alkyl, lower alkoxy, and halogenated alkyl; R8 is selected from H, lower alkyl, halo, lower alkoxy, and halogenated alkyl; Y and Y1 are the same or different and independently selected from the group consisting of: monodentate and bidentate ligands: and n is 0 or 1. Preferred monodentate ligands for the oxovanadium complex include H,O, 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' 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. 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; (diaqua)( 1 , 10-phenantnroline)oxovanadium(l V) 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; (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 (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:
Figure imgf000025_0001
where R, Ri, R2, R3, and I * are independently hydrogen, F, Cl, Br, or I, and where at least one of R, Ri, R2, R3, and j is F, Cl, Br, or I. Preferably, one of R, Ri, R , R . and t in structure above is F or Cl. Some, but not all ofthe suitable halogen- substituted PBT derivatives ofthe invention are listed below:
N-[2-(2-fmorophenethyl)]-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.
One ofthe more preferred PBT derivatives is N-[2-(2-fluorophenethyl)]- N'-[2-(5-bromopyridyl)]-thiourea (F-PBT) which has the chemical structure shown below:
Figure imgf000025_0002
F-PBT PBT derivatives can be synthesized as described in Vig et al.. BlOORG. MED. CHEM.. 6:1789-1797 (1998). In brief, 2-amino-5-bromopyridine is condensed with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl derivative. Further reaction with appropriately halogen-substituted phenylethylamine gives the target halogenated PBT derivatives.
Examples of suitable DABO derivatives include those ofthe formula, or a pharmaceutically acceptable salt thereof:
Figure imgf000026_0001
where Ri and R2 are alike or different, and are hydrogen, halo, alkyl, alkenyl. hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl. Preferably, one or more of Ri and R2 is an alkyl having 1 to 3 carbonatones, ( - C3), such as methyl (Me), ethyl (Et), or isopropyl ( -Pr). Preferably, Ri is alkyl, alkenyl, ROH, or RNH2. R2 is preferably halo, alkyl, or -C3 alkoxy;
Y is S or O, and is preferably S. R3 is alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R is alkyl , and is preferably -C3 alkyl.
Preferred DABO derivatives include compounds having the chemical structure shown below, or a pharmaceutically acceptable salt thereof:
Figure imgf000026_0002
where Ri is Me, Et, or /-Pr and R-> is H or Me. Some, but not all. ofthe suitable DABO derivative compounds include compounds (a) through (d) listed below, or a pharmaceutically acceptable salt thereof: (a) 5-methyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4- 1 H- one,
(b) 5-ethyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-lH- one, (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 ofthe more preferred DABO derivatives is the compound 5-isopropyl- 2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-( 1 H)-ones (S-DABO) , and pharmaceutically acceptable salts thereof, which is exemplified by the chemical structure shown below:
Figure imgf000027_0001
S-DABO
DABO derivatives can be prepared as described in descrioed 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:
Figure imgf000028_0001
Figure imgf000028_0002
Reagents and conditions a) R CHBrCOOEt/Zn/THF, b) HCI(aq), c) (H2N^CS Na EtOH, d) DMF, KCQi, Chioromethyl methyl sulfide, 15h.
Briefly, ethyl-2-alkyl-4-(phenyl)-3-oxobutyrates la-d were obtained from commercially available phenyl acetonitrile. The β-ketoesters were condensed with 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., Ada 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).
Subsequent reaction of thiouracil with methylchloromethyl sulfide in N.N-dimethylformamide (DMF) in the presence of potassium carbonate afforded compounds 3 a-d in moderate yields. A mixture of thiouracil compound 2 (1 mmol), methylchloromethyl sulfide (lmmol), and potassium carbonate (lmmol) 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 ), filtered and concentrated in vacuo to give the crude products 3 a-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 compounds include complexes having the following structural formula, or pharmaceutically acceptable salts thereof:
Figure imgf000029_0001
wherein R , R , R and R are the same or different and are independently selected from H, halo, OH2, O3SCF , N3, CN, OCN, SCN, SeCN, NO2, Cj-C4 alkyl, C|-C alkoxy, and aryl; and n is 0 or 1 ; and R5 and R6 are the same or different and are either monodentate ligands or R5 and R6 together comprise a bidentate ligand.
Suitable monodentate ligands include, for example, aryl, halo, H2O, O SCF3, N3, COOH, CN, OCN, SCN. SeCN, NO2, C,-C4 alkyl, -C 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 O,O' bidentate ligands. Examples of suitable N, N' bidentate ligands include diamines and other such known suitable N, N' 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. 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.
Some preferred vanadium (IV) catacholate complexes include ''bent sandwich" vanadocene monocatacholate complexes having the following structure formula, or pharmaceutically acceptable salts thereof:
Figure imgf000030_0001
wherein Cp is unsubstituted cyclopentadienyl, or cyclopentadieneyl substituted with one or more substituents selected from substituted or unsubstituted aryl, - alkyl, d-C4 alkoxy, halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, NO2. Preferably, Cp is unsubstituted cyclopentadienyl.
R , R , R and R4 are the same or different and are independently selected from H, halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN. NO2, C,-C4 alkyl, and C,- C alkoxy Preferably, electron donating groups, for example electron donating alkyl groups, are present as substituents of the catacholate ring in positions R , R , R and/or R4.
Particularly preferred such compounds include vanadocene catacholate, vanadocene mono-tertbutyl catacholate, and vanadocene 1 , 3-diisopropyl 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:
Figure imgf000031_0001
where R7 and R8 are the same or different and are independently selected from: H, aryl, -C4 alkyl, halo, Cι~C alkoxy, carboalkoxy (e.g. C2-C6), cyano, and nitro; n is 0 or 1 ; R1, R 2, R3 and R4 are the same or different and are independently selected from H, halo, OH2, O3SCF , N3, CN, OCN, SCN, SeCN, NO2, C,-C4 alkyl, and C,-C4 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:
Figure imgf000032_0001
where R9 and R10 are the same or different and are selected from H, aryl, Cj- C4 alkyl, halogen, C[-C4 alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C2-C6) and nitro; Z is selected from O, CH2, CH2-CH2, and CH=CH; n is 0 or 1; and halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, NO2, -C, alkyl, and Cj-C alkoxy.
Another example of vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is a bridged bipyridyl, and the bridged bipyridyl is phenanthroline, has the general formula shown below, or pharmaceutically acceptable salts thereof:
Figure imgf000032_0002
where R11, R12 and R13 are the same or different and are independently selected from: H, aryl, Cι~C4 alkyl, halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C2-C6) and nitro; n is 0 or 1; and R1, R2, R3 and R are the same or different and are independently selected from H, halo, OH2, 03SCF3, N3, CN. OCR SCN, SeCN. NO2, C,-C4 alkyl, and C,-C4 alkoxy.
Another example of vanadium (IV) catacholate complexes include complexes having a bidentate ligand wherein the bidentate ligand is an O,O' bidentate ligand, and the complex has the general formula VI, is shown below, or pharmaceutically acceptable salts thereof:
Figure imgf000033_0001
where R14 and R16 are the same or different and are independently selected from: H, aryl, -C alkyl, and C]-C alkoxy, and halogenated alkyl; R15 is selected from H, Cι~C4 alkyl, halo, Cι~C4 alkoxy, and halogenated alkyl; n is 0 or 1; and R1, R2, R3 and R4 are the same or different and are independently selected from H, halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN. NO2, Cj-C4 alkyl, and -C4 alkoxy.
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 formulated and prepared in substantially the same way as the primary gel- microemulsions discussed above, with the only difference being the addition ofthe additional therapeutic agent. The amount of additional ingredient added is dependent upon the desired effective amount ofthe ingredient in the final gel- microemulsion formulation. It is still desirable to provide 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. 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. % ofthe ingredients in reference to the total weight ofthe formulation.
Table 3
Figure imgf000034_0001
In some preferred embodiments, the formulation includes the specific constituent concentrations for base components as found in Table 4, wherein the values are given in wt. % ofthe ingredients in reference to the formulation weight.
Table 4
Figure imgf000034_0002
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 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 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.
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 as the gel-microemulsion spermicide use discussed above. However, it is contemplated that formulations including other theraputic agents, for example antiviral 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 effectuate the desired theraputic effect. For example, in anti-microbial applications, the formulation is preferably administered to a site appropriate for desired antimicrobial 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 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 ofthe preferred embodiments, and not to limit the invention. EXAMPLES Example 1: Synthesis of Gel-Microemulsion Formulations
Materials: Captex 300 was obtained from ABITEC Coφ., Janeswille,
WI. Cremophor EL was from BASF Coφ., Mount Olive, NJ. Phospholipon 90G was purchased from American Lecithin Co., Danbury, CT. PEG-200 was from Union Carbide Coφ., Danbury, CT while propylene glycol was obtained from Spectrum Quality Products Inc., New Brunswick, NJ. Seaspan and viscarin carrageenan were obtained from FMC Coφ., 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: 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 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 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, 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 ofthe microemulsion components used for the preparation of GM-4 is shown in Figure 1. The non-grid area represents the single phase microemulsion region. The asterisk represents the microemulsion which was used for GM-4 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 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
Engineering Laboratories, Spoughton, MA).
Table 5: Components of GM-4 Formulation
Figure imgf000037_0001
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 activity ofthe pharmaceutical excipients used in the GM-4 formulation, a highly 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:\ 18-30.; D'Cruz OJ, et al., Biol Reprod, 1998; 59:503-15.). All donor specimens were obtained after informed consent and in compliance with the guidelines ofthe Parker Hughes Institute
Institutional Review Board. Highly motile fraction of sperm _ 10x10 /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 (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 benzoate (0.025%> - 0.2%). After 3 h incubation at 37°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 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 (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 ofthe test compound was expressed as EC50 (the final concentration ofthe compound in the medium that decreased the proportion of 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, seaspan carrageenan. viscarin carrageenan, and sodium benzoate, demonstrated little 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 ofthe swimming pattern, linearity ofthe sperm tracks, beat-cross frequency, and the amplitude of 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, 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
Methods and Materials
The effect of duration of incubation on spermicidal activity in the presence of each ofthe 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 concentrations contained in GM-4. At timed intervals of 15, 30, 45 and 60 min, 5- μl samples were transferred to two 20-μm 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 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 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— μl sample was transferred to a 20 μm Microcell chamber (Conception Technologies) and examined immediately under a phase contrast microscope (Olympus BX-20; Olympus Coφoration, Lake Success, NY) attached to a CCD camera (Hitachi Deneshi Ltd., Tokyo, Japan) and a videomonitor. The time required for sperm immobilization was recorded in seconds. This test was performed in six separate trials, with semen obtained from six different donors.
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 ofthe individual components of GM-4 formulation revealed that none ofthe eight 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 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.
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:
Figure imgf000040_0001
Chemical name: (5R,6R)-and (5S,6S)-5-bromo-6-methoxy-5,6-dihydro- AZT-5'-(para- bromophenyl methoxyalaninyl phosphate). Molecular weight:
698
WHI-07 was synthesized using the following synthetic scheme. Synthetic Scheme for WHI-07
Figure imgf000041_0001
Chemical Characterization:
Melting Point : 59-60°C; Rf : 0.56 ( 10%MeOH/ 90% CHCI3 ); 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 (CDCI3) d 8.66 (IH, 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, m, -CH at C-1 '), 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 (CDCI3) 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, 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.; 31P NMR (CDCI3) d 2.70, 2.60, 2.54, 2.32.; MS (Cl, m/e) 700.6 (M+, 81Br+81Br), 698.6 (M+, 81Br+79Br), 696.6 (M+, 79Br+79Br), 588.8 (M+ - Br-OCH3, 81Br), 586.9 (M+ - Br-OCH3, 79Br).; HPLC: 39.06, 40.28, 45.33, & 49.25 min (Column: LiChrospher 100 RP-18e (5 μm); Flow rate: 1 mL/min; Solvent: H2O (0.1% TEA + 0.1% TFA) : CH3CN = 62:38). Appearance: White solid at room temperature; Solubility: < 0.003%o solubility in water, soluble in oil (medium chain triglyceride, 4.1%), and quite soluble in some hydrophilic cosol vents such as polyethylene glycol 300 (13.1%). It is also soluble in several organic solvents including chloroform, ethanol, methanol, and DMSO.; 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 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 partition coefficient.
Table 6
Figure imgf000042_0001
The average octanol/water partition coefficient is: Log KD = 2.05
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.
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. 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 ofthe calibration curve is shown in Curve 1.
Table 7
Figure imgf000043_0001
Curve 1
WHI-07 Cone mg/ml = 0 0134 + 0 912 x Correlation coefficient RΛ2 = 0999
Abs (272 nm)
Sample preparation and assay
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 μm filter, and diluted with ethanol for UV-Vis measurement. The results are shown in Table 8.
Table 8
Figure imgf000044_0001
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- 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 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 was heated to 70 C. 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 measurement. The particle diameter ofthe 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 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 gel formulations, however, are not totally soluble in either ethanol or acetonitrile because ofthe polymers in the formulation. The following WHI-07 analytical methods were developed for the gel formulations. The accuracy and precision ofthe methods meet pharmaceutical requirements.
WHI-07 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 50 C until the drug was completely dissolved and the mixture was clear. The vial was removed from the hot plate and 3.5 ml ofthe 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. The control (drug-free) gel microemulsion was prepared by mixing 1.2 ml of the microemulsion with 2.8 ml ofthe polymer suspension. The calibration curve was prepared as following:
Five standard solutions with varying WHI-07 contents were prepared as 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 φm. The vials were carefully removed from the centrifuge and 0.5 ml ofthe supernatant was pipetted out from each vial for HPLC analysis.
The condition for HPLC analysis (WHI-07) was as followed:
Column: RP18e (5μm) Lot # L228433
Eluent: Acetonitrile / (0.1% TFA and 0.1% TEF) = 45 / 55
Eluent flowrate: 1 ml/min
Sample injection volume: 20 μl 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 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
Vial # WHI-07 gel Control gel ACN added WHI-07 concentration Peak(20 min) added (IT »0 added (ml) (ml) (mg/ml) (mAU*s)
(20.26 m g/ml)
1 0 0.50 4.0 0 0
2 0.05 0.45 4.0 0.225 1 121.2
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 Curve 2: Calibration curve for WHI-07 gel formulation
WHI-07 Cone mg/ml = 0 0022 + 1 924 x Peak area / Correlation coefficient RΛ2 =
2000 4000 HPLC Peak areas
Precision ofthe analytical method To test the precision ofthe 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 ofthe 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 C until the drug was dissolved and the mixture was clear. The vial was removed from the hot plate and 3.5 ml ofthe 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 ofthe microemulsion with 2.8 ml ofthe polymer suspension. To each of the 10 vials, 0.2 ml ofthe WHI-07 gel, 0.3 ml ofthe 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 φm. The vials were carefully removed from the centrifuge and 0.5 ml ofthe supernatant was pipetted out from 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 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
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
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
9 2332.8 38.04
10 2303.2 8.44
Average 2294.76 14.08 0.61%
Procedures for WHI-07 formulation sample preparation:
240 mg of WHI-07 was weighed in a 10 ml glass shell vial. 4.0 ml ofthe 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 Corning stir/hot plate for mixing. The dials for both stir and heat were set at #3. The mixture 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 ofthe concentrated WHI-07 microemulsion with 4 ml ofthe polymer suspension in a 10 ml glass shell vial. 1% WHI-07 gel was prepared by mixing 1 ml ofthe concentrated WHI-07 microemulsion with 1 ml ofthe microemulsion and 4 ml ofthe polymer suspension. 0.5% WHI-07 gel was prepared by mixing 0.5 ml ofthe concentrated WHI-07 microemulsion with 1.5 ml ofthe microemulsion and 4 ml ofthe polymer suspension. The control gel was prepared by mixing 2 ml ofthe microemulsion and 4 ml ofthe polymer suspension.
WHI-07 formulation concentration analysis: Two samples, 100 μl each, were taken from each preparation for concentration analysis. The samples were placed in 5 ml glass shell vials. 4.2 ml of acetonitrile was added to each ofthe sample vials. The vials were hand shaken for 10 seconds and were placed in a shaker at 200 φm for 30 min. The vials were hand shaken again for 10 seconds and span for 10 min at 2000 φm in the Beckman GS-6 Centrifuge. The vials were carefully removed from the centrifuge and 1 ml each ofthe supernatant was pipette out for HPLC analysis. The results were shown in Table 11. All ofthe samples meet pharmaceutical requirement (RSD less than 5.0%).
Table 11
Vial # Sample ID WHI-07 Average RSD 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%
5 2.0% WHI-07 1.88%
6 2.0% WHI-07 1.99% 1.94 3.0%
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: Table 12
Figure imgf000050_0001
The result indicates that WHI-07 was stable in the gel microemulsion 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 was a translucent gel with the following composition:
% (by weight)
WHI-07 2.0
Captex 300 10.7
Phospholipon 90G 5.0
Cremophor EL 7.5
Propylene glycol 4.2
PEG 200 4.2
SeaSpan Carrageenan 0.9
Viscarin Carrageenan 0.9
Sodium benzoate 0.2
Water 64.4
The viscosity of the GM-4 formulation with and without WHI-07 was determined using a Brookfield DV-E Viscometer with spindle #3 (speed: 10 φm). Drug-free GM-4 formulation had a viscosity of 301.9 centripoises. The GM-4 formulation containins 2% WHI-07 had a viscosity of 64.7. Although a viscosity of 64.7 appeared to be sufficient for the utility ofthe
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: 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:
% (by weight)
WHI-07 2.0
Captex 300 10.7
Phospholipon 90G 5.0
Cremophor EL 7.5
Propylene glycol 4.2
PEG 200 4.2
SeaSpan Carrageenan 0.9
Xantural 0.5
Sodium benzoate 0.2
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.
EXAMPLE 4: Preclinical Studies:
Methods and Materials
Rabbits: Fifty nine female and 12 male, sexually mature (> 6 months old; > 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 ± 2°C with relative humidity of 50 ± 20% and a 12-h fluorescence light cycle. The 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. Mice: Twenty, female B6C3F1 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°C with relative humidity of 50 ± 10% and a 12-h 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. Semen was obtained from bucks (n = 12) of proven fertility via a prewarmed (45°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
7 and 0.5 ml (>30 x 10 sperm/ml) aliquots were transferred to 1 ml tuberculin syringes. Two ml of a GM-4 formulation or a commercial 2% N-9 formulation (Gynol II; Ortho Pharmaceutical Coφ., 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 ofthe test agent followed by the application of semen dose (0.5 ml) which was deposited within 1-2 min by inserting approximately 8 cm ofthe syringe into the vagina for the delivery ofthe 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 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 repeated a second time. 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 1 1 and the reproductive tract was examined grossly and microscopically after completion ofthe study (Eckstein P, et al.,J Reprod Fertil,
1969;20:85-93.). The vaginal tissues were rapidly removed and parts ofthe caudal, mid, and distal regions of each vagina were fixed in 10%> buffered formalin. Tissues were embedded in paraffin, sectioned at 4-6 μm and stained with hematoxylin and eosin and examined under x200 and χ400 magnification using a Leica light 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 analysis. Each ofthe 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., J Reprod 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, 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 μl ofthe GM-4 formulation intravaginally for 5 days per week for 13 consecutive weeks. Ten mice 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 ofthe study, animals were sacrificed 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 analyzer (Abbot Laboratories. Abbot Park, IL) which was standardized for mouse blood. This instrument uses flow cytometric techniques to provide the hemograms for anticoagulated whole blood samples: red blood cell count (RBC; 10 /μl), total and differential leukocyte count (lymphocytes [LYM], neutrophils [NEU], monocytes [MONO], eosinophils [EOS], and basophils [BASO] as 10 /μl or %), hemoglobin concentration (HGB; g/dl), hematocrit (HCT; %>), mean coφuscular volume (MCV; fl), mean cell hemoglobin content (MCH; pg), mean cell hemoglobin concentration (MCHC; g/dl), red cell distribution width (RDW; %), platelet count
(PLT; 103/μl), and mean platelet volume (MPV; fl).
Clinical Chemistry Profiles: Biochemical analyses were performed using a 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 (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 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, skeletal muscle, skin, spinal cord, and urinary bladder were fixed in 10%> buffered formalin solution, trimmed, embedded in paraffin, sectioned at 4-6 μm, and stained with hematoxylin and eosin. Complete histopathological examination of all tissues was performed on mice from the control and GM-4 group.
Statistical Analysis: Group means and standard deviations were calculated from initial and terminal body weights, organ weights, hematology and clinical chemistry parameters. Statistical significance ofthe 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 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 (FlVβang)- Cats were challenged with FIV by inoculating 7x10 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 ofthe 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. 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 cells/culture) were cocultured for 3 weeks with 5x10 T- cell enriched PBMCs from specific pathogen-free (SPF) cats in a total volume of 5
2 ml in a 25 cm flask. Culture supematants 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 supematants 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 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 ofthe following criteria was met during the 18-week observation period: (1) Serum samples from two different bleeding dates were positive by Western blot analysis; (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.
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 standard rabbit model. Gynol II, a commercial contraceptive containing 2% N-9, 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
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 ofthe 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%) 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 68.7%, ? < 0.05, Fisher's exact test).
Table 13 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
Figure imgf000056_0001
Aliquots (0.5 ml) of fresh, pooled semen obtained from fertile bucks (n = 12) 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 of hCG and allowed to complete term pregnancy.
Significantly different from control by Fisher's exact test, (p < 0.0001) 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 ofthe vaginal tissue after daily intravaginal application of GM-4 for 10 consecutive days showed lack of significant vaginal irritation in all seven rabbits examined (mean individual scores 0-1; total score 2, 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 (Eckstein P, et al., J Reprod 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 ofthe epithelial lining, and an inflammatory response with influx of leukocytes, consistent with previously published observations in rats (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
GM-4 GM-4 + 4% N-9
(n = 7) (n = 4)
Epithelial ulceration 0 3 ± 2 "
Lamina propria thickness 1 + 1 2 ± 1
Leukocyte Infiltration 1 + 1 3 ± 2
Vascular congestion 0 1 + 1
Total score 2 ± 1 9 ± 2
Seven rabbits were administered intravaginally with 1 ml of GM-4 and 4 rabbits were exposed to GM-4 containing 4% N-9. Mean ± SD values representing the caudal, middle, and distal sections of vagina from each rabbit. ^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 >11 unacceptable.
Lack of Systemic Toxicity from GM-4 in Mice: Female B6C3F1 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 ofthe study. Mean body weight gain and final 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 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 to GM-4 formulation.
Table 15 Hematological Findings for B6C3F1 Mice Given GM-4
Intravaginally for 13 Weeks
Parameter Control GM-4
RBC (x lONμl) 949 ± 62 905 ± 61
WBC (x lONμl) 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 Others (%)T 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(χ lONμl) 358 ±226 413 ±327
MPV (fl) 7.4 ± 2.7 6.2 ± 0.7
.Mean ± SD for groups of 5 mice. TOthers = MONO, EOS, AND BASO.
RBC, red blood cells; WBC, white blood cells; LYM, lymphocytes; NEU, neutrophils; MONO, monocytes; EOS, eosinophils; BASO, basophils; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean coφuscular volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; RDW, red cell distribution width; PLT, platelets; MPV, mean platelet volume.
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 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/1 125 ±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
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).
13- Week Necropsy/Organ Weights and Histopathology: Table 17 summarizes the terminal body weight, terminal absolute and relative organ weights observed at the conclusion ofthe 13-week study. No statistically significant 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 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 ofthe lipophilic and spermicidal GM-4 formulation.
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 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). 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 7x10 FIVBang-infected FeT-J cells mixed in 0.2 mL of infected 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).
Table 18 WHI-07(2%)/GM-4 Gel Formulation Prevents Vaginal
Transmission of FIV in Cats
Cat No. Treatment WB Virus Isolation PCR Overall Protection
Serum PBMC PBMC Conclusion Rate
1 None 3X 3X 3X +
2 None 5X 4X 2X +
3 None IX 2X 2X + Not applicable
4 None 5X 5X 6X +
5 None 4X 2X 2X +
6 WHI-07/GM-4 0 0 0 - 7 7 WHI-07/GM-4 0 0 0 -
8 WHI-07/GM-4 5X 5X 4X + 60%
9 WH1-07/GM-4 0 0 0 -
10 WHI-07/GM-4 5X 6X 5X +
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.
Table 19 WHI-07(2%)/GM-4 Gel Formulation Prevents Rectal
Transmission of FIV in Cats
Cat No. Treatment WB Virus Isolation PCR Overall Infection
Serum PBMC PBMC Conclusion Rate
1 None 6X 6X 6X 2 2 N Noonnee 0 0 0 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
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. 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 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 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 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 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, 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 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 studies suggest that this is most likely due to incomplete mixing of semen with N-9 gel or inadequate distribution ofthe 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 ofthe potent in vitro and in vivo spermicidal activity of GM-4 formulation, it was 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 ofthe rabbit despite the fact that it was a potent 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 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., Cancer Res, 1990;50:4199-203.; Nerurkar MM, et al., Pharmaceutical Research, 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. In short-term toxicity studies, intravaginal administration ofthe 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. 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 ofthe intravaginally applied GM-4 on the long-term health and reproductive performance of test animal species are currently in progress.
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 ingredients. In some embodiments ofthe 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 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. The microemulsion-based lipophilic and vaginal spermicide, GM-4, appears to offer several benefits for vaginal delivery including increased absoφtion, improved contraceptive efficacy, and decreased toxicity. Under the described conditions of its intended use, a 13-week intravaginal application of GM-4 formulation in B6C3F1 mice did not result in systemic toxicity and no other specific target organs were identified. Therefore, the spermicidal GM-4 formulation shows 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 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 heφes, gonorrhea and chlamydia.
All publications, patents, and patent documents described herein are incoφorated 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 ofthe invention, as claimed below.

Claims

Claims:
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 C6-Cι 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 hydrogel 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; 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 C6-Ci2 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 C6-Cι2 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 C6-Ci2 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.
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:
Figure imgf000070_0001
wherein R, is H, N3, halo, CN, COOH or NH,; R2 is halo; R3 is alkoxy; and R' is a group that facilitates the passage ofthe 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 ofthe following formula, or a pharmaceutically acceptable salt thereof:
Figure imgf000071_0001
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:
Figure imgf000071_0002
in which R6 is purine or pyrimidine; and R7, R8, Rg, R10, R,,, and R12 are independently hydrogen, hydroxy, halo, azido, -NO2, -NR13R14, or -N(OR15)R,6, in which RI3, R,4, R15, and R16 are independently hydrogen, acyl, alkyl, or cycloalkyl;
R3 is hydrogen, acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino acid; or R3 and R4 may be taken together to form the side chain of pro line or hydroxyproline; and R5 is hydrogen, alkyl, cycloalkyl, or aryl.
24. The composition of claim 23, wherein the nucleoside derivative is selected from those of the formulas:
Figure imgf000071_0003
wherein X is an electron withdrawing group.
25. The composition of claim 24, wherein X is bromo or chloro.
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:
Figure imgf000073_0001
wherein R , R2, R3 and R4 are the same or different and are independently selected from H, halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, NO , C,-C4 alkyl, Cj-C alkoxy, and aryl; and n is 0 or 1 ; and R3 and R6 are the same or different and are either monodentate ligands or R5 and R6 together comprise a bidentate ligand.
34. The composition of claim 33, wherein the vanadium (IV) complex comprises complexes having the structural formula:
Figure imgf000073_0002
wherein Cp is unsubstituted cyclopentadienyl, or cyclopentadieneyl substituted with one or more substituents selected from substituted or unsubstituted aryl, C,-C4 alkyl, C,-C4 alkoxy, halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, NO2;
R , R , RJ and R4 are the same or different and are independently selected from H. halo, OH2, O3SCF3, N3, CN, OCN, SCN, SeCN, NO2, C,-C4 alkyl, and Cr C4 alkoxy
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 ofthe following chemical formula, or a pharmaceutically acceptable salt thereof:
Figure imgf000074_0001
where R, Ri, R2, R3, and R are independently hydrogen, F, Cl, Br, or I, and where at least one of R, Ri, R2, R3, and * 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 ofthe following chemical formula, or a pharmaceutically acceptable salt thereof:
Figure imgf000075_0001
where R\ and R2 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 R3 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-l H-one, 5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-l 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 ofthe therapeutic agent.
43. The composition of claim 42, wherein the gel-microemulsion 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; 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 Cό-Cι2 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 C6-Cι2 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 C6-Cι2 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 ofthe therapeutically active agent; administering the gel-microemulsion to the subject.
50. The method of claim 49, wherein the therapeutically active agent is an antiviral agent.
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US6667339B2 (en) 1999-03-19 2003-12-23 Parker Hughes Institute Vanadium (IV) complexes containing catecholate ligand and having spermicidal activity
WO2000056302A3 (en) * 1999-03-19 2001-08-16 Parker Hughes Inst Vanadium (iv) complexes containing catacholate ligand and having spermicidal activity
US6465450B2 (en) 1999-03-19 2002-10-15 Parker Hughes Institute Vanadium (IV) complexes containing catecholate ligand and having spermicidal activity
WO2000056302A2 (en) * 1999-03-19 2000-09-28 Parker Hughes Institute Vanadium (iv) complexes containing catacholate ligand and having spermicidal activity
WO2001021153A2 (en) * 1999-09-23 2001-03-29 Hexal Ag Pharmaceutical gel containing active substances
WO2001021153A3 (en) * 1999-09-23 2001-10-11 Hexal Ag Pharmaceutical gel containing active substances
WO2001066084A2 (en) * 2000-03-07 2001-09-13 Rush-Presbyterian-St. Luke's Medical Center Compositions and methods for trapping and inactivating pathogenic microbes and spermatozoa
WO2001066084A3 (en) * 2000-03-07 2002-02-28 Rush Presbyterian St Luke Compositions and methods for trapping and inactivating pathogenic microbes and spermatozoa
US7811594B2 (en) 2002-03-28 2010-10-12 Beiersdorf Ag Crosslinked oil droplet-based cosmetic or pharmaceutical emulsions
WO2003082182A2 (en) * 2002-03-28 2003-10-09 Beiersdorf Ag Cross-linked cosmetic or pharmaceutical phospholipid-containing gels and emulsions based on ethylene oxide-containing or propylene oxide-containing emulsifiers
WO2003082223A2 (en) * 2002-03-28 2003-10-09 Beiersdorf Ag Cross-linked-oil-droplet-based cosmetic or pharmaceutical emulsions
WO2003082223A3 (en) * 2002-03-28 2004-02-05 Beiersdorf Ag Cross-linked-oil-droplet-based cosmetic or pharmaceutical emulsions
WO2003082182A3 (en) * 2002-03-28 2003-12-18 Beiersdorf Ag Cross-linked cosmetic or pharmaceutical phospholipid-containing gels and emulsions based on ethylene oxide-containing or propylene oxide-containing emulsifiers
US9040088B2 (en) 2002-04-12 2015-05-26 Alkermes Pharma Ireland Limited Nanoparticulate megestrol formulations
US9107827B2 (en) 2002-04-12 2015-08-18 Alkermes Pharma Ireland Limited Nanoparticulate megestrol formulations
US9101540B2 (en) 2002-04-12 2015-08-11 Alkermes Pharma Ireland Limited Nanoparticulate megestrol formulations
US9101549B2 (en) 2002-04-12 2015-08-11 Alkermes Pharma Ireland Limited Nanoparticulate megestrol formulations
WO2005105090A1 (en) * 2004-05-04 2005-11-10 Aq+ Plc Compositions for progeny gender control
GB2430371A (en) * 2004-05-04 2007-03-28 Aq & Plc Compositions for progeny gender control
US9278990B2 (en) 2010-09-22 2016-03-08 Alios Biopharma, Inc. Substituted nucleotide analogs
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8980865B2 (en) 2011-12-22 2015-03-17 Alios Biopharma, Inc. Substituted nucleotide analogs
US9605018B2 (en) 2011-12-22 2017-03-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
US9394330B2 (en) 2012-03-21 2016-07-19 Alios Biopharma, Inc. Solid forms of a thiophosphoramidate nucleotide prodrug
US9856284B2 (en) 2012-03-21 2018-01-02 Alios Biopharma, Inc. Solid forms of a thiophosphoramidate nucleotide prodrug
US9012427B2 (en) 2012-03-22 2015-04-21 Alios Biopharma, Inc. Pharmaceutical combinations comprising a thionucleotide analog
CN105418680A (en) * 2015-12-11 2016-03-23 重庆康施恩生物科技有限公司 Medicine sofosbuvir midbody capable of resisting hepatitis C virus and preparation method thereof
WO2020006191A1 (en) * 2018-06-29 2020-01-02 The Procter & Gamble Company Vaginal care compositions

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