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WO2025147262A1 - Device system and use for hpv infection and cervical intraepithelial neoplasia - Google Patents

Device system and use for hpv infection and cervical intraepithelial neoplasia Download PDF

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Publication number
WO2025147262A1
WO2025147262A1 PCT/US2024/010538 US2024010538W WO2025147262A1 WO 2025147262 A1 WO2025147262 A1 WO 2025147262A1 US 2024010538 W US2024010538 W US 2024010538W WO 2025147262 A1 WO2025147262 A1 WO 2025147262A1
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WO
WIPO (PCT)
Prior art keywords
compound
retaining device
certain embodiments
vaginal
administered
Prior art date
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Pending
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PCT/US2024/010538
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French (fr)
Inventor
Oranee DANIELS
Elaine Y. CHIEN
Catherine Marie BEHRENS
Ramakrishna GADIRAJU
Barry Aldous
Sarah Walter
Gail MADERIS
Ankush Argade
Ravichandran Mahalingam
Zhengle ZHAO
Zhaoyang QIN
Siyi Jiang
Runyan LI
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Antiva Biosciences Inc
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Antiva Biosciences Inc
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Priority to PCT/US2024/010538 priority Critical patent/WO2025147262A1/en
Priority to PCT/US2025/010220 priority patent/WO2025147598A1/en
Publication of WO2025147262A1 publication Critical patent/WO2025147262A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/664—Amides of phosphorus acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/2027—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • 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
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds

Definitions

  • HPV Papillomaviruses are a group of non-enveloped DNA viruses, which in humans infect keratinocytes of skin and mucous membranes including in the cervical area. HPV infections can cause cellular transformations in the human patient that have not yet progressed to cancer but have reached the stage of neoplasia.
  • HPV-induced neoplasia forms include cervical intraepithelial neoplasia (“CIN”), anal intraepithelial neoplasia (“AIN”), perianal intraepithelial neoplasia (“PAIN”), vulvar intraepithelial neoplasia (“VIN”), penile intraepithelial neoplasia (“PIN”) and vaginal intraepithelial neoplasia (“VAIN”).
  • Cancers caused by HPV include cervical, anal, perianal, penile, vaginal, vulvar, and oropharyngeal cancer. Thus, HPV can cause viral infection, neoplasia and cancer.
  • HPV and cervical intraepithelial neoplasia are adjunctive only.
  • Commonly used drug therapies include trichloroacetic acid, 5-fluorouracil, imiquimod and podofilox.
  • Imiquimod (Aldara TM , Zyclara TM ) stimulates the immune system to clear the infection through toll-like receptor signaling and causes redness and swelling.
  • Podofilox (Condylox TM ) destroys tissues by destabilizing microtubules which prevents host cell replication.
  • the cervical epithelium is composed of several layers of tissue and is referred to as stratified squamous epithelium. The layers are the superficial cell layer, the intermediate cell layer, the parabasal cell layer and the basal cell layer.
  • Cervical intraepithelial neoplasia is most often treated by observation (the wait and see approach) or by excision or ablation of the cervical transformation zone.
  • Techniques include cryotherapy, laser therapy, loop electrosurgical procedure (LEEP) and cone biopsy. All of these surgical procedures damage the affected areas and can lead to scarring.
  • LEEP loop electrosurgical procedure
  • Cervical high-grade squamous intraepithelial lesions (cHSIL), sometimes referred to as CIN2 and CIN3, is a disease caused by the abnormal hyperproliferation (dysplasia) of squamous cells in the cervical epithelium (Waxman, A. G., et. al 2012. “Revised terminology for cervical histopathology and its implications for management of high-grade squamous intraepithelial lesions of the cervix”. Obstet Gynecol, 120, 1465-71).
  • Hyperproliferation usually occurs where the simple columnar, endometrial-type epithelium of the endocervix transitions to the stratified squamous epithelium of the ectocervix; this region is referred to as the “transformation zone” (Sellors, J. W. & Sankaranarayanan, R. 2003. An introduction to the anatomy of the uterine cervix. Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginners' Manual). Cervical HSIL is classified as a pre-cancerous condition because apoptosis is impaired in these hyperproliferating cells, which can lead to the accumulation of genetic alterations that transform the cells into cancer.
  • Artesunate is an artemisinin derivative with cytotoxic activity. Artesunate is a known WHO-approved anti-malarial agent. The cytotoxic agent is delivered in the trial at a dosage, for example, of 50 to 200 mg for a 5-day cycle on weeks 0, 2 and 4. The artesunate vaginal inserts are self-administered at bedtime with a vaginal applicator, followed by use of a tampon, which is removed in the morning. Artesunate has a cytotoxic effect but is not an anti-viral agent, so does not directly stop the HPV replication.
  • artesunate cannot be used to treat patients who have an HPV infection that has not progressed to cervical intraepithelial neoplasia. Also, artesunate does have some systemic exposure under these conditions. See generally Trimble, et al., “A first- in-human proof-of-concept trial of intravaginal artesunate to treat cervical intraepithelial neoplasia 2/3 (CIN 2/3)”, Gynecologic Oncology 157 (2020)188-194 as well as U.S. Patents 6,586,464; 8,394,849; 8,940,787 and 7,989,491.
  • Cidofovir a pyrimidine based acyclic phosphonate nucleoside, which has broad spectrum activity against DNA viruses, is recognized as one of the effective treatments for HPV lesions that have not become cancerous. It is a DNA terminator and causes cell death via apoptosis of HPV transformed cells and regression of HPV-induced tumors. Cidofovir has been tested as a topical treatment of CIN2 and CIN3. See Van Pachterbeke, et al., “Topical treatment of CIN 2+ by cidofovir: Results of a phase II, double-blind, prospective, placebo-controlled study”, Gynecologic Oncology 115 (2009) 69-74.
  • Snoeck, et al “Cidofovir, a New Approach for the Treatment of Cervix Intraepithelial Neoplasia Grade III (CIN III)” Journal of Medical Virology 60:205-209 (2000).
  • Snoeck, et al. except for two patients, patients had at least partial responses and half had a total response. In the partial responses, the transformed cells persisted in the deep tissues that can lead to neoplasia. Therefore, because of imperfect bioavailability, modifications were needed. The clinical trial thus had mixed results and was not progressed through completed clinical trials to an approved product.
  • Patent Nos.10,702,532; 10,213,430; 9,493,493; and 9,801,884, with a priority date of September 15, 2014 PCT/US2015/050202; published as WO 2016/044281
  • U.S. Patent No. 11,014,950 and 10,377,782 with a priority date of September 15, 2015.
  • the drug must be lipophilic enough to pass through the tissue layers and be metabolized if necessary to the active agent in a sufficient concentration to kill the pathogenic cells. It is an object of the present invention to provide an improved method for the treatment of cervical or vaginal HPV infection and related conditions such as HPV-induced cervical or vaginal intraepithelial neoplasia in a female in need thereof, that is effective against the infection or related conditions and also minimize toxicity to nearby tissues and cells.
  • the use of a retaining device is advantageous to minimize potential adverse effects on surrounding tissue that can be uncomfortable or even painful for the patient and possibly damaging to normal tissue.
  • the invention includes a device system and therapeutic use to effectively treat HPV cervical or vaginal HPV infection or cervical or vaginal intraepithelial neoplasia.
  • a particularly advantageous treatment for cervical or vaginal HPV infection or cervical or vaginal intraepithelial neoplasia is achieved by the administration of an effective amount of the PMEG prodrug Compound I or a pharmaceutically acceptable salt thereof such as the monofumarate or hemifumarate salt, or other solid form including in particular Compound II or Compound III, by steps comprising (i) administering an effective amount of the anti-HPV therapeutic agent Compound I (or a pharmaceutically acceptable salt thereof) or Compound II or III, typically in solid tablet form (although semi-solid formulations may also be acceptable), to the cervix using a vaginal applicator and then (ii) inserting a retaining device that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post-treatment leakage of vaginal fluids which may contain, for example, remaining therapeutic agent or its metabolite such as PMEG, in a manner that minimizes toxicity-causing damage to non
  • the lubricant can be coated on the retaining device for ease of placement as well as to help maintain the device in position.
  • a pharmaceutical salt of Compound I below sometimes referred to as “ABI-2280”
  • PMEG a prodrug of PMEG
  • HPV human papillomavirus
  • HSIL high-grade squamous intraepithelial lesions
  • hrHPV high-risk HPV
  • Compound I is an acyclic nucleotide phosphonate that metabolizes to a known potent antiviral compound (PMEG; ((9-[2-phosphonomethyoxy)ethyl)guanine])), but PMEG has poor cellular permeability and use- limiting systemic toxicity. Applicant has discovered how to improve the prodrug to be delivered topically in a manner that it is rapidly taken up into epithelial cells, a challenging task to date and one that ABI-1968 failed.
  • PMEG potent antiviral compound
  • the female patient optionally coats the vagina with a lubricant and then inserts a vaginal tablet into the base of the cervical area with a vaginal applicator (optionally that has been coated with a lubricant), and then removes the vaginal applicator.
  • the female then inserts the retaining device (also which has been optionally coated with a lubricant) and leaves it in place for at least 4-12 hours, such as 6-10 hours or 5-8 hours and typically at least 6 hours. It is advantageous to insert the vaginal tablet and then place the retaining device in the evening and leave it in overnight, for at least 5 or 6 hours or more.
  • retaining device In the morning, the retaining device is removed and cleaned for the next use if reusable, or disposed of if not.
  • the topical medication is allowed to penetrate into the intraepithelial tissue while the patient is in a supine position with little movement, and the retaining device protects surrounding healthy tissue from the PMEG prodrug toxicity.
  • retaining devices are those that are conventionally used for period protection or those normally used for birth control.
  • Nonlimiting examples of retaining devices normally used for period protection are: a menstrual cup, a menstrual disc, and a tampon. These devices are commercially available without a prescription.
  • a menstrual cup is a cup that is placed in the vagina below the cervix that stays in place and normally holds blood flow.
  • menstrual cups are commercially available with different shapes and sizes, and are typically bell-shaped, V-shaped, round or asymmetrical.
  • a menstrual cup is typically made from medical grade silicone or latex rubber. Menstrual cups can typically be used for up to 8 to 12 hours, which is well within the time needed for the Compound I, II, or III or PMEG fluid retainment. It should be thoroughly cleaned between uses, with an antibiotic soap or in boiling water.
  • Some menstrual cups have a tapered shape that can make insertion and removal easy.
  • Nonlimiting examples of current commercially available products include FlexTM, CoraTM, SaaltTM, LenaTM, LummaTM, TampaxTM, ShordyTM and DivaTM.
  • a menstrual disc is a product that is worn inside the vaginal canal that is flat and disc- shaped. It is placed differently in the vaginal canal than a menstrual cup. It is designed to fit at the base of the cervix, where it creates a seal to prevent leaks.
  • the menstrual disc comes is a range of sizes, shapes and materials to fit the comfort of the patient.
  • Menstrual cups sit lower in the vaginal canal than menstrual discs and are usually designed to create a suction.
  • Nonlimiting examples of current commercially available menstrual disc products include, but are not limited to, those made by nixitTM, CoraTM, SaaltTM, LenaTM, LummaTM, ShordyTM and DivaTM.
  • a tampon is a well-known device that is a pocket-sized absorbent material that can optionally have a cardboard or plastic applicator that is inserted into the body normally to collect period fluid. It is normally removed every few hours up to once a day, for example every 4-6 hours. It is sold in many different varieties by many different vendors.
  • Non-limiting birth control devices include, but are not limited to a diaphragm, a cervical cap, and a sponge. The diaphragm and cervical cap devices often need a doctor’s prescription as part of gynecological healthcare for birth control, while sponges do not.
  • a diaphragm is a shallow bendable cup that is placed in the vagina typically as a means for contraception.
  • the separate vaginal applicator and diaphragm can be combined into one device by including a contoured surface in the diaphragm that the vaginal tablet can be placed in.
  • a cervical cap is smaller than a diaphragm and can be left in place longer. It covers the cervix and should be covered with a spermicide.
  • a nonlimiting example of a current commercially available cervical cap product is the FemcapTM.
  • Sponges can also be used according to this invention. They are typically made of polyurethane or natural sea sponges. It is soft, spongy and usually round. It is placed against the cervix and typically contains applied spermicide (when used as a birth control device but not in the present invention). Often it has a loop attached for easy displacement and removal.
  • Nonlimiting examples of products include, but are not limited to, those made by Safe-TTM and Today SpongeTM.
  • a female diagnosed with HPV or cervical or vaginal squamous intraepithelial lesions is treated with a PMEG prodrug, for example, ABI-2280 vaginal tablet in a dose strength of 0.1 mg to 1 mg, for example 0.1, 0.2, 0.3, 0.4 or 0.5 mg, once daily on a schedule of 2, 3 or 4 times a week optionally for 1, 2, 3, 4, 5 or 6 weeks with the use of a retaining device to adequately protect surrounding tissue.
  • the dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form.
  • Compound I (or a pharmaceutically acceptable salt thereof), Compound II or Compound III is therefore expected to eliminate HPV infection in productively infected cells typical of low-grade lesions and to induce apoptosis in advanced lesions, which often have integrated HPV genomes and express elevated levels of HPV oncoproteins.
  • Compound I ethyl (((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)- (benzyloxy)phosphoryl)-L-alaninate) has two chiral centers, one at the phosphorus atom and one in the amino acid moiety, either of which can be in the R or S stereoconfiguration.
  • Compound I exists as four stereoisomers, or two diastereomeric pairs: (R P , S C )/(S P , S C ) and (R P , RC)/(SP, RC). While U.S. Patent Nos. 9,801,884 and 11,344,555 describe Compound I generally, the patents do not address the potential stereochemistry of the phosphorus atom. It has been discovered that the stereoisomer of Compound I with R-stereochemistry at the phosphorus and S- stereochemistry at the amino acid carbon has advantageous properties over the other three stereoisomers.
  • the advantageous salt (for example fumarate) of Compound I is used as a mixture of (R,S) and (S,S) diastereomers, wherein the first R/S designates the stereochemistry at the phosphorus atom and the second S is the stereochemistry of the carbon in the amino acid moiety (corresponding to the L-alanine residue having S-configuration). While any ratio of the diastereomers can be used that provides the desired results, the (R,S) diastereomer stands out. In other embodiments, the ratio is approximately 1:1 of the R to S enantiomer at the phosphorus atom.
  • the compound is used with R- stereoconfiguration at the chiral carbon and wherein the R-stereoconfiguration is greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or more.
  • a pharmaceutically acceptable salt of the enantiomerically pure (Rp,Sc, or simply “R,S”) version of Compound I is a principal embodiment in the disclosed therapeutic use device therapy as disclosed herein.
  • An enantiomerically pure Compound II is at least 90% free of the opposite enantiomer.
  • the free base parent compound is an oil, not a solid, and thus would not have been selected as the active ingredient for the topical formulation.
  • Compound II exhibits superior stability properties over its stereoisomer, ethyl ((S)-((2-(2-amino-6-methoxy-9H- purin-9-yl)ethoxy)methyl)(benzyloxy) phosphoryl)-L-alaninate monofumarate (Compound III). This is important for the success of the topical application to the cervix or vagina.
  • the formulation of the dosage form is very important for adequate administration of the active agent into the intraepithelial tissue.
  • Tablet formulation should display the properties of mucoadhesion and substantivity and include excipients that have solubilizing, erosion-generating (for disintegration), porosity (for water uptake) and viscosity enhancing (to keep the drug at the target site) properties.
  • excipients that will cause rapid disintegration of a solid dosage form to cover the cervix or vaginal areas include, but are not limited to mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid or fumaric acid.
  • excipients that can enhance disintegration and coverage of the affected area include but are not limited to sodium starch glycollate, pregelatinized starch, crospovidone and croscarmellose sodium.
  • Mucoadhesive excipients that are useful in the present invention include but are not limited to microcrystalline cellulose, polycarbophil, hydroxymethyl cellulose, hypromellose, hydroxypropyl cellulose, and PVP.
  • a nonlimiting example of a tablet formulation includes, but is not limited to, microcrystalline cellulose, crospovidone, magnesium stearate, silicon dioxide, polyethylene oxide and mannitol.
  • Another non-limiting example of a tablet formulation has microcrystalline cellulose, magnesium stearate and mannitol.
  • Semi solid dosage forms may include, for example, a mucoadhesive polymer, a solubility/penetration enhancer, a lipophilic solubilizer and a penetration enhancer.
  • the mucoadhesive polymer for example, may be, but is not limited to, a carbomer, polyethylene glycol, crospovidone, hypromellose, polycarbophil and/or hydroxyethyl cellulose.
  • the solubility/penetration enhancer can be, for example, but not limited to, a mixture of polyoxyl 6 stearate Type I, ethylene glycol stearate and polyoxy 32 stearate Type I, cetyl alcohol, stearyl alcohol, polysorbate 80, sodium lauryl sulphate, mono and di-glycerides, sorbitan monostearate, glyceryl isostearate, polyoxy 15 hydroxystearate, poly15 hydroxystearate, polyoxy 40 hydrogenated castor oil, octyl dodecanol, and/or soybean lecithin.
  • Lipophilic solubilizers include, but are not limited to light mineral oil, mineral oil, white wax and silicone fluid.
  • Penetration enhancers include but are not limited to propylene glycol, transcutol, oleic acid, isopropyl myristate, propylene glycol glycerol monooleate, propylene glycol monocaprylate, PEG-8 Bees wax, cetyl alcohol, stearic acid, cetyl palmitate and/or cetostearyl alcohol.
  • a non-limiting example of a semi-solid formulation includes, for example, one or more of a carbomer, propylene glycol, sorbic acid, EDTA and water.
  • a semi solid formulation includes one or more of a carbomer, mineral oil, a mixture of polyoxy 6 stearate Type I, ethylene glycol stearate, polyoxy 32 stearate Type I, parabens, propylene glycol, EDTA and/or water.
  • Films can be produced, for example, with, but not limited to, hypromellose, polyethylene glycol, polymethacrylates, microcrystalline cellulose, xanthan gum, guar gum and/or polyvinylpyrrolidone.
  • a pessary (vaginal suppository) can be formulated with, for example but not limited to, hard fat (such as Ovucire, Witepsol, Supposi-Base), polyethylene glycol, macrogols, cocoa butter and glycerol.
  • hard fat such as Ovucire, Witepsol, Supposi-Base
  • polyethylene glycol such as polyethylene glycol
  • macrogols such as polyethylene glycol
  • cocoa butter and glycerol glycerol
  • a non-limiting example of a pessary can be made from Witepsol H 15 or Ovucire WL 3264.
  • the present invention includes at least the following features: (i) A method to treat a cervical or vaginal HPV-induced infection or an associated condition in a female in need thereof, including but not limited to cervical or vaginal intraepithelial neoplasia, comprising administering to a host in need thereof an effective amount of Compound I or a pharmaceutically acceptable salt thereof such as the monofumarate or hemifumarate, or other solid form, or Compound II or Compound III, in a pharmaceutical composition, in a vaginal tablet or semisolid formulation with a vaginal applicator in combination with the use of a retaining device for sufficient time to collect the post-treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non- diseased tissue.
  • a method to treat a cervical or vaginal HPV-induced infection, intraepithelial neoplasia, or an associated condition in a human in need thereof comprising (i) administering an effective amount of the anti-HPV therapeutic agent Compound I or a pharmaceutically acceptable salt thereof, such as the monofumarate or hemifumarate, or other solid form or Compound II or Compound III typically in tablet form, to the cervix using a vaginal applicator (which is optionally coated with a lubricant) and then (ii) inserting a retaining device (which is optionally coated with a lubricant) that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post-treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue.
  • a vaginal applicator which is optionally coated with a lubricant
  • (xii) The method of (i)- (xi) that provides a dosage form containing of from 0.1 mg to 30 mg, from 0.05 to 0.3 mg, from 0.5 mg to 20 mg, from 1 mg to 20 mg, from 1 mg to 15 mg, from 1 mg to 10 mg of a compound of embodiments (i)-(v) and in certain embodiments about or at least 0.03, 0.05, 0.1 mg, 0.3 mg.0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45mg or 50 mg of the active agent.
  • (xiii) The method of (i)-(xi) that provides a dosage form containing from about 0.05 mg to about 1.0 mg, such as 0.1 to 0.5 mg, for example 0.2, 0.3 or 0.4 mg of a compound of embodiments (i)-(v).
  • (xiv) The method of (i)-(xiii) wherein the retaining device is held in place for between 4- 12 hours, 5-10 hours or 6-9 hours and then removed.
  • (xv) The method of (i)-(xiv) wherein the topical formulation with retaining device is administered once a day for one, two, three or four days a week for as long as necessary to achieve the desired results.
  • kits that includes a dosage form with a therapeutic compound as described herein and a retaining device.
  • the kit of (xxiv) that also includes a vaginal applicator.
  • the kit of (xxiv) wherein the retaining device is selected from a menstrual cup, a menstrual disc, a tampon, diaphragm, cervical cap and a sponge.
  • the retaining device is used as both the vaginal applicator and a retaining means.
  • FIG.1A and FIG 1B depict the molecular structure of Compound I monofumarate pattern 1 as determined by the single crystal X-ray diffraction analysis of Example 21. There exists an intermolecular interaction between protonated N5-atom of free base and O7-atom of fumaric acid anion (N(5)–H(5) ⁇ O(7)) in the single-crystal form of Compound II.
  • FIG.2 is an in vitro tissue permeation test in vaginal tissue comparing ABI-2280 fumarate salt to ABI-1968. Bar A shows the tissue penetration of a 0.1% ABI-2280 gel in porcine vaginal tissue. Bars B and C show the tissue penetration of a 0.1% ABI-2280 gel in human cervical tissue.
  • Bar D shows the tissue penetration of a 1% formulation of ABI-1968 in 6% NMP into porcine vaginal tissue.
  • Bar E shows the tissue penetration of a 1% nanosuspension of ABI-1968 in porcine vaginal tissue.
  • Bar F shows the tissue penetration of a 3% formulation of ABI-1968 in 6% NMP into porcine vaginal tissue.
  • Bar G shows the tissue penetration of a 3% formulation of ABI-1968 in 20% NMP into porcine vaginal tissue.
  • ABI-1968 penetrates the tissue to a substantially smaller degree, which hinders the ability of the compound to reach the cells which are infected with HPV. This may be a contributing factor to the performance of ABI-1968 in clinical studies.
  • FIG. 3 shows a flow diagram for the process of preparing a topical cream formulation described in Example 6.
  • FIG. 4 shows a flow diagram for the process of preparing a topical gel formulation described in Example 6.
  • FIG. 5 shows a flow diagram for the process of preparing a tablet formulation described in Example 7.
  • FIG.6 is a bar graph comparing the tissue penetration of a topical gel and a topical tablet dosage form as described in Example 15. The tablet dosage form produces similar tissue penetration to the topical gel, with an average of 58 ng/mg of compound in the tissue.
  • FIG.8 depicts the structure of Compound I monofumarate.
  • the synthesis of Compound I and stereoisomers and pharmaceutically acceptable salts thereof is provided in Examples 1-5.
  • FIG. 9 is a comparison of XRPD diffractograms of Compound I hemifumarate Pattern 1 and Compound I monofumarate Pattern 1 (small scale preparation) obtained in Example 23.
  • FIG.10 is a DSC thermogram of Compound I hemifumarate Pattern 1 obtained in Example 23.
  • FIG.11 is a TGA thermogram of Compound I hemifumarate Pattern 1 obtained in Example 23.
  • FIG. 12A is a DSC thermogram of Compound I monofumarate Pattern 1 (small scale preparation, Example 23), recorder at heating rate of 10°C/min.
  • FIG. 12B is a DSC thermogram of Compound I mono-fumarate Pattern 1 (small scale preparation, Example 23), recorder at heating rate of 2°C/min.
  • FIG. 12C is a DSC cycle of Compound I mono-fumarate Pattern 1 (DSC cycle, 0-150oC, 150-0oC, 0-250oC, 10oC/min) for the small-scale preparation sample (Example 23).
  • FIG.13 is TGA thermogram of Compound I mono-fumarate Pattern 1 for the small-scale preparation sample (Example 23).
  • FIG.14 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 obtained in stability test experiments at 25°C/84%RH (2 days, open container), 25°C/92%RH (1 week, open container), 40°C/75%RH (1 week, open container), and 60°C (1 week, tight container) as described in Example 24 with original sample of Compound I monofumarate Pattern 1 before the test.
  • FIG. 15 is a Dynamic Vapor Sorption (DVS) plot and DVS change in mass plot for Compound I Pattern 1(Example 26).
  • DVDS Dynamic Vapor Sorption
  • FIG.16 is a comparison of XRPD diffractograms of Compound I Pattern 1 before and after DVS study (Example 26).
  • FIG. 17 is a Dynamic Vapor Sorption (DVS) plot and DVS change in mass plot for Compound I hemifumarate Pattern 1 (Example 26).
  • FIG.18 is a comparison of XRPD diffractograms of Compound I hemifumarate Pattern 1 before and after DVS study (Example 26).
  • FIG. 19 is a Dynamic Vapor Sorption (DVS) plot and DVS change in mass plot for Compound I monofumarate Pattern 1 (Example 26).
  • FIG.20 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 before and after DVS study (Example 26).
  • FIG.21A is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 obtained in Example 27 (large scale) before and after heating to 106°C.
  • FIG. 21B is a DSC thermogram of Compound I monofumarate Pattern 1 (Example 27), recorded at heating rate of 10°C/min.
  • FIG. 21C is a DSC thermogram of Compound I monofumarate Pattern 1 (large scale preparation, Example 27), recorded at heating rate of 2°C/min.
  • FIG. 21A is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 obtained in Example 27 (large scale) before and after heating to 106°C.
  • FIG. 21B is a DSC thermogram of Compound I monofumarate Pattern 1 (Example 27), recorded at
  • 21D is a DSC thermogram of Compound I monofumarate Pattern 1 (obtained in Example 27).
  • FIG.22 is TGA thermogram of Compound I monofumarate Pattern 1 (obtained in Example 27).
  • FIG.23 is a comparison of XRPD diffractograms of Compound I mono-fumarate Pattern 1, hemi-fumarate Pattern 2, hemi-fumarate Pattern 3, Pattern 4 obtained from equilibration experiment in water at 25°C for 2 weeks and fumaric acid pattern (obtained in Example 28).
  • FIG.24 is a comparison of XRPD diffractograms of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 2 and 3 weeks (obtained in Example 28).
  • FIG. 25 is a DSC thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 2 weeks (obtained in Example 28).
  • FIG. 26 is a DSC thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 3 weeks (obtained in Example 28).
  • FIG. 27 is a TGA thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 2 weeks (obtained in Example 28).
  • FIG. 28 is a TGA thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 3 weeks (obtained in Example 28).
  • a method to treat a cervical or vaginal HPV-induced infection, intraepithelial neoplasia, or an associated condition in a human in need thereof comprising: (i) administering an effective amount of the compound or pharmaceutical composition for use as described in any one of embodiments 1-42, to the cervix using a vaginal applicator (which is optionally coated with a lubricant); and then (ii) inserting a retaining device (which is optionally coated with a lubricant) that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post- treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue.
  • a vaginal applicator which is optionally coated with a lubricant
  • a retaining device which is optionally coated with a lubricant
  • the retaining device is selected from a menstrual cup, a menstrual disc, a tampon, diaphragm, cervical cap and a sponge.
  • the retaining device is used as both the applicator and a retaining means to collect the post-treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue.
  • the retaining device is a menstrual cup.
  • the retaining device is a menstrual disc. 49.
  • the method of embodiments 43 or 44, wherein the retaining device is a tampon. 50.
  • the method of embodiments 43, 44, or 46, wherein the retaining device is a diaphragm. 51.
  • the method of embodiments 43 or 44, wherein the retaining device is a cervical cap. 52.
  • the method of embodiments 43 or 44, wherein the retaining device is a sponge. 53.
  • the method of embodiment 46, wherein the retaining device used as both an applicator and a retaining means is a diaphragm.
  • the method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is atypical squamous cells of undetermined significance (ASC-US). 55.
  • any one of embodiments 43-53 wherein the condition caused by a human papillomavirus is high grade squamous intraepithelial lesions (HSIL). 59. The method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is adenocarcinoma in situ (AIS). 60. The method of any one of embodiments 43-53, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia. 61. The method of embodiment 60, wherein the cervical intraepithelial neoplasia is Grade 1 cervical intraepithelial neoplasia. 62.
  • HSIL high grade squamous intraepithelial lesions
  • the method of embodiment 60, wherein the cervical intraepithelial neoplasia is Grade 2 cervical intraepithelial neoplasia.
  • the method embodiment 60, wherein the cervical intraepithelial neoplasia is Grade 3 cervical intraepithelial neoplasia.
  • 64. The method of any one of embodiments 43-53, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia.
  • Isotopic Substitution includes but is not limited to compounds, pharmaceutical compositions, and the use of any of the active compounds described herein in combination with the retaining device, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as fumarate, Compound II or Compound III, with desired isotopic substitutions of atoms at amounts above the natural abundance of the isotope, i.e., enriched.
  • Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons.
  • isotopes of hydrogen for example, deuterium ( 2 H) and tritium ( 3 H) may be used anywhere in described structures.
  • isotopes of carbon e.g., 13 C and 14 C
  • isotopes of carbon e.g., 13 C and 14 C
  • a preferred isotopic substitution is deuterium for hydrogen at one or more locations on the molecule to improve the performance of the drug.
  • the deuterium can be bound in a location of bond breakage during metabolism (an ⁇ -deuterium kinetic isotope effect) or next to or near the site of bond breakage (a ⁇ -deuterium kinetic isotope effect).
  • Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280) describes deuteration of nucleotides to improve their pharmacokinetic or pharmacodynamic, including at the 5-position of the molecule.
  • isotopically-labeled refers to an analog that is a "deuterated analog", a " 13 C-labeled analog,” or a “deuterated/ 13 C-labeled analog.”
  • deuterated analog means a compound described herein, whereby a H-isotope, i.e., hydrogen/protium ( 1 H), is substituted by a H-isotope, i.e., deuterium ( 2 H).
  • Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium.
  • the isotope is 90, 95 or 99% or more enriched in an isotope at any location of interest.
  • a method for the treatment of HPV infection or HPV-induced intraepithelial neoplasia includes (i) administering an effective amount of the anti-HPV therapeutic agent such as ABI-2280 or a pharmaceutically acceptable salt thereof, or other compound or its salt described herein, optionally in morphic form, and typically in tablet form, to the cervix using a vaginal applicator and then (ii) inserting a retaining device that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post- treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue as described in detail above.
  • the anti-HPV therapeutic agent such as ABI-2280 or a pharmaceutically acceptable salt thereof, or other compound or its salt described herein
  • the patient can be instructed to remain supine for several hours, for example, 3, 4, 5, 6, 7 or 8 hours with minimal activities and may resume normal activities after that time-period.
  • the patient should repeat this procedure once daily for a total of 2, 3 or 4 applications in a week as instructed. It is preferred to carry out the two step administration process in the evening.
  • Patients typically administer three doses on alternate days over a 1-week period.
  • Patients can optionally be instructed to use the following preventative measures to protect against leakage of drug or metabolite-containing fluid to healthy tissue: • Minimize amount of water-based lubricant (e.g.,KY gel) used for dosing. • Do not bathe the genital area until after the instructed administration dosage time period.
  • water-based lubricant e.g.,KY gel
  • a non-water based barrier gel for example zinc oxide
  • Peripads can be used and can be changed frequently to avoid continued exposure to any leaked drug or metabolite containing fluid.
  • Dosings are preferably initiated and completed prior to anticipated starting date of the period.
  • suitable schedules include days 1, 3, and 5 followed by days 8, 10 and 12, followed by days 15, 17 and 19. Patients can combine this protocol with other standard of care procedures who have screening biopsy results of HSIL (CIN2 or CIN3).
  • the patient may opt to undergo a LLETZ procedure or another procedure described in the Background section above.
  • the Compound I, or a pharmaceutically acceptable salt thereof such as Compound II or Compound III, vaginal tablet is self-administered.
  • one or more doses of the compound are administered by a physician.
  • the first dose is administered by a physician or medical provider and the remaining doses, for example doses on days 3, 5, 8, and 10, are administered at home.
  • a formulation for the treatment of intraepithelial neoplasia is a dosage form that contains from about 0.01 mg to about 10 mg, from about 0.05 to about 5 mg, from about 0.05 to about 0.15 mg, from about 0.15 mg to about 0.45 mg, or from about 0.5 to about 1.5 mg of any of the active compounds described herein including but not limited to Compound I or a pharmaceutically acceptable sale such as a monofumarate, hemifumarate, or other solid form, or Compound II or Compound III.
  • a formulation for the treatment of intraepithelial neoplasia is a dosage form that contains about or at least 0.01, 0.03, 0.05, 0.1 mg, 0.3 mg.0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg or 20 mg, of Compound I monofumarate, Compound II or Compound III, optionally in a selected morphic form.
  • the dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form.
  • the topical formulation is administered once a day, or several days a week (such as 2 or 3 days a week) in combination with the retaining device, as long as necessary to achieve the desired results. In certain embodiments, the topical formulation is administered on a weekly schedule for one, two, three, four, five, six or more weeks. In certain aspects, the topical formulation is administered on a schedule of three dosages a week for two, three, four, five, or six weeks. In certain embodiments, the compound can be administered in one or more therapeutic cycles comprising a treatment cycle and a rest cycle, wherein the treatment cycle comprises administering the compound as described herein in combination with the retaining device, followed by a rest cycle (comprising a period of no treatment) before the next treatment cycle.
  • the rest cycle is from about one day to about six months. In certain embodiments the rest cycle is one, two, three, four, five, six, seven, eight or more weeks before the next treatment cycle. In certain embodiments, multiple therapeutic cycles are administered, for example one, two, three, four, five, or six therapeutic cycles.
  • Dosage forms which do not adhere well to the target site may be dislodged, interfering with treatment. Dosage forms have been discovered that adhere to the target site and dissolve rapidly in low fluid volumes. Adhesion to the target site also prevents exposure to healthy tissues, which may limit toxicity and side effects. Dosage forms which soften, break down, and/or disintegrate quickly in low fluid volumes are advantageous to cause a rapid release of the active compound to the target tissue.
  • Dosage forms that disintegrate in, for example, less than about 50 ⁇ L, less than about 100 ⁇ L, less than about 125 ⁇ L, less than about 150 ⁇ L, less than about 175 ⁇ L, less than about 200 ⁇ L, or less than about 250 ⁇ L fluid particularly facilitate drug penetration into the target site.
  • the dosage form is a gel.
  • the dosage form is a cream.
  • the dosage form is a tablet.
  • the dosage form disintegrates in about one to about ten seconds. In certain embodiments, the dosage form disintegrates in about ten seconds to one minute. In certain embodiments, the dosage form disintegrates in about one minute to about one hour.
  • the dosage form disintegrates in about one to six hours.
  • the physical dimensions of the dosage form can impact the effectiveness of the dosage form.
  • a tablet that is thinner provides a greater surface area to volume ratio and may degrade quicker and cover the target area better.
  • the dosage form is less than about 6, 5, 4, 3, or 2 millimeters thick in its smallest dimension.
  • the formulation of the dosage form is important for adequate administration of the active agent into the intraepithelial tissue.
  • the formulation for example, can be prepared for use as a tablet, a reconstituted powder, a dry powder, a semi solid dosage form, a film or a pessary (i.e., a vaginal suppository).
  • Some embodiments disclosed herein include the use of an effective amount of Compound I monofumarate, Compound II or Compound III, in the manufacture of a medicament for ameliorating or treating a human papillomavirus infection, wherein the infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA.
  • Other embodiments disclosed herein include the use of an effective amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II or Compound III, for ameliorating or treating a human papillomavirus infection, wherein the human papillomavirus infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA.
  • Certain nonlimiting embodiments disclosed herein include a method for ameliorating or treating a human papillomavirus infection that can include contacting a cell infected with the human papillomavirus in a subject with an effective amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound I hemifumarate, or other solid form of Compound I, Compound II or Compound III, wherein the infection is ameliorated or treated by inhibiting the synthesis of viral DNA.
  • Yet still other embodiments disclosed herein include a method for ameliorating or treating a human papillomavirus infection that can include administering to a subject infected with the human papillomavirus an effective amount of Compound I monofumarate, Compound II or Compound III, wherein the human papillomavirus infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA.
  • Some embodiments disclosed herein relate Compound I monofumarate, Compound II or Compound III, for use in ameliorating or treating a human papillomavirus infection, wherein the human papillomavirus infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA.
  • the human papillomavirus can be a high-risk human papillomavirus, such as those described herein.
  • the high-risk human papillomavirus can be selected from HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV- 56, HPV-58, HPV-59, HPV-68, HPV-73 and HPV-82.
  • the human papillomavirus can be HPV-16.
  • the human papillomavirus can be HPV-11.
  • the human papillomavirus can be HPV-18.
  • the human papillomavirus can be one or more of the following high-risk types: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73 and HPV-82.
  • HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73 and HPV-82 can be detected using the Papanicolaou test (Pap smear) and/or DNA probe testing (for example, HPV DNA probe testing for one or more high-risk HPV types).
  • an effective amount of Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III
  • a subject diagnosed with an HPV infection in combination with the retaining device for example a high-risk HPV infection, by a DNA test.
  • an effective amount of Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III can be provided to a subject diagnosed with an HPV infection, or a disease associated with HPV infection, as identified by a Papanicolaou test.
  • an effective amount of Compound I monofumarate, Compound II, or Compound III may be provided to a subject with a Papanicolaou test result that does not indicate the disease has progressed to cervical cancer.
  • the Bethesda system is a standardized scoring system for reporting pap smear test results and assigns a grade of 1-3 based on severity. Grade 1 CIN (CIN 1) indicates mild dysplasia. Grades 2 and 3 CIN (CIN 2, CIN 3) are more serious and typically require intervention.
  • Compound I monofumarate, Compound II or Compound III is used to treat CIN 1 (Grade 1 cervical intraepithelial neoplasia).
  • Compound I monofumarate, Compound II or Compound III is used to treat CIN 2 (Grade 2 cervical intraepithelial neoplasia) in combination with the retaining device.
  • Compound I monofumarate, Compound II or Compound III is used to treat CIN 3 (Grade 3 cervical intraepithelial neoplasia) in combination with the retaining device.
  • a pharmaceutical composition comprising Compound I monofumarate, Compound II or Compound III, is used in the manufacture of a system for the treatment of CIN 1 (Grade 1 cervical intraepithelial neoplasia) that includes the use of a retaining device, as described further herein.
  • a pharmaceutical composition comprising Compound I monofumarate, Compound II or Compound III, is used in the manufacture of a system for the treatment of CIN 2 (Grade 2 cervical intraepithelial neoplasia) that includes the use of a retaining device as described further herein.
  • a pharmaceutical composition comprising Compound I monofumarate, Compound II or Compound III, is used in the manufacture of a medicament for the treatment of CIN 3 (Grade 3 cervical intraepithelial neoplasia).
  • Compound I monofumarate, Compound II or Compound III is used to treat a condition selected from the group consisting of atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells (AGC), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cells (cannot exclude high grade squamous intraepithelial lesion) (ASC-H), high grade squamous intraepithelial lesions (HSIL), adenocarcinoma in situ (AIS), and cervical cancer (e.g. squamous cell carcinoma or adenocarcinoma).
  • ASC-US atypical squamous cells of undetermined significance
  • ASC atypical glandular cells
  • LSIL low-grade squamous intraepithelial lesions
  • ASC-H atypical squamous cells (cannot exclude high grade squamous intraepithelial lesion)
  • Compound II is administered in combination with the use of a retaining device twice a week for up to 12 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 10 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 8 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 6 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 4 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 2 weeks.
  • Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 12 weeks. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 10 weeks. In certain embodiments Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 8 weeks.
  • Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 6 weeks. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 4 weeks. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 2 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device three times a week for up to 1 week.
  • Compound I monofumarate is administered in combination with a retaining device daily. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 12 weeks or indefinitely as instructed by a healthcare provider. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 10 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 8 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 6 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 4 weeks.
  • Compound I monofumarate is administered in combination with a retaining device daily for up to 2 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 1 week. In certain embodiments, Compound I monofumarate, or other solid form Compound II or Compound III may be administered in combination with a retaining device three, four, five or six times a week. In certain embodiments, Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device once per day. In certain embodiments, Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device twice per day.
  • Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device three, four, or more times per day. In certain embodiments, Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device daily. In certain embodiments, the compound can be administered in combination with a retaining device in one or more therapeutic cycles comprising a treatment cycle and a rest cycle, wherein the treatment cycle comprises administering the compound as described herein, followed by a rest cycle (comprising a period of no treatment) before the next treatment cycle. In certain embodiments, the rest cycle is from about one day to about six months.
  • the rest cycle is one, two, three, four, five, six, seven, eight or more weeks before the next treatment cycle.
  • multiple therapeutic cycles are administered, for example one, two, three, four, five, or six therapeutic cycles.
  • a number of compounds have been investigated for the treatment of HPV-induced neoplasia, however none has been approved yet.
  • investigated approaches see Ahn W.S., et al. Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions. Eur. J. Cancer Prev.2003;12:383–390; Ashrafian L,et al.
  • lopimune lopinavir/ritonavir
  • vaginal tablets can be packaged for example in bottles with a desiccant.
  • a desiccant Nonlimiting examples are high density polyethylene bottles with a silica gel desiccant canister and rayon, induction sealed and capped with a child-resistant closure.
  • the vaginal tablet can be administered using the single-use vaginal tablet applicator or retaining device. It is preferred that tablets are stored refrigerated (2-8°C) in bottles with desiccant. Prior to administration, the bottle should be brought to room temperature for at least 30 minutes before opening and removing the tablet. Tablets are typically stored refrigerated (2-8°C) in bottles with desiccant.
  • compositions used in the present invention comprise an anti-HPV effective amount of Compound II described herein, optionally in a morphic form, in combination with a pharmaceutically acceptable carrier, additive, or excipient, further optionally in combination with at least one other antineoplastic agent or antiviral agent, such as an anti-HPV agent in combination with the retaining device.
  • the pharmaceutical composition includes Compound II in combination with a second antiviral drug in combination with the retaining device.
  • the pharmaceutical composition includes Compound II in combination with an anticancer drug in combination with the retaining device.
  • a therapeutically effective amount will vary with the infection or condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetic of the agent used, as well as the patient or subject (animal or human) to be treated, and such therapeutic amount can be determined by the attending physician or specialist.
  • the prodrug form of the compounds especially including acylated (acetylated or other), and ether (alkyl and related) derivatives, phosphate esters, thiophosphonamidates, phosphonamidates, and various salt forms of the present compounds, may be used to achieve the desired effect.
  • the amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II, or Compound III included within the therapeutically active formulation according to the present invention is an effective amount to achieve the desired outcome according to the present invention in combination with the retaining device, for example, for treating the HPV infection, reducing the likelihood of a HPV infection or the inhibition, reduction, and/or abolition of HPV or its secondary effects, including disease states, conditions, and/or complications which occur secondary to HPV infection.
  • Compound I monofumarate, Compound II, or Compound III may be administered in a gel in combination with the use of a retaining device.
  • the gel contains from about 0.001% to about 10%, from about 0.01% to about 10%, from about 0.05% to about 5%, from about 0.1 to about 3% from about 0.1 to about 2% Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II, or Compound III (weight/weight). In certain embodiments the gel contains from about 0.001% to about 0.05% Compound I monofumarate, Compound II, or Compound III. In certain embodiments, the gel contains from about 0.01% to about 0.5% Compound I monofumarate, Compound II, or Compound III. In certain embodiments, the gel contains from about 0.1% to about 5% Compound I monofumarate, Compound II, or Compound III.
  • any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II, or Compound III is administered topically via a tablet, capsule, suspension, liquid, emulsion, implant, particle, sphere, cream, ointment, suppository, pessary, transdermal form, gel, mucosal, and the like in combination with the retaining device.
  • the dosage form may also be a bilayer tablet, in which the full dose of active compound is released one direction (for example towards the target tissue).
  • the dosage form can soften, disintegrate, and/or release the drug in low fluid volumes. In certain embodiments, the dosage form softens and begins to release the drug immediately.
  • the dosage form softens and begins to release the drug gradually. In certain embodiments, the dosage form softens and begins to release the drug within one hour. In certain embodiments, the dosage form softens and begins to release the drug within two hours.
  • the dosage form may be prepared to maximize surface area, facilitating disintegration. In certain embodiments, the dosage form is a round tablet. In certain embodiments, the dosage form is an oval tablet. In certain embodiments, the dosage form is a caplet.
  • the tablet width is the largest dimension, and the tablet thickness is the smaller dimension. In certain embodiments, the dosage form is twice as wide as it is thick. In certain embodiments, the dosage form is three times as wide as it is thick. In certain embodiments, the dosage form is four or more times as wide as it is thick.
  • the dosage form is from about 0.1 mm thick to about 5 mm thick. In certain embodiments, the dosage form is from about 1 mm to about 2 mm thick. In certain embodiments, the dosage form is from about 2 mm to about 3 mm thick. In certain embodiments, the dosage form is from about 3 mm to about 4 mm thick. In certain embodiments the dosage form is from about 4 mm to about 5 mm thick. In certain embodiments the tablet is from about 5 mm to about 15 mm thick. In certain embodiments, the dosage form is less than 5 grams. In certain embodiments, the dosage form is from about 0.05 gram to about 0.15 gram. In certain embodiments, the dosage form is from about 0.1 to about 1 gram.
  • the dosage from about 0.75 grams to about 2 grams. In certain embodiments, the dosage form is from about 1 gram to about 5 grams.
  • the dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form. In certain embodiments, the dose form is not easily removed, dislodged, or moved from the target site. These desirable properties may be achieved by inclusion of a mucoadhesive polymer into the pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises a mucoadhesive polymer or mucoadhesive excipient.
  • mucoadhesive polymers and excipients include: Hypromellose, lectin, thiolated polymers (e.g. chitosan–iminothiolane, poly(acrylic acid)–cysteine, poly(acrylic acid)–homocysteine, chitosan– thioglycolic acid, chitosan–thioethylamidine, alginate– cysteine, poly(methacrylic acid)–cysteine and sodium carboxymethylcellulose–cysteine), polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidinone, polyacrylic acid (Carbopol®), polyheroxyethyl methacrylate, chitosan, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, sodium carboxymethyl cellulose, aminated corn starch, cellulose derivatives, poly (acrylic acid) polymers, poly (hydroxy(acrylic
  • the pharmaceutical composition comprises from about 0 to about 10% mucoadhesive polymer excipients selected from the list consisting of carbomer, polyethylene glycol, crospovidone, polycarbophil, hypromellose, and hydroxyethyl cellulose. In certain embodiments, the pharmaceutical composition comprises from at least about 0.1% to about 90, about 92%, about 93%, about 95%, about 98% about 97%, about 98%, or about 99% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 0.1% to about 1% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 0.5% to about 5% mucoadhesive polymer.
  • the pharmaceutical composition comprises from about 1% to about 10% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 5% to about 20% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 10% to about 50% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 20% to about 75% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 50% to about 90% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 75% to about 99% mucoadhesive polymer.
  • the pharmaceutical composition comprises about or at least about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 percent mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises no more than about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 percent mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises 0% mucoadhesive polymer. In this instance, the adhesion to the target site is achieved by use of other pharmaceutically acceptable excipients.
  • a therapeutically effective amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II, or Compound III optionally in morphic form according to the present invention is often admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose.
  • a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques.
  • any of the usual pharmaceutical media may be used.
  • suitable carriers and additives including water, glycols, oils, alcohols, preservatives, and the like may be used.
  • the pharmaceutical composition comprises propylene glycol. In certain embodiments, the pharmaceutical composition comprises carboxypolymethylene. In certain embodiments, the pharmaceutical composition comprises ethylenediaminetetraacetic acid (EDTA). In certain embodiments, the pharmaceutical composition comprises sorbic acid. In certain embodiments, the pharmaceutical composition comprises carbomer. In certain embodiments, the pharmaceutical composition comprises hydroxyethyl cellulose. In certain embodiments, the pharmaceutical composition comprises polyethylene glycol.
  • suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose, and related carriers, diluents, granulating agents, lubricants, binders, mucoadhesive polymer, disintegrating agents, and the like may be used.
  • the tablets or capsules may be coated or sustained release by standard techniques. The use of these dosage forms may significantly enhance the bioavailability of the compounds in the patient.
  • the pharmaceutical composition comprises mannitol.
  • the pharmaceutical composition comprises magnesium stearate.
  • the pharmaceutical composition comprises microcrystalline cellulose.
  • the pharmaceutical composition comprises polycarbophil. In certain embodiments, the pharmaceutical composition comprises polyethylene oxide. In certain embodiments, the pharmaceutical composition comprises colloidal silicon dioxide. In certain embodiments, the pharmaceutical composition comprises povidone. In certain embodiments, the pharmaceutical composition comprises isopropyl alcohol. In certain embodiments, the pharmaceutical composition comprises sodium starch glycolate. In certain embodiments, the pharmaceutical composition comprises croscarmellose sodium. In certain embodiments, the pharmaceutical composition comprises crospovidone. In certain embodiments, the pharmaceutical composition comprises hydroxypropylmethylcellulose. In certain embodiments, the pharmaceutical composition comprises lactose.
  • a powder pharmaceutical composition comprises one or more excipient from the group consisting of xanthan gum, microcrystalline cellulose, polyethylene oxide, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose sodium, povidone, mannitol, colloidal silicon dioxide, sodium benzoate, sodium starch glycolate, sodium lauryl sulfate, poloxamer 407, polyoxypropylene- polyoxyethylene copolymers, and the like.
  • the pharmaceutical composition comprising an effective amount of a fumarate salt of any of the active compounds described herein including but not limited to Compound I, further comprises a pharmaceutically acceptable excipient selected from the list consisting of Acacia, agar, alginic acid, ascorbyl palmitate, bentonite, benzoic acid, butylated hydroxyanisole, butylated hydroxytoluene, butylene glycol, calcium acetate, calcium hydroxide, canola oil, carob bean gum, carrageenan, castor oil, cellulose, corn starch, disodium edetate, erythorbic acid, ethyl lactate, ethylcellulose, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, hypromellose, lactic acid, lauric acid, lecithin, lin
  • the pharmaceutical composition comprises pharmaceutically acceptable excipients for use as a pessary.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III further comprises up to 99.9% pessary excipient selected from the group consisting of hard fat, PEG, macrogols, cocoa butter, and glycerol.
  • Non limiting examples of hard fat include Ovucire® (mono-, di- and triglyceride esters of fatty acids (C 10 to C 18 ), the triester fraction being predominant and ethoxylated fatty alcohols), Witepsol® (glycerol esters of vegetable saturated fatty acids, such as lauric acid), and Supposi-baseTM (a blend of saturated polyglycolysed glycerides).
  • Ovucire® mono-, di- and triglyceride esters of fatty acids (C 10 to C 18 ), the triester fraction being predominant and ethoxylated fatty alcohols
  • Witepsol® glycerol esters of vegetable saturated fatty acids, such as lauric acid
  • Supposi-baseTM a blend of saturated polyglycolysed glycerides
  • the pharmaceutical composition comprising an effective amount of any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III further comprises a pharmaceutically acceptable excipient that enhances the penetration, disintegration, film forming and/or controlled release properties of the composition.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III further comprises a penetration enhancing excipient.
  • the penetration enhancing excipient is selected from the group consisting of oleic acid, eucalyptol, Caprylol, Labrafil, Labrasol, Lauroglycol, diethylene glycol monomethyl ether (Transcutol), propylene glycol, sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, poloxamer (231, 182, 184), Tween 20, 40, 60, 80, fatty acids and fatty acid esters, isostearic acid, glycerin, and chitosan.
  • the pharmaceutical composition comprising Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III contains from 0% to about 20% penetration enhancing excipients selected from the group consisting of cetyl alcohol, propylene glycol, transcutol P, oleic acid, isopropyl myristate, propylene glycol dicaprylate, glyceryl monooleate, propylene glycol monocaprylate, PEG-8 bees wax, cetyl alcohol, stearic acid, cetyl palmitate, and cetosteryl alcohol.
  • penetration enhancing excipients selected from the group consisting of cetyl alcohol, propylene glycol, transcutol P, oleic acid, isopropyl myristate, propylene glycol dicaprylate, glyceryl monooleate, propylene glycol monocaprylate, PEG-8 bees wax, cetyl alcohol, stearic acid, cetyl palmitate, and
  • the pharmaceutical composition comprises from about 0 to about 25% penetration enhancing excipients selected from the list consisting of stearyl alcohol, polysorbate 80, sodium lauryl sulfate, mono and diglycerides, sorbitan monostearate, glyceryl isostearate, polyoxyl 15 hydroxystearate, polyoxyl 40 hydrogenated castor oil, octyl dodecanol, and soybean lecithin.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises a film forming excipient.
  • the pharmaceutical composition comprising any of the active compounds described herein including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III contains from 0% to about 99% film forming excipients selected from the group consisting of hypromellose, polyethylene glycol, polymethacrylates, microcrystalline cellulose, guar gum, xanthan gum, and polyvinylpyrrolidone.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises and excipient which allows for controlled release of the active compound.
  • the controlled release pharmaceutical composition comprises ethylcellulose, hypromellose, microcrystalline wax, polycarbophil, beeswax. Percentage ranges of excipients and other components of the pharmaceutical composition are given as a percent by weight, unless otherwise specified.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises a disintegration enhancing excipient.
  • the disintegration enhancing excipient is selected from the group consisting of cellulose, guar gum, crospovidone, polyplasdone, soy polysaccharides, calcium silicate, gelatin, cation exchange resins, bentonite, citrus pulp, alginic acid, calcium alginate, methylcellulose, microcrystalline cellulose, sodium carboxymethylcellulose, croscarmellose, solka floc, corn starch, sodium starch glycolate (Explotab, Primojel), glycine, hydroxypropyl starch, and starch 1500.
  • the pharmaceutical composition comprises up to about 99% disintegration enhancing excipient such as mannitol and/or microcrystalline cellulose.
  • the pharmaceutical composition comprises from about 0 to about 70% disintegration enhancing excipients selected from the list consisting of lactose, sucrose, and calcium phosphate. In certain embodiments, the pharmaceutical composition comprises from about 0 to about 50% disintegration enhancing excipients selected from the list consisting of sodium bicarbonate, citric acid, maleic acid, adipic acid, and fumaric acid. In certain embodiments, the pharmaceutical composition comprises from about 0 to about 20% disintegration enhancing excipients selected from the list consisting of sodium starch glycollate, pregelatinized starch, crospovidone, and croscarmellose sodium.
  • the pharmaceutical composition comprising any of the active compounds described herein including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 70% mannitol, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%.
  • the pharmaceutical composition comprising Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 70% lactose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 70% sucrose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 70% microcrystalline cellulose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 20% sodium starch glycolate, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 20% pregelatinized starch, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 20% crospovidone, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 20% croscarmellose sodium, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% xanthan gum, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% polycarbophil, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% polyethylene oxide, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% hydroxyethylmethyl cellulose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%.
  • the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% hypromellose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%.
  • a tablet used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 250 mg of microcrystalline cellulose, about 20 mg of crospovidone, about 5 mg of magnesium stearate, about 5 mg of silicon dioxide, about 5 mg of polyethylene oxide, and about 100 mg of mannitol.
  • a tablet used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV further comprises about 155 mg microcrystalline cellulose, about 1.75 mg of magnesium stearate, and about 17.5 mg of mannitol.
  • a semi-solid formulation used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 15 mg of carbomer, about 50 mg of propylene glycol, about 10 mg of sorbic acid, about 5 mg of EDTA, and about 920 mg of water.
  • a semi-solid formulation used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 20 mg of carbomer; about 70 mg of mineral oil; about 80 mg of a mixture of polyoxyl 6 stearate Type I, ethylene glycol stearates and polyoxyl 32 stearate type 1; about 5 mg parabens; about 60 mg propylene glycol; about 5 mg EDTA; and about 760 mg water.
  • a dry powder for reconstitution is used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 15.5 mg xanthan gum, about 19.8 mg mannitol, about 5 mg silicon dioxide, and about 0.5 mg sodium benzoate.
  • Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 15.5 mg xanthan gum, about 19.8 mg mannitol, about 5 mg silicon dioxide, and about 0.5 mg sodium benzoate.
  • cryotherapy laser therapy
  • loop electrosurgical procedure LEEP
  • cone biopsy cone biopsy. All of these surgical procedures damage the affected areas and can lead to scarring.
  • LEEP loop electrosurgical procedure
  • the treatments described herein in combination with the retaining device are used to lessen, ameliorate or substitute for the use of these conventional practices.
  • the treatments described herein can be used in combination with a surgical technique.
  • a patient in need thereof can receive surgery before, during and/or after administration of an effective amount of a compound described herein.
  • the compound is administered in combination with the retaining device about or less than about 8 months, 6 months, 4 months, 3 months, 2 months, or 1 month prior to surgery. In certain embodiments, the compound is administered in combination with the retaining device about or at least about 1 month, 2 months, 3 months, 4 months, 6 months, or 8 months after surgery.
  • the surgical procedure can be an excision of the target and/or diseased tissue, including but not limited to loop electrosurgical excision procedure (LEEP), large loop excision of the transformation zone (LLETZ), knife conization, cold knife conization, knife cone biopsy, or laser conization.
  • the surgical procedure can be ablation, including but not limited to laser ablation or cryoablation.
  • the efficacy of a drug against an HPV infection or neoplasia may be prolonged, augmented, or restored by administering the compound in combination or alternation with another, and perhaps even two or three other, antiviral compounds that induce a different mutation or act through a different pathway, from that of the principal drug.
  • the pharmacokinetic, biodistribution, half-life, or other parameter of the drug can be altered by such combination therapy (which may include alternation therapy if considered concerted).
  • combination therapy with anticancer therapeutics can provide better outcomes for patients.
  • the disclosed Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III are DNA Polymerase inhibitors, it may be useful to administer the compound to a host in need thereof in combination with, for example: a) a protease inhibitor; b) another DNA polymerase inhibitor; c) an inhibitor of E6 or E6AP such as MEDI0457, luteolin, CAF-24 or gossypetin; d) an inhibitor of E7; e) an inhibitor of E1 or E2, including inhibitors of the E1-E2 protein interaction; f) L2 lipopeptides; g) an inhibitor or degrader of L1 or L2; h) an HDAC inhibitor such as vorinostat; i) degraders of tetraspanins such as CD9, CD63 or CD151; j) immunotherapeutics such as T-cell therapies (including adoptive T-cell therapies) and checkpoint inhibitors; k) anti-proliferative drugs
  • Solid-State Forms Any of the materials described herein that include Compound I in any stereochemical designation, or mixtures thereof, along with one or more additional compounds in any selected ratio, such as but not limited to an acid as described herein or water (or other solvent) in a solid form, can be used for HPV anti-viral or intraepithelial neoplasia therapy.
  • Such solid forms when they contain Compound I so described and one or more additional compounds, may be considered multicomponent solids and, when crystalline, multicomponent crystalline forms.
  • “crystalline A B” would be considered a multicomponent crystalline form of compounds A and B.
  • a salt is an example of a multicomponent solid, and a crystalline salt is a multicomponent crystalline form.
  • Salts typically exhibit proton transfer between an acid and a base.
  • Other multicomponent crystalline forms include cocrystals.
  • a cocrystal is typically a crystalline solid of two or more neutral species. Because charge balance is not necessary for neutral species, the stoichiometry of cocrystals cannot be determined based on charge balance.
  • one component is typically an active drug or prodrug and the other (or others) is usually termed a coformer, and it interacts nonionically with the active drug or prodrug in the crystal lattice.
  • the solid form may be both a salt and a cocrystal.
  • the active drug or prodrug may be in the form of a salt with a counterion and interact nonionically with a coformer.
  • a solid form would be a multicomponent solid and, when crystalline, a multicomponent crystalline form.
  • cocrystals when the coformer is volatile includes solvates.
  • a solid form may exist in a single crystalline solid form or be polymorphic where more than one crystalline form of the same solid form exists. Polymorphism is typically determined by use of x-ray powder diffraction.
  • Other examples of multicomponent solids are hydrates and solvates.
  • a hydrate is a solid form that includes water in the solid form.
  • the water may form part of the unit cell of the crystal in which case it is often a stoichiometric hydrate.
  • a solvate is a solid form that includes one or more solvent molecules and, when crystalline, may form part of the unit cell of the crystal in which case it is often a stoichiometric solvate.
  • a hydrate or a solvate can also be used in the therapy described generally herein.
  • the solid form may be partially crystalline, disordered, or amorphous..
  • any of the solid forms described above that comprise Compound I can be used in an effective amount in the treatment of HPV infection or cervical or vaginal intraepithelial neoplasia in a patient in need thereof, as generally taught herein.
  • a multicomponent crystal comprising Compound I can include a salt form of Compound I, a cocrystal form of Compound I and/or a mixture of a salt and cocrystal form of Compound I.
  • the solid form formed can be a function of the synthesis and crystallization or solidification conditions used, among other factors.
  • an effective amount of a multicomponent crystal comprising Compound I and a pharmaceutically acceptable coformer is used in the treatment of cervical or vaginal intraepithelial neoplasia in a patient in need thereof. In certain embodiments, an effective amount of a multicomponent crystal comprising Compound I and a pharmaceutically acceptable coformer is used in combination with a retaining device to treat cervical or vaginal intraepithelial neoplasia in a patient in need thereof.
  • the crystalline form formed by addition of about 1 equivalent of fumaric acid to a solution of Compound I optionally in isopropyl alcohol and heptane, optionally in a pharmaceutically acceptable carrier, is used in the treatment of cervical or vaginal intraepithelial neoplasia, optionally in combination with a retaining device.
  • Any solid form based on desired properties that includes Compound I can be used in an effective amount with or without a retaining device to deliver therapy to the HPV-infected patient in need thereof.
  • the dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form.
  • Example 1 Preparation of Fumarate Salts
  • Compound I Hemifumarate About 200 mg of Compound I free base was added to 0.5 mL of EtOH. While stirring, 0.5 molar amount of fumaric acid was added at 50°C and the mixture was stirred for 2 hours. A clear solution was obtained. The solution was then cooled to 25°C within 1 hour. Hemi-fumarate seeds of Sample RC13 (Example 5, Table 7) were added, followed by addition of 2.5 mL of heptanes to induce precipitation. An oil was obtained and stirred at 25°C for about 4 days. After 4 days, the resulting suspension was cooled to 5°C.
  • Mono-fumarate seeds of Sample RC 18 (Table 7) were added to the mixture, followed by addition of 4 mL of heptanes as an antisolvent. The mixture was stirred at 25°C for 4 days. The precipitated material was collected by filtration and dried at 40°C under vacuum for about 2 hours. As a result, 208 mg of mono- fumarate solids were obtained in yield of 69%.
  • Example 2 Large Scale Preparation of Mono-Fumarate About 4.16 g of Compound I free base was dissolved in 11 mL of IPA. Then, 1.0 eq. of fumaric acid was added to the yellow clear solution under stirring at 50°C. After about 1 hour, some solids precipitated out.
  • Step 1 Preparation of diethyl-((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)- phosphonate (3) g, 0.296 mol, 1 equiv.), Cs 2 CO 3 (96.37 g, 0.296 mol, 1 equiv.) and DMF (250 mL) under N 2 atmosphere at room temperature. To this at room temperature and under stirring was added diethyl 2- chloroethoxymethyl phosphonate 2 (74.85 g, 0.325 mol, 1.1 equiv.) in a drop-wise manner.
  • the reaction was stirred at 40-50°C for 0.5 to 1.5 hours, heated to 60-70°C and stirred for 0.5-1.5 hours, and then stirred at 75 to 85°C for 18-24 h. After bringing the reaction temperature to 20- 30°C, the reaction mixture was filtered and the resulting cake was washed with DMF (100 mL x 2). The combined filtrate was concentrated to a half volume below 70°C, diluted with n-heptane (250 mL) and again concentrated to a half volume below 75°C. The resulting solution was poured into DCM (1 L), stirred at 20 to 30°C for 20-40 min., then aqueous 10% Na2SO4 solution ( ⁇ 100 mL) was added.
  • the resulting bi-phasic solution was stirred for 20-40 minutes then filtered through diatomite and the wet cake was washed with DCM ( ⁇ 100 mL). From the filtrate, the aqueous phase was separated and the organic phase was again washed with aqueous 10% Na 2 SO 4 solution ( ⁇ 100 mL). The combined aqueous phases upon washing (back extraction) with DCM (200-300 mL), the organic phases were combined and concentrated. The resulting crude product 3 was then purified by silica gel column chromatography using DCM to 1% MeOH in DCM. The fractions containing products were combined and the solvent was evaporated below 40°C.
  • the solid product 3 was treated with the repeated dilution with MTBE and concentration (up to 1/3 rd volume). The resulting slurry was then diluted with MTBE (400-500 mL) and agitated at 40-50°C for 4-6 h and at 15-25°C for 8-15 h. The suspension was filter and washed with MTBE and dried at 35-40°C for 15-20 h to afford the desired product, diethyl-((2-(2-amino- 6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonate 3 in 43.4% (48.66 g) isolated yield with 91.8 % purity by HPLC.
  • Step 4a Preparation of a mixture of (R,S)- and (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H- purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (8)
  • Example 4 Preparation of (+)-Compound I Monofumarate ((+)-(2S)-Ethyl-2-((((2-(2- amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate monofumarate) (9) To a solution of (+)-(2S)-ethyl-2-((((2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (Compound I) 8 (29.72 g, 0.06 mol, 1 equiv.) in IPA was added a solution of fumaric acid (7.66 mol, 1.1 equiv.) in IPA through a filter at 45-55°C and stirring was continued for 1-2 h.
  • the stereoisomers are separated using HPLC or SFC with achiral or chiral stationary phases.
  • chiral stationary phases which may be used include Chiralpak AD, Chiralpak AS, Chiralcel OG, and Chiralcel OJ.
  • the individual isomers can be synthesized asymmetrically.
  • asymmetric synthesis of phosphonamidates see Numan, A et al. “Asymmetric Synthesis of Stereogenic Phosphorus P(V) Centers Using Chiral Nucleophilic Catalysis”, Molecules 2021, 26, 3661 and Ambrosi, A. et al.
  • Step 1b Chiral Separation of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)- ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (S,S)-ethyl-2-((((2-(2- amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate Separation of (R,R)-ethyl-2-((((2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)
  • Step 1c Chiral Separation of (R P )-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)- ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (SP)-ethyl-2-((((2-(2- amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate Separation of (RP)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)-phosphoryl)-amino)-propionate and (SP)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy
  • Step 2a Preparation of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate (Compound II)
  • n-heptane ⁇ 30 mL
  • stirring was continued for another 8-15h at 20-30°C and 0-5°C for 8-15h.
  • the solid observed was filtered and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, ⁇ 5 mL).
  • Step 2b Preparation of (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate (13), also referred as Compound III
  • the seeds of the compound 13 were added to the reaction mixture and stirring was continued for 1-2 h at 45-55°C. After allowing the reaction mixture to settle at 20-30°C for 4-6 h, a drop-wise addition of n-heptane ( ⁇ 30 mL) was performed and stirring was continued for another 8-15h at 20-30°C and 0-5°C for 8-15h. The solid observed was filtered and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, ⁇ 5 mL).
  • Step 2c Preparation of (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (S,R)-ethyl-2-((((2- (2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate monofumarate Synthesis of (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (S,R)-ethyl-2-((((2-(2-amino-6- methoxy-9H-pur
  • Step 2d Preparation of (R P )-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (SP)-ethyl-2-((((2- (2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate monofumarate - - - - - (benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (S P )-ethyl-2-((((2-(2-amino-6- methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate can be
  • a topical cream formulation can be prepared, for example, by emulsifying an oil phase and an aqueous phase along with an active pharmaceutical ingredient.
  • the oil phase of the cream was prepared by mixing light mineral oil, propylparaben and Tefose® 63.
  • the aqueous phase of the cream was prepared by mixing water, EDTA, methylparaben, and Carbopol®974P.
  • the oil and water phases were then emulsified.
  • To the emulsified mixture was added the active pharmaceutical ingredient and propylene glycol. The mixture was pH adjusted and then filled into tubes.
  • a topical gel formulation can be prepared, for example, by mixing an aqueous gel carrier with an active pharmaceutical ingredient.
  • the aqueous phase of the topical gel was prepared by mixing water, EDTA, methylparaben (or sorbic acid) and Carbopol®974P.
  • the active pharmaceutical ingredient and propylene glycol was added to this solution, mixed and pH adjusted, then filled into tubes.
  • from about 0.001% w/w to about 10% w/w active pharmaceutical ingredient is added to the semisolid formulation.
  • from about 0.0025% w/w to about 2.5% w/w such as 0.003%, 0.01%, 0.03%, 0.1%, 0.3% or 1%.
  • Blend the active and the excipients, and screen to remove chunks Take 49.5 grams of the excipients blend and add 2.12 grams of Compound I monofumarate 3. Blend the active and the excipients, and screen to remove chunks 4. To this blend add 148.5 grams of the excipients blend 5. Blend the active blend and the excipients and screen to remove chunks 6. To this blend add 247.5 grams of the excipients blend 7. Blend the active blend and the excipients and screen to remove chunks 8. To this blend add the remaining 495 grams of the excipients blend 9. Blend the active blend and the excipients and screen to remove chunks Final Blending 10. Add magnesium stearate to the diffusion blender and mix the contents. 11. Discharge and reconcile blend. Compression 1.
  • Packaging 1. Package bulk tablets into double lined re-closable clear PE bags with desiccants between the bags and further into an aluminum foil pouch with desiccant and heat sealed.
  • Illustrative excipients for a vaginal tablet formulation Tablet formulations are optionally selected to display the properties of mucoadhesion and substantivity and include excipients that have solubilizing, erosion-generating (for disintegration), porosity (for water uptake) and viscosity enhancing (to keep the drug at the target site) properties.
  • excipients that will cause rapid disintegration to cover the cervix or vaginal areas include, but are not limited to mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid or fumaric acid.
  • excipients that can enhance disintegration and coverage of the affected area include but are not limited to sodium starch glycollate, pregelatinized starch, crospovidone and croscarmellose sodium.
  • Mucoadhesive excipients that are useful in the present invention include but are not limited to microcrystalline cellulose, polycarbophil, hydroxymethyl cellulose, hypromellose, hydroxypropyl cellulose, and PVP.
  • a tablet formulation may for example comprises the active pharmaceutical ingredient, microcrystalline cellulose and may contain mannitol.
  • the tablet formulation comprises one or more excipients selected from the rapid disintegrant category (left column of Table 3).
  • the tablet formulation comprises one or more excipients selected from the disintegration enhancement category (middle column of Table 3).
  • the tablet formulation comprises one or more excipients selected from the mucoadhesive excipient category (right column of Table 3). Table 3. Excipients for Tablets Rapidly disintegrating To Enhance Mucoadhesive polymers d i t t Dii t ti d e o Example 8. Illustrative excipients for a reconstitution powder or dry powder formulation A reconstitution powder or dry powder formulation may improve the shelf stability of a pharmaceutical agent or formulation. In certain nonlimiting embodiments, the dry powder formulation may be mixed with saline, propylene glycol or other aqueous carrier shortly before it is administered, minimizing the time for degradation.
  • the dry powder formulation is mixed with an oil, cream, or other nonaqueous carrier shortly before it is administered.
  • the reconstitution powder or dry powder formulation rapidly covers the infected or diseased tissue in the cervix, vulva or vagina.
  • Excipients which enhance the rapid coverage of the cervix, vulva or vagina include but are not limited to mannitol, lactose, sucrose, calcium phosphate, and microcrystalline cellulose.
  • the excipient for rapid coverage of the cervix, vulva or vagina is mannitol.
  • the reconstitution powder or dry powder formulation has good coverage of the cervix, vulva or vagina.
  • Nonlimiting examples of excipients which enhance the coverage of the cervix, vulva or vagina include sodium starch glycollate, pregelatinized starch, crospovidone, and croscarmellose sodium.
  • the reconstitution powder or dry powder formulation contains mucoadhesive properties once it has been reconstituted. This prevents smearing of the dosage form or otherwise exposing healthy tissues to the active pharmaceutical ingredient.
  • the dry powder or reconstitution powder formulation comprises one or more excipients selected from the rapid coverage category (left column of Table 4). In certain non-limiting embodiments, the dry powder or reconstitution powder formulation comprises one or more excipients selected from the coverage enhancement category (middle column of Table 4). In certain non-limiting embodiments, the dry powder or reconstitution powder formulation comprises one or more excipients selected from the mucoadhesive excipient category (right column of Table 4). Table 4. Excipients for Reconstitution Powders of Dry Powder Dosage Forms Rapidly covering To Enhance coverage Mucoadhesive polymers PVP (0 to 50%) e Example 9.
  • the formulation for a vaginal tablet dosage form comprises the ingredients in Table 7.
  • the formulation for a tablet dosage form comprises the ingredients in Table 7.
  • An illustrative process for combining these ingredients into a tablet dosage form can be found in Example 7.
  • Table 7. Example Tablet Formulation Material Amount in tablet Table 8.
  • Illustrative semisolid formulations In certain non-limiting embodiments, the formulation for a gel semisolid dosage form comprises the ingredients in Table 9.
  • the formulation for a cream semisolid dosage form comprises the ingredients in Table 10. An illustrative process for combining these ingredients into a cream or gel semisolid dosage form can be found in Example 6 above.
  • In-vitro cytotoxicity testing Compounds Three compounds (Compound I, Compound II and Compound III) were solubilized at 40 mM in DMSO and stored at -20°C. The test compounds were evaluated using a high test concentration of 50 ⁇ M. Serial half-logarithmic dilutions were performed for the in vitro cytotoxicity assays. Tamoxifen citrate was purchased from Sigma-Aldrich (St. Louis, MO).
  • Tamoxifen citrate was solubilized in DMSO at 40 mM and used as a positive control compound at a high test concentration of 100 ⁇ M for the cytotoxicity assays.
  • In Vitro Cytotoxicity Evaluations Cells listed in Table 13 were enumerated by Trypan Blue Dye exclusion method and seeded in the interior wells of a 96 well flat bottom microtiter plate at 100 ⁇ L/well. Proliferating cells were incubated overnight at 37°C/5% CO2 to allow the cells to adhere to the plates at approximately 70% confluency. Tissue culture medium was removed and replaced with 100 ⁇ L/well of fresh medium.
  • Table 70 lists the cell line, type of cell, source of cell stock, base tissue culture medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, and microtiter plate seeding density.
  • Table 13 Cell Culture for in vitro Cytotoxicity Cell Seeding Cell Line Cell Type Cell Source C lt D ity l) 4 Cytotoxicity XTT: Following incubation at 37°C in a 5% CO2 incubator for five days, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5- sulfopheny1)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product. XTT solution was prepared daily as a stock of 1 mg/ml in RPMl1640.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/ml in PBS and stored in the dark at - 20°C.
  • XTT/PMS stock was prepared immediately before use by adding 40 ⁇ L of PMS per ml of XTT solution. Fifty microliters of XTT/PMS were added to each well of the plate and the plate was reincubated for 4 hours at 37°C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader. Data Analysis and Evaluation: Microsoft Excel 2010 was used to analyze and graph the raw data.
  • CCso 50% reduction in cell viability values are tabulated and provided.
  • Raw data for cytotoxicity with a graphic representation of the data are provided in a printout summarizing the compound effect on cell viability.
  • In Vitro Cytotoxicity Evaluations Compounds I, II, and III were evaluated for cytotoxicity to proliferating Hs27, HeLa, C33A and HEK293 cells by measuring cell viability using XTI tetrazolium dye following five days in culture (Table 14). The CC 50 values calculated from these assays are summarized in the below tables. Tamoxifen citrate was evaluated in parallel as a control compound.
  • CC50 value for tamoxifen citrate in proliferating C33A, HeLa, Hs27, and HEK293 cells was 17.2, 19.9, 21.2 and 21.3 ⁇ M, respectively. Compound I and its two isomers were similarly cytotoxic when evaluated in parallel against each of the four cell lines.
  • CC50 values for the three test compounds ranged from approximately 0.1 to 0.28 ⁇ M in C33A cells. In HeLa cells, CC50 values for the three test compounds ranged from 15.1 to 18.6 ⁇ M. The CC 50 values for the three test compounds ranged from approximately 7.62 to 23.2 ⁇ M in Hs27 cells. In HEK293 cells, CC50 values for the three test compounds was approximately 0.1 ⁇ M.
  • Example 15 In vitro Cytotoxicity Data Compound C33A HeLa Hs27 HEK293 )
  • Example 15 Ex Vivo permeation and penetration of antiviral drugs across porcine vaginal tissue Preparation of Porcine Vaginal Tissue Freshly harvested porcine vaginal tissue was procured from local slaughterhouse in an ice box. The vaginal tissue was cut open to expose the mucosal surface and tissue was cleaned by gentle flow of PBS pH 7.4. The porcine vaginal tissue was cut into circular portions (approximately 2 cm 2 ) with help of a telemetric punch.
  • Porcine vaginal tissue mounted on Franz diffusion cells
  • the circular portion of tissue was sandwiched between two chambers of a Franz diffusion cell with an active diffusion area of 1 cm 2 , and the mucosal layer was exposed to the donor chambers.
  • the resistance across porcine vaginal tissue was measured using a wave form generator to ensure the integrity of the tissue segment used for the permeation study.
  • Porcine vaginal tissue with resistance of ⁇ 3 K ⁇ .cm 2 was used for study.
  • the receiver chamber was filled with 8 ml of 5% solutol PBS 7.4 pH, which was stirred at 600 rpm with a 3 mm magnetic stir bar and the temperature was maintained at 37 °C with a circulating water bath.
  • the tissue was minced into smaller pieces on a dish with a precooled surgical blade.
  • the minced tissue was transferred to sample tubes and dish was rinsed with 1 ml of 5% solutol in PBS 7.4 pH and transferred to same tissue sample tube. These tubes were stored in -70 ⁇ C until analysis.
  • Preparation of receptor fluid for analysis The samples stored at -20 °C was removed and thawed at room temperature for 30 min.
  • the drug from receptor fluid was centrifuged at 13000 rpm for 5 min and to 200 ⁇ L of supernatant equal volume of extraction solvent was added. These samples were centrifuged at 13000 rpm for 5 min and supernatant was transferred into vials for analysis.
  • Vagina and Cervix Many animals, including sham and placebo controls, had microscopic infiltrates of a mixed cell population noted within the lamina intestinal of sections of vulva and vagina after repeated administration of Compound I vaginal tablets. Within the vagina, microscopic findings in the two studies included minimal to mild epithelial degeneration, mixed cell inflammation and luminal exudate in animals administered tablets ⁇ 0.03 mg Compound I. In the first study, two cases of minimal arterial degeneration/necrosis were seen at the 2.9 mg dose, which was characterized by disruption of the arterial wall with karyorrhectic (nuclear) debris and/or fibrinous change. In addition, one animal treated at 2.9 mg had moderate ulceration in the middle portion of the vagina.
  • Compound I administered by the IV route in safety pharmacology studies evaluated the potential for adverse effects on the respiratory, cardiovascular, and central nervous systems. No adverse effects were observed.
  • Single-dose IV toxicity studies were also conducted to describe potential systemic toxicity. In rabbits, single doses of 0 (vehicle control), 0.03, 0.3 and 3.0 mg/kg Compound I were given intravenously and animals were monitored for 7 or 23 days before sacrifice for necropsy and microscopic examination of tissues. Compound I was well tolerated with no signs of toxicity at any dose and all animals survived to the scheduled termination. There were no changes in chemistry or coagulation parameters.
  • Karyomegaly is an enlargement of the cell nucleus that has been proposed to be a response to chemical/drug exposure or insult (Hard, 2018). As indicated, the NOAEL for this study was 0.3 mg/kg which is approximately 12-fold greater exposure (by AUC) than the plasma exposure in rabbits dosed intravaginally at the 0.29 mg dose strength. Genotoxicity A standard battery of genotoxicity studies to assess the potential mutagenicity of Compound I was completed. A bacterial reverse mutation (Ames) study was negative. A mammalian chromosomal aberrations study was positive in the presence of metabolic activation at the highest concentration tested; however, there was no evidence of mutagenicity in an in vivo micronucleus assay.
  • Part B consisted of 2 multiple ascending dose cohorts administering Compound I vaginal tablet at 0.1 mg and 0.3 mg or placebo (3 doses over 1-week period). The study completed in June 2023. A total of 35 subjects were enrolled in the study: 19 subjects in Part A (13 received Compound I; 6 received placebo) and 16 subjects in Part B (12 received Compound I; 4 received placebo). In Part A and the 0.1 mg dose cohort of Part B, Compound I was safe and well tolerated. No dose limiting toxicities (DTLs) were reached or serious adverse events (SAEs) reported. All AEs were mild or moderate in severity. After review of safety data, it was determined that DLT at the 0.3 mg dose in Part B was reached.
  • DTLs dose limiting toxicities
  • SAEs serious adverse events
  • Compound I vaginal tablets can be administered with a vaginal applicator, followed by application of a retaining device. In certain embodiments, Compound I vaginal tablets can be administered with a diaphragm or menstrual cup.
  • Example 19 Exemplary Instructions For Self-Administration Of Compound I vaginal tablets With Vaginal Applicator Tablet administration is preferably carried out at bedtime to reduce displacing medication that may occur when standing or walking. 1. Place all supplies on a clean flat surface 2.
  • gloves for administration vaginal tablet applicator, water-based lubricant (such as KY Jelly, Surgilube or similar), and bottle of Compound I monofumarate vaginal tablets are assembled. 3. Wash and dry hands, and put on gloves. 4. Remove the applicator from its packaging. 5. Remove the Compound I monofumarate vaginal tablet from the bottle and place it into the end of the applicator. 6. To load the tablet into the applicator, retract the plunger slightly and insert the tablet into the barrel of the applicator until it is securely seated. Illustrations of the tablet placed in the barrel of the applicator are shown in FIG 7A. If loaded properly, the tablet will be firmly in the barrel and will not drop even when the applicator is held with tablet facing down.
  • water-based lubricant such as KY Jelly, Surgilube or similar
  • the tablet can be released by pushing in the plunger. 7.
  • a small amount about a teaspoon, which should be enough to cover/coat the tablet and applicator
  • the lubricant may be provided in individual packets if available; in that case, open the packet and dispense the entire amount of lubricant onto the Compound I monofumarate vaginal tablet.
  • the female can position her body in one of two ways: a. Stand upright (standing with her feet apart and her knees bent). It may also be helpful to raise one leg up on a chair or the bed. b. Lie on her back with her knees bent and legs slightly apart. 9.
  • the diaphragm should be a flexible device that is created to be inserted through the vagina and placed to cover the cervix.
  • the retaining device is used to help prevent leakage from the dissolved Compound I monofumarate vaginal tablet.
  • the device will form a seal against the cervix.
  • the diaphragm with the vaginal tablet is inserted, and then removed approximately 5-10 hours after insertion, and more typically 5-8 hours, for example around or at least 6 hours.
  • vaginal tablet administration should be preferably done at bedtime to reduce displacing medication that may occur when standing or walking.
  • FIG.7B Prior to self-administration, remove the Compound I monofumarate vaginal tablet from the bottle and place it inside the diaphragm first (FIG.7B) 5. Place a small amount (about a teaspoon or 5 mL) of water-based lubricant into the diaphragm to cover the Compound I monofumarate vaginal tablet.
  • the lubricant may be provided in individual packets if available; in that case, open the packet and dispense the amount of lubricant onto the Compound I monofumarate vaginal tablet (FIG.7C). 6. Hold the diaphragm with one hand by placing the thumb and index finger on the grip dimples along the rim. The arrow should point towards her body. See FIG.7D. 7.
  • the vaginal tablet will be located on either the left or right side of the fold (See FIG.7E). 8. Alternatively, if the lubricant and vaginal tablet rise up too close to the rim when folding, and appear to be coming out, the patient can push down on the diaphragm dome while folding to allow for one big fold (See FIG.7F). 9. For insertion of the diaphragm the patient can position her body in one of three ways: kneeling down in a sitting position; lying down with legs bent; or standing with one leg elevated. 10. Using her free hand, spread the labia.
  • Example 21 Single-Crystal X-Ray Diffraction (SC-XRD) study of Compound II Pattern 1
  • SC-XRD Single crystals of Compound II Pattern 1 suitable for SC-XRD study were obtained in a temperature cycling experiment in MeOH.
  • Example 22 Synthesis of Salts of Compound I Compound I Sulfate Two methods were used to synthesize the sulfate salt of Compound I. Method A To a solution of Compound I (0.049 g, 0.1 mmol) in dry THF (1 mL) at 0-10°C was added a solution of sulfuric acid (1N in THF) slowly.
  • Compound I sesquifumarate salt can be synthesized by washing Compound I sesquifumarate salt with additional MTBE and drying under high vacuum.
  • Compound I Pattern 1 is an anhydrate and has a melting peak at T onset of 75.0°C with an enthalpy of about 64 J/g. It shows about 0.3% weight loss at about 70°C. KF shows it contains about 1.7% of water. About 0.7% of MTBE (by weight) residue was detected by 1 H NMR.
  • Compound I hemifumarate Pattern 1 is an anhydrate. The stoichiometry of free form: fumaric acid is about 1:0.5 based on 1 H NMR result. It has a melting peak at Tonset of 85.2°C with an enthalpy of about 37 J/g. It shows about 1.0% weight loss at about 85°C. KF shows it contains about 1.7% of water.
  • Compound I monofumarate Pattern 1 is an anhydrate.
  • the stoichiometry of free form: fumaric acid is about 1:1.0 based on 1 H NMR result. It has a melting peak at T onset of 107.2°C with split peaks and with an enthalpy of about 78 J/g.
  • the two thermal events cannot be resolved with 2 K/min and 0.5 K/min heating rate by DSC as well. It shows about 0.3% weight loss at about 107°C.
  • KF shows it contains about 1.2% of water.
  • About 0.7% of IPA and 2.2% of heptanes (by weight) residue was detected by 1 H NMR.
  • Compound I hemifumarate Pattern 1 tends to degrade more than Compound I monofumarate Pattern 1 in the two conditions mentioned above.
  • Table 19 Bulk stability of Compound I free base Pattern 1, Compound I monofumarate Pattern 1 and Compound I hemifumarate Pattern 1 Compound I Compound I Compound I Compound I Compound I Storage Conditions free base Hemifumarate Monofumarate Example 25. Solubility of Compound I free base Pattern 1, Compound I Monofumarate Pattern 1 and Compound I Hemifumarate Pattern 1 About 4 mg of Compound I Pattern 1 was added into 2 mL of buffer solution.
  • Solubility of Compound I free base Pattern 1, Compound I monofumarate Pattern 1 and Compound I hemifumarate Pattern 1 Compound I free base
  • Compound I Compound I Pattern 1 Hemifumarate Monofumarate Solubility was tested in five media, pH 3.0 citrate buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, water, simulated vaginal fluid (pH 4.2) at 37°C for 0.5 h and 2 h. All the three candidates are highly soluble in the media (>2 mg/mL).
  • Example 28 Polymorph Screening Study of Compound I fumarate salts The polymorph screening study was performed for fumarate salts of Compound I (mixture of diastereomers). Their polymorphic behaviors were investigated by equilibration, precipitation by addition of antisolvent, slow cooling, fast cooling and slow evaporation experiments.
  • Table 32 Results of equilibration with IPA/toluene (1:1 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks) XRPD diffractogram is shown in FIG.35.
  • Table 33 Results of equilibration with IPA/MTBE (1:3 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks) XRPD diffractogram is shown in FIG.35. Table 34.
  • Solvent XRPD 1 Acetone Pattern 1 with medium crystallinity Crystallization from hot saturated solutions by slow cooling About 30 mg of Compound I monofumarate Pattern 1 was dissolved in the minimal amount of selected solvents at 50°C. Obtained solutions were cooled to 5°C at the rate of 0.1°C/min. Precipitates were collected by filtration. The solid part (wet cake) was investigated by XRPD. When differences were observed, additional investigations were performed (e.g., NMR, DSC, TGA). If no precipitation was obtained, the solutions were put in a -20°C freezer for crystallization. After storing in -20°C freezer for about 5 days, no precipitation was obtained, heptane was added as antisolvent.
  • Precipitates were collected by filtration.
  • the solid part (wet cake) was investigated by XRPD. Results are presented in Table 42. XRPD diffractograms are shown in FIG. 50 and FIG. 51. Table 42. Crystallization from hot saturated solutions by slow cooling Exp. Solvent XRPD Comments Residual solvent: 2.0% (by weight) ACN solvent DSC onset (enthal ): 740°C (89 J/ ) at N ar ar Crystallization from hot saturated solutions by fast cooling About 30 mg of Compound I monofumarate Pattern 1 was dissolved in the minimal amount of selected solvents at 50°C. Obtained solutions were put into an ice bath and agitated. Precipitates were collected by filtration.
  • the solid part (wet cake) was investigated by XRPD. When differences were observed, additional investigations were performed (e.g., NMR, DSC, TGA). If no precipitation was obtained, the solutions were put in -20°C freezer for crystallization. After storing in -20°C freezer for about 7 days, no precipitation was obtained, heptanes was added as antisolvent. Precipitates were collected by filtration. The solid part (wet cake) was investigated by XRPD. Results are presented in Table 43. XRPD diffractograms are shown in FIG. 52 and FIG. 53. Table 43. Crystallization from hot saturated solutions by fast cooling Exp.
  • Cycle 2 0°C to 130°C at 10°C /min; 130°C to 0°C at 10°C /min; reheat from 0°C to 250°C at 10°C /min.
  • Table 44 Behavior of Compound I monofumarate Pattern 1 under heating and cooling Exp. Heating rate Thermal events 1 Cycle 1: 0°C to 106°C at 10°C/min; 106°C Heating: Behavior under compression About 10 mg of Compound I monofumarate Pattern 1 was compressed for 5 minutes at 10 MPa with a hydraulic press. XRPD characterization was performed to investigate the polymorphic behavior under compression. According to the XRPD, no form change was observed.
  • Pattern 1 used in the study was prepared from Compound I free base according to Example 7.
  • the initial form of monofumarate used in the polymorph study described below (Pattern 1) is an anhydrate of monofumarate with HPLC purity of about 99.3%.
  • the ratio of free form and fumaric acid is about 1: 0.96 by 1 H NMR. It has two melting peaks with Tonset of about 98.5°C with an enthalpy of about 14 J/g and 109.6°C with an enthalpy of about 25 J/g, as measured by differential scanning calorimetry (DSC).
  • Pattern 1 shows about 0.5% weight loss at about 98°C and 0.6 % weight loss from 98°C to 140°C. About 1.0% (by weight) heptanes and 0.2% (by weight) IPA residue were detected by 1 HNMR. Karl Fischer titration shows it contains about 1.3% water. During the polymorph study, four new patterns were identified. Although monofumarate was used as initial physical form obtained new patterns showed different stoichiometric ratio. Pattern B, Pattern C and Pattern E are hemifumarate salts, and Pattern D is a degradation product. Pattern B is an anhydrate of hemifumarate with HPLC purity of about 99.6%.
  • the ratio of free base form to fumaric acid is about 1: 0.52 by 1H-NMR. It has two thermal events with T onset of about 77.4°C with an enthalpy of about 71 J/g and 88.4°C with an enthalpy of about 18 J/g. It shows about 0.7% weight loss at about 77°C and 4.2% weight loss from 77°C to 130°C. About 4.6% (by weight) MEK residue was detected by 1 H NMR.
  • Pattern C is not a monofumarate. It has two thermal events with T onset of about 74.0°C with an enthalpy of about 89 J/g and T onset of about 90.6°C with an enthalpy of about 15 J/g. It shows about 0.4% weight loss at about 73°C and 2.1% weight loss from 73°C to 144°C.
  • Pattern D is a degradation product with HPLC purity of about 0.2%. It was obtained in water by equilibration experiment experiments. It has two thermal events with T onset of about 41.4°C with an enthalpy of about 67 J/g and Tonset of about 72.1°C with an enthalpy of about 29 J/g. It shows about 0.6% weight loss at about 41°C and 8.5% weight loss from 41°C to 178°C.
  • Pattern E is an anhydrate of hemifumarate with HPLC purity of about 98.9%. It was obtained in acetone/toluene by equilibration experiment.
  • the ratio of free base form to fumaric acid of the mixture is about 1: 0.69 by 1 H NMR. It has two thermal events with Tonset of about 53.1°C with an enthalpy of about 33 J/g and T onset of about 96.5°C with an enthalpy of about 34 J/g. It shows about 1.0% weight loss at about 53°C and 3.6% weight loss from 53°C to 96°C. About 0.6% (by weight) acetone residue was detected by 1 H NMR. Results of the study on Compound I mono- and hemifumarate polymorphs are summarized in Table 45 below.
  • the salt ratio is the ratio between Compound I free base and the salt counterion.
  • AS indicates the form can be prepared by antisolvent addition, using the solvent/antisolvent pairs listed in the table.
  • EQ indicates the form can be prepared through equilibration in the listed solvent.
  • SC indicates the form can be prepared by slowly cooling a solution of Compound I monofumarate in the listed solvent.
  • FC indicates t the form can be prepared by quickly cooling a solution of Compound I monofumarate in the listed solvent. Table 45. Summary of identified Compound I fumarate salt Patterns A, B, C, and E Melting Polymorph Salt Temperatures Salt ratio Preparation E th l s, Example 29: Preparation of Compound II Pattern 1 Experiment 1.
  • XRPD diffractogram of Compound II Pattern 1 is shown in FIG.56.
  • Large-scale preparation Three grams of R P Compound I free base and 9 mL of IPA were added into a glass vial. To it was added 1.0 equiv. of fumaric acid and the resulting mixture was stirred at 50°C for 5 min. About 21 mg of Compound II Pattern 1 seeds from Experiment 1 were added into the mixture.30 mL of heptanes was added into the mixture.
  • Example 30 Preparation of Compound III Pattern 1 SP Compound I free base (100 mg) and 0.3 mL of IPA were added into a glass vial. To it was added 1.0 equiv. of fumaric acid and the mixture was stirred at 50°C for 15 min, most of the material was precipitated out.

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Abstract

A pharmaceutically acceptable salt of an acyclic nucleotide phosphonamidate to treat HPV and related conditions including neoplasia, as well as pharmaceutical compositions, morphic forms and dosage forms thereof.

Description

DEVICE SYSTEM AND USE FOR HPV INFECTION AND CERVICAL INTRAEPITHELIAL NEOPLASIA FIELD OF THE INVENTION The present invention provides a device and therapeutic system for the topical delivery of the PMEG prodrug Compound I or its pharmaceutically acceptable salt such as monofumarate and hemifumarate salts thereof, Compound II, Compound III, or one or more morphic forms of the foregoing to treat a female in need thereof with papilloma virus (HPV) infection or a related disorder such as HPV-induced cervical or vaginal intraepithelial neoplasia, in a manner that reduces toxicity to nearby cells and tissues. BACKGROUND OF THE INVENTION According to the U.S. Center for Disease Control, there is no direct cure for human papilloma virus. HPV is so common that most sexually active people have been infected at some point in their lives. Papillomaviruses are a group of non-enveloped DNA viruses, which in humans infect keratinocytes of skin and mucous membranes including in the cervical area. HPV infections can cause cellular transformations in the human patient that have not yet progressed to cancer but have reached the stage of neoplasia. Forms of HPV-induced neoplasia include cervical intraepithelial neoplasia (“CIN”), anal intraepithelial neoplasia (“AIN”), perianal intraepithelial neoplasia (“PAIN”), vulvar intraepithelial neoplasia (“VIN”), penile intraepithelial neoplasia (“PIN”) and vaginal intraepithelial neoplasia (“VAIN”). Cancers caused by HPV include cervical, anal, perianal, penile, vaginal, vulvar, and oropharyngeal cancer. Thus, HPV can cause viral infection, neoplasia and cancer. Most of the cancer-causing HPV types are from the alpha-7 and alpha-9 species and include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. The most common cancer-causing HPV types are 16 and 18. The causal role of HPV in cancer of the cervix has been firmly established biologically and epidemiologically. Persistent infection with high-risk HPV is necessary to promote progression of pre-malignant stages to invasive cancer. Oncogenic HPV types are detected in almost all cervical cancer specimens, with types 16 and 18 accounting for about 70% of cervical cancers and about 50% of high-grade lesions. The majority of venereal warts are caused by the low-risk HPV types 6 and 11. The current therapeutic options for HPV and cervical intraepithelial neoplasia are adjunctive only. Commonly used drug therapies include trichloroacetic acid, 5-fluorouracil, imiquimod and podofilox. Imiquimod (AldaraTM, ZyclaraTM) stimulates the immune system to clear the infection through toll-like receptor signaling and causes redness and swelling. Podofilox (CondyloxTM) destroys tissues by destabilizing microtubules which prevents host cell replication. The cervical epithelium is composed of several layers of tissue and is referred to as stratified squamous epithelium. The layers are the superficial cell layer, the intermediate cell layer, the parabasal cell layer and the basal cell layer. It is essential that a topical drug for the treatment of cervical intraepithelial neoplasia is able to penetrate these multiple layers of tissue to adequately reach and treat the transformed cells. This is a formidable task because the cells are tightly bound and without blood vessels. Cervical intraepithelial neoplasia is most often treated by observation (the wait and see approach) or by excision or ablation of the cervical transformation zone. Techniques include cryotherapy, laser therapy, loop electrosurgical procedure (LEEP) and cone biopsy. All of these surgical procedures damage the affected areas and can lead to scarring. The most common intervention, LEEP, is effective in 60-90% of cases, however, it may be associated with a significantly increased risk of miscarriage, ectopic pregnancies, and negative psychological outcomes. Despite extensive research, no drug has been approved to replace or combine with these surgical methods. Cervical high-grade squamous intraepithelial lesions (cHSIL), sometimes referred to as CIN2 and CIN3, is a disease caused by the abnormal hyperproliferation (dysplasia) of squamous cells in the cervical epithelium (Waxman, A. G., et. al 2012. “Revised terminology for cervical histopathology and its implications for management of high-grade squamous intraepithelial lesions of the cervix”. Obstet Gynecol, 120, 1465-71). Hyperproliferation usually occurs where the simple columnar, endometrial-type epithelium of the endocervix transitions to the stratified squamous epithelium of the ectocervix; this region is referred to as the “transformation zone” (Sellors, J. W. & Sankaranarayanan, R. 2003. An introduction to the anatomy of the uterine cervix. Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginners' Manual). Cervical HSIL is classified as a pre-cancerous condition because apoptosis is impaired in these hyperproliferating cells, which can lead to the accumulation of genetic alterations that transform the cells into cancer. The probability that the patient’s immune system will be sufficiently activated to clear lesions diminishes over time and is contingent upon a variety of factors including the patient’s immunocompetency, lifestyle (e.g., smoking), and nutritional status (Schiffman, M., et al 2011. “Human papillomavirus testing in the prevention of cervical cancer”. J Natl Cancer Inst, 103, 368-83). For women already infected with HPV, there is no approved drug therapy to treat cHSIL and thereby, to prevent the progression of HSIL to cervical cancer. Frantz Viral Therapies, LLC is conducting clinical trials to assess the use of artesunate vaginal inserts for the treatment of women with cervical high grade intraepithelial neoplasia (CIN2/3). Artesunate is an artemisinin derivative with cytotoxic activity. Artesunate is a known WHO-approved anti-malarial agent. The cytotoxic agent is delivered in the trial at a dosage, for example, of 50 to 200 mg for a 5-day cycle on weeks 0, 2 and 4. The artesunate vaginal inserts are self-administered at bedtime with a vaginal applicator, followed by use of a tampon, which is removed in the morning. Artesunate has a cytotoxic effect but is not an anti-viral agent, so does not directly stop the HPV replication. Therefore artesunate cannot be used to treat patients who have an HPV infection that has not progressed to cervical intraepithelial neoplasia. Also, artesunate does have some systemic exposure under these conditions. See generally Trimble, et al., “A first- in-human proof-of-concept trial of intravaginal artesunate to treat cervical intraepithelial neoplasia 2/3 (CIN 2/3)”, Gynecologic Oncology 157 (2020)188-194 as well as U.S. Patents 6,586,464; 8,394,849; 8,940,787 and 7,989,491. Cidofovir, a pyrimidine based acyclic phosphonate nucleoside, which has broad spectrum activity against DNA viruses, is recognized as one of the effective treatments for HPV lesions that have not become cancerous. It is a DNA terminator and causes cell death via apoptosis of HPV transformed cells and regression of HPV-induced tumors. Cidofovir has been tested as a topical treatment of CIN2 and CIN3. See Van Pachterbeke, et al., “Topical treatment of CIN 2+ by cidofovir: Results of a phase II, double-blind, prospective, placebo-controlled study”, Gynecologic Oncology 115 (2009) 69-74. Researchers found that cidofovir cannot replace conization, but may be used for topical chemotherapy. See also Snoeck, et al, “Cidofovir, a New Approach for the Treatment of Cervix Intraepithelial Neoplasia Grade III (CIN III)” Journal of Medical Virology 60:205-209 (2000). According to Snoeck, et al., except for two patients, patients had at least partial responses and half had a total response. In the partial responses, the transformed cells persisted in the deep tissues that can lead to neoplasia. Therefore, because of imperfect bioavailability, modifications were needed. The clinical trial thus had mixed results and was not progressed through completed clinical trials to an approved product. The Regents of the University of California, with Karl Hostetler, et. al, as named inventors, has filed a series of patents on various acyclic nucleotide derivatives to treat papilloma infections, including (i) U.S. Patent Nos. 8,835,603; 9,629,860; 9,156,867; 10,449,207; 10,195,222; 10,076,533; 10,076,532; 9,775,852; 9,387,217 with a priority date of March 15, 2013; (ii) U.S. Patent Nos.10,702,532; 10,213,430; 9,493,493; and 9,801,884, with a priority date of September 15, 2014 (PCT/US2015/050202; published as WO 2016/044281); and (iii) U.S. Patent No. 11,014,950 and 10,377,782 with a priority date of September 15, 2015. Some of these patents are licensed to Antiva Biosciences, Inc., which is developing novel therapeutics to treat pre-cancerous lesions caused by HPV. Antiva Biosciences carried out human clinical trials with the phosphonate ABI-1968 to assess its ability to adequately penetrate the various layers of cervical epithelium and release the antiviral agent PMEG ((9-[2-phosphonomethyoxy)ethyl)guanine]). PMEG is phosphorylated to PMEGpp (PMEG polyphosphate), which is the active compound. It was determined that ABI- 1968, when used even up to a 3% dose, does not reach 15 ng/mg of tissue concentration for ABI- 1968, (See Bar F and G in FIG. 2) and thus is not suitable as a topical drug to treat cervical intraepithelial neoplasia. The clinical trials were terminated as not successful. It was administered with or without a diaphragm or cervical cap retaining device.
Figure imgf000005_0001
It is thus a formidable challenge to topically dose HPV-infected epithelial stratified tissue in an effective manner that destroys the neoplasia cells in the multiple epithelial layers. The drug must be lipophilic enough to pass through the tissue layers and be metabolized if necessary to the active agent in a sufficient concentration to kill the pathogenic cells. It is an object of the present invention to provide an improved method for the treatment of cervical or vaginal HPV infection and related conditions such as HPV-induced cervical or vaginal intraepithelial neoplasia in a female in need thereof, that is effective against the infection or related conditions and also minimize toxicity to nearby tissues and cells. SUMMARY OF THE INVENTION It has been discovered that an effective composition for the treatment of cervical or vaginal HPV infection and cervical or vaginal intraepithelial neoplasia, requires the combination of the selection of a number of aspects that work together to achieve the desired results. It was essential to select the right compound with advantageous lipophilic and tissue penetrating properties combined with a selected pharmaceutically acceptable salt or other solid form optionally in an advantageous morphic form to achieve the long-sought ability to penetrate the deep epithelial stratified tissues in an effective amount to deliver the active agent. It required years of research to solve this problem, after many failures, to the benefit of patients globally suffering from interepithelial neoplasia that may become cancerous. In addition, Applicant has found that the use of a retaining device is advantageous to minimize potential adverse effects on surrounding tissue that can be uncomfortable or even painful for the patient and possibly damaging to normal tissue. Thus, the invention includes a device system and therapeutic use to effectively treat HPV cervical or vaginal HPV infection or cervical or vaginal intraepithelial neoplasia. Specifically, it has been discovered that a particularly advantageous treatment for cervical or vaginal HPV infection or cervical or vaginal intraepithelial neoplasia is achieved by the administration of an effective amount of the PMEG prodrug Compound I or a pharmaceutically acceptable salt thereof such as the monofumarate or hemifumarate salt, or other solid form including in particular Compound II or Compound III, by steps comprising (i) administering an effective amount of the anti-HPV therapeutic agent Compound I (or a pharmaceutically acceptable salt thereof) or Compound II or III, typically in solid tablet form (although semi-solid formulations may also be acceptable), to the cervix using a vaginal applicator and then (ii) inserting a retaining device that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post-treatment leakage of vaginal fluids which may contain, for example, remaining therapeutic agent or its metabolite such as PMEG, in a manner that minimizes toxicity-causing damage to non-diseased tissue. In some embodiments, the retaining device is held in place for 3- 10 hours, for example 4-8 hours or 5-7 hours or about or at least around 6 hours. The retaining device is then removed. The retaining device is used after every topical application of the anti- HPV therapeutic agent to prevent undesired PMEG toxicity to surrounding tissue or cells. The retaining device can be, in non-limiting embodiments, a retaining period device, a birth control device, or other type of device that adequately separates the cervix from the vagina, or part of the vagina. Alternatively, it can be any device that forms an adequate barrier between the inserted tablet and surrounding vaginal tissue for a time period of at least 6 hours, and more generally, 7, 8, 9, 10, 11 or 12 hours. For treatment of vaginal HPV infection or vaginal intraepithelial neoplasia potentially without cervical involvement, the patient can, in non-limiting embodiments, insert the retaining device lower in the vaginal canal. In some non-limiting embodiments, a semisolid dosage form such as a gel or cream may be used for the treatment of vaginal HPV infection or vaginal intraepithelial neoplasia, in combination with a retaining device placed such that the infected tissue is exposed to the active PMEG metabolite but uninfected tissues are not. Alternatively, the vaginal tablet can be inserted below the cervix or just above the infected parts of the vaginal canal to treat an infection in the vaginal canal. In certain embodiments, the separate vaginal applicator and retaining device can be combined into one device that can be used to both insert a vaginal tablet and act as a retaining device to protect surrounding tissue. In principal embodiments, the combined device and applicator is similar in structure to a diaphragm with a contoured surface that the vaginal tablet can be placed in for administration. In an alternative embodiment, instead of using a vaginal tablet, a gel or cream formulation with the active therapeutic, as described further below, can be administered and then used in combination with the retaining device. While the vaginal tablet is preferred for its solid structure and physical properties, a gel or cream with sufficient rigidity to deliver the active therapeutic and not unduly escape to surrounding tissue is also appropriate. A lubricant or other suitable material can be applied to the retaining device to hold the vaginal tablet in place. In certain embodiments, the vaginal applicator is first lubricated with a water-based gel such as for example, a glycerol based lubricant, hydroxyethylcellulose-based lubricant, KY jelly or Surgilube to help maintain the vaginal tablet in place during application and for patient comfort. The lubricant may help the vaginal tablet remain in place for effective disintegration. Alternatively or in addition, a water-based gel can also be applied to the vaginal area optionally up to the cervix prior to vaginal tablet administration. In addition or alternatively, the lubricant can be coated on the retaining device for ease of placement as well as to help maintain the device in position. The Applicant is now in clinical development of a pharmaceutical salt of Compound I below, sometimes referred to as “ABI-2280”, a prodrug of PMEG, for topical, intravaginal treatment of several precancerous and potentially precancerous conditions related to human papillomavirus (HPV) infection, including high-grade squamous intraepithelial lesions (HSIL) of the cervix and high-risk HPV (hrHPV) infection in women. Specifically, it was discovered that the key compound is a specific salt of:
Figure imgf000008_0001
. Compound I is (ethyl(((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)- phosphoryl)-L-alaninate). U.S. Patent Nos.9,801,884 and 11,344,555 assigned to the Regents of the University of California claim Compound I and pharmaceutically acceptable salts generally, as well as methods of using the same for treating a papillomavirus infection. Compound I is an acyclic nucleotide phosphonate that metabolizes to a known potent antiviral compound (PMEG; ((9-[2-phosphonomethyoxy)ethyl)guanine])), but PMEG has poor cellular permeability and use- limiting systemic toxicity. Applicant has discovered how to improve the prodrug to be delivered topically in a manner that it is rapidly taken up into epithelial cells, a challenging task to date and one that ABI-1968 failed. Advantageous salt forms of ABI-2280 include
Figure imgf000009_0001
At high local exposures, inhibition of DNA replication by ABI-2280 or its pharmaceutically acceptable salt (for example in the form of Compound II) in healthy epithelial tissues may lead to immune cell infiltration, edema, epithelial thinning, and inflammation at sites of administration. Such effects have been observed in rabbits in toxicology studies evaluating repeated intravaginal administration over 2 weeks given a maximal dose volume of gel containing relatively high concentrations of ABI-2280. These findings were dose level-dependent, with evidence of recovery observed 4 weeks following cessation of treatment. Adverse events (AEs) that were observed to be related to ABI-2280 vaginal tablet have mostly involved the lower genitourinary tract that were graded as mild or moderate in severity. Genitourinary AEs appeared approximately 1 to 2 weeks following the first dose, were persistent for 1 to 2 weeks after onset, and were reversible. In the multiple dose escalation portion of a clinical trial in healthy participants, multiple doses (3 doses over a 1-week period) of ABI-2280 vaginal tablet (0.1 mg) were safe and well tolerated, however, it is important to further minimize these negative side effects of the very potent therapy using a retaining device, as described in detail herein. As a non-limiting example, the female patient optionally coats the vagina with a lubricant and then inserts a vaginal tablet into the base of the cervical area with a vaginal applicator (optionally that has been coated with a lubricant), and then removes the vaginal applicator. The female then inserts the retaining device (also which has been optionally coated with a lubricant) and leaves it in place for at least 4-12 hours, such as 6-10 hours or 5-8 hours and typically at least 6 hours. It is advantageous to insert the vaginal tablet and then place the retaining device in the evening and leave it in overnight, for at least 5 or 6 hours or more. In the morning, the retaining device is removed and cleaned for the next use if reusable, or disposed of if not. By administering before evening sleep, the topical medication is allowed to penetrate into the intraepithelial tissue while the patient is in a supine position with little movement, and the retaining device protects surrounding healthy tissue from the PMEG prodrug toxicity. Nonlimiting examples of retaining devices are those that are conventionally used for period protection or those normally used for birth control. Nonlimiting examples of retaining devices normally used for period protection are: a menstrual cup, a menstrual disc, and a tampon. These devices are commercially available without a prescription. A menstrual cup is a cup that is placed in the vagina below the cervix that stays in place and normally holds blood flow. A variety of menstrual cups are commercially available with different shapes and sizes, and are typically bell-shaped, V-shaped, round or asymmetrical. A menstrual cup is typically made from medical grade silicone or latex rubber. Menstrual cups can typically be used for up to 8 to 12 hours, which is well within the time needed for the Compound I, II, or III or PMEG fluid retainment. It should be thoroughly cleaned between uses, with an antibiotic soap or in boiling water. Some menstrual cups have a tapered shape that can make insertion and removal easy. Nonlimiting examples of current commercially available products include Flex™, Cora™, Saalt™, Lena™, Lumma™, Tampax™, Shordy™ and Diva™. A menstrual disc is a product that is worn inside the vaginal canal that is flat and disc- shaped. It is placed differently in the vaginal canal than a menstrual cup. It is designed to fit at the base of the cervix, where it creates a seal to prevent leaks. The menstrual disc comes is a range of sizes, shapes and materials to fit the comfort of the patient. Menstrual cups sit lower in the vaginal canal than menstrual discs and are usually designed to create a suction. Nonlimiting examples of current commercially available menstrual disc products include, but are not limited to, those made by nixit™, Cora™, Saalt™, Lena™, Lumma™, Shordy™ and Diva™. A tampon is a well-known device that is a pocket-sized absorbent material that can optionally have a cardboard or plastic applicator that is inserted into the body normally to collect period fluid. It is normally removed every few hours up to once a day, for example every 4-6 hours. It is sold in many different varieties by many different vendors. Non-limiting birth control devices include, but are not limited to a diaphragm, a cervical cap, and a sponge. The diaphragm and cervical cap devices often need a doctor’s prescription as part of gynecological healthcare for birth control, while sponges do not. A diaphragm is a shallow bendable cup that is placed in the vagina typically as a means for contraception. It covers the cervix during sex to prevent pregnancy. It is typically made of a soft silicone and is used in combination with a spermicide. Nonlimiting examples of current commercially available diaphragm products include, but are not limited to, those made by Caya™, Milex™ and Cooper Surgical™. In an alternative embodiment, the separate vaginal applicator and diaphragm can be combined into one device by including a contoured surface in the diaphragm that the vaginal tablet can be placed in. A cervical cap is smaller than a diaphragm and can be left in place longer. It covers the cervix and should be covered with a spermicide. A nonlimiting example of a current commercially available cervical cap product is the Femcap™. Sponges can also be used according to this invention. They are typically made of polyurethane or natural sea sponges. It is soft, spongy and usually round. It is placed against the cervix and typically contains applied spermicide (when used as a birth control device but not in the present invention). Often it has a loop attached for easy displacement and removal. Nonlimiting examples of products include, but are not limited to, those made by Safe-T™ and Today Sponge™. In certain embodiments, a female diagnosed with HPV or cervical or vaginal squamous intraepithelial lesions (SIL, either low-grade and high-grade lesions) is treated with a PMEG prodrug, for example, ABI-2280 vaginal tablet in a dose strength of 0.1 mg to 1 mg, for example 0.1, 0.2, 0.3, 0.4 or 0.5 mg, once daily on a schedule of 2, 3 or 4 times a week optionally for 1, 2, 3, 4, 5 or 6 weeks with the use of a retaining device to adequately protect surrounding tissue. The dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form. Short-term treatment with the vaginal tablet that includes Compound I (or a pharmaceutically acceptable salt thereof), Compound II or Compound III is intended to exert two effects on cervical or vaginal lesions caused by HPV. First, the selected therapeutic has an anti-viral effects in HPV-infected cells by inhibiting viral genome replication. Second, it selectively and effectively induces apoptosis in hyperproliferating lesional cells, while the retaining device protects the surrounding normal tissue. The therapeutic effects of Compound I (or a pharmaceutically acceptable salt thereof), Compound II or Compound III (and other compounds of the same class) are mediated by inhibition of cellular DNA polymerases. Inhibition of HPV DNA replication in productively infected cells results in elimination of the replicating episomal viral DNA accompanied by arrest of supra basal cell DNA synthesis and attendant cellular apoptosis. Cells in high-grade lesions that are rapidly proliferating and deficient in normal cell cycle regulatory mechanisms should be highly sensitive to the pro- apoptotic effects of these therapeutic compounds and salts thereof that result from DNA synthesis blockage. Normal cells can typically withstand this temporary insult with well characterized checkpoints and DNA regulatory mechanisms, and the use of the retaining device further assures protection. Compound I (or a pharmaceutically acceptable salt thereof), Compound II or Compound III is therefore expected to eliminate HPV infection in productively infected cells typical of low-grade lesions and to induce apoptosis in advanced lesions, which often have integrated HPV genomes and express elevated levels of HPV oncoproteins. Compound I (ethyl (((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)- (benzyloxy)phosphoryl)-L-alaninate) has two chiral centers, one at the phosphorus atom and one in the amino acid moiety, either of which can be in the R or S stereoconfiguration. Therefore, Compound I exists as four stereoisomers, or two diastereomeric pairs: (RP, SC)/(SP, SC) and (RP, RC)/(SP, RC). While U.S. Patent Nos. 9,801,884 and 11,344,555 describe Compound I generally, the patents do not address the potential stereochemistry of the phosphorus atom. It has been discovered that the stereoisomer of Compound I with R-stereochemistry at the phosphorus and S- stereochemistry at the amino acid carbon has advantageous properties over the other three stereoisomers.
In a non-limiting embodiment, the advantageous salt (for example fumarate) of Compound I is used as a mixture of (R,S) and (S,S) diastereomers, wherein the first R/S designates the stereochemistry at the phosphorus atom and the second S is the stereochemistry of the carbon in the amino acid moiety (corresponding to the L-alanine residue having S-configuration). While any ratio of the diastereomers can be used that provides the desired results, the (R,S) diastereomer stands out. In other embodiments, the ratio is approximately 1:1 of the R to S enantiomer at the phosphorus atom. In aspects, the compound is enantiomerically enriched with the R chirality at the phosphorus atom, wherein the amount of R by weight is for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or more. The S-stereoconfiguration at the chiral carbon corresponding to the natural amino acid configuration is advantageous in the present invention. In certain aspects, the amount of S by weight is for example, greater than about 50%, or equal to or greater than about 60%, 70%,75%, 80%, or even 85% or more. In alternative embodiments, the compound is used with R- stereoconfiguration at the chiral carbon and wherein the R-stereoconfiguration is greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or more. A pharmaceutically acceptable salt of the enantiomerically pure (Rp,Sc, or simply “R,S”) version of Compound I is a principal embodiment in the disclosed therapeutic use
Figure imgf000013_0001
device therapy as disclosed herein. An enantiomerically pure Compound II is at least 90% free of the opposite enantiomer. Surprisingly, the free base parent compound is an oil, not a solid, and thus would not have been selected as the active ingredient for the topical formulation. This is especially true because the racemic mixture or enantiomerically enriched R,S with S,S as a free base is a solid. However, when formed as the fumarate salt, the R,S enantiomerically pure Compound I becomes a highly crystalline material and the most advantageous for intraepithelial topical administration. Thus, the monofumarate salt of Compound I exhibits unexpected stability, processability, and thus has therapeutic advantages over the free base Compound I. Compound II as referred to herein and illustrated below is the enantiomerically enriched or pure embodiment that has predominately R-stereochemistry at the phosphorus atom and S- stereochemistry at the amino acid carbon atom. In an enantiomerically pure form, Compound II exhibits superior stability properties over its stereoisomer, ethyl ((S)-((2-(2-amino-6-methoxy-9H- purin-9-yl)ethoxy)methyl)(benzyloxy) phosphoryl)-L-alaninate monofumarate (Compound III). This is important for the success of the topical application to the cervix or vagina.
Figure imgf000014_0001
Other advantageous salts of Compound I that have been discovered include the hemifumarate salts ethyl ((R)-((2-(2-amino-6-methoxy-9H-purin-9- yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alaninate hemifumarate (Compound IV) and ethyl ((S)-((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alaninate hemifumarate (Compound V).
In aspects of the invention, active compounds or pharmaceutically acceptable salts thereof are used in a morphic form described in detail below. There are many strains of HPV, some of which are strongly associated with the development of cancer and are known as high-risk strains. Compound I fumarate or Compound II can be used to treat the high-risk types of HPV, including HPV-16 and HPV-18. The present invention in certain aspects provides Compound II and in particular the isolated morphic form Compound II Pattern 1, pharmaceutical compositions containing such compound, methods of treating an HPV infection or intraepithelial neoplasia related to HPV infection using the selected morphic form described herein, and methods of preparing such compound and morphic form which is used in the therapeutic delivery with retaining device invention. In particular, it has been surprisingly discovered that the monofumarate salt of ethyl ((R)- ((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alaninate (Compound II) has very high tissue penetration when administered topically to the target tissue. Topical administration avoids toxicity associated with systemic administration of the drug. Because precancerous and/or cancerous cells that are infected with HPV are several layers into the epithelium, the compound must have high penetration into the tissue to reach and treat these affected cells. Compound I monofumarate has superior tissue permeation and penetration in both porcine and human vaginal tissues over ABI-1968, which failed in clinical trials despite also being a phosphonate ester of an acyclic purine nucleoside. Compound I monofumarate can reach concentrations of about 40-85 ng/mL in vaginal tissue at a 0.1% dose. ABI-1968, even when used in a 3% dose does not reach 15 ng/mL concentration (See FIG. 2). The significant improvement in tissue penetration, especially considering the decrease in dose, could not have been predicted in advance. Compound I monofumarate is surprisingly stable (Example 23) in comparison to other salt forms of the compound. As shown in Table 17, Compound I monofumarate melts at 100°C, whereas the next highest melting salt, the sulfate, has a 15% lower melting point of 85°C. A higher melting point generally correlates with higher stability. Careful selection of each aspect of the invention in combination with the use of a retaining device to protect surrounding tissue was key to achieving the desired results. One important aspect of the invention is the selection of the formulation, with detailed descriptions provided herein. Nonlimiting advantageous solid dosage forms includes a vaginal tablet, which can be inserted into the affected area. Alternative topical formulations as used herein include semisolid dosage forms such as gels, creams or ointments which are stable enough to have integrity at body temperature to deliver the active agent in the target area in a time period that allows deep tissue penetration. It has been discovered that Compound I or its pharmaceutically acceptable salt, or other solid form or Compound II or Compound III can be prepared in a solid dosage form for topical administration. In some embodiments, the tablet formulation provides similar tissue penetration of the gel formulation (55-85 ng/mg for gel and 44-79 ng/mg for the tablet, FIG.6). In exemplary non-limiting embodiments, a method for the treatment of HPV-induced intraepithelial neoplasia is provided that includes administering an effective amount of one or a combination of the active compounds as described herein in a topical formulation that is sufficient to treat the neoplasia in combination with the use of a retaining device as described further herein. In certain embodiments, a formulation to use in the vaginal tablet (or acceptable gel, cream or other semisolid formulation with sufficient integrity) for the treatment of intraepithelial neoplasia is a dosage form containing from about 0.01 milligram to about 0.03 milligram, from about 0.03 milligrams to about 0.25 milligrams, from about 0.20 milligrams to about 0.5 milligrams, from about 0.4 milligrams to about 1 milligram, from about 0.75 milligram to about 3 milligrams, from about 1 milligram to about 10 milligrams, from about or 5 milligrams to about 20 milligrams. In certain embodiments, a formulation for the treatment of intraepithelial neoplasia is a dosage form contains about or at least 0.03, 0.05, 0.1 mg, 0.3 mg.0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45mg or 50 mg of Compound I monofumarate, Compound II or Compound III. The dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form. A particular dosage is 0.05 mg, 0.1 mg, 0.2 mg, or 0.3 mg of Compound I monofumarate, Compound II or Compound III. In certain embodiments, the 0.05 mg, 0.1 mg, 0.2 mg, or 0.3 mg dosage of Compound I monofumarate, Compound II or Compound III is administered once, twice, or three times per week as needed. In certain embodiments the 0.1-1.0 mg dosage, such as 0.1 to 0.3 dosage of Compound I monofumarate, Compound II or Compound III is administered for the prescribed time instructed by the healthcare provider, including daily dosing, in combination with the retaining device. In certain embodiments, the compound can be administered in one or more therapeutic cycles comprising a treatment cycle and a rest cycle, wherein the treatment cycle comprises administering the compound as described herein, followed by a rest cycle (comprising a period of no treatment) before the next treatment cycle. In certain embodiments, the rest cycle is from about one day to about six months. In certain embodiments the rest cycle is one, two, three, four, five, six, seven, eight or more weeks before the next treatment cycle. In certain embodiments, multiple therapeutic cycles are administered, for example one, two, three, four, five, or six therapeutic cycles. Dosage forms which do not adhere well to the target site may be dislodged, interfering with treatment. The present invention provides dosage forms that adhere to the target site and dissolve rapidly in low fluid volumes. Adhesion to the target site also minimizes exposure to non-target tissues, which may limit toxicity, unwanted systemic exposure, and side effects, especially when used in combination with the retaining device. Dosage forms which soften, break down, and/or disintegrate quickly in low fluid volumes are advantageous to cause a rapid release of the active compound to the target tissue. Dosage forms that disintegrate in, for example, less than about 50 µL, less than about 100 µL, less than about 125 µL, less than about 150 µL, less than about 175 µL, less than about 200 µL, less than about 250 µL, less than about 500 μL, less than about 1 mL, or less than about 2 mL fluid facilitate drug penetration into the target site. In certain embodiments, the dosage form is a vaginal tablet. In certain embodiments, the dosage form is a semisolid such as a gel or cream. In certain embodiments, the dosage form disintegrates in about one to about ten seconds. In certain embodiments, the dosage form disintegrates in about ten seconds to one minute, in certain embodiments, the dosage form disintegrates in about one minute to about one hour. In certain embodiments, the dosage form disintegrates in one to six hours. The physical dimensions of the dosage form can impact the effectiveness of the dosage form. A tablet that is thinner provides a greater surface area to volume ratio and may degrade quicker and cover the target area better. In certain embodiments the dosage form is less than 3 millimeters thick in its smallest dimension. The formulation of the dosage form is very important for adequate administration of the active agent into the intraepithelial tissue. Tablet formulation should display the properties of mucoadhesion and substantivity and include excipients that have solubilizing, erosion-generating (for disintegration), porosity (for water uptake) and viscosity enhancing (to keep the drug at the target site) properties. Examples of excipients that will cause rapid disintegration of a solid dosage form to cover the cervix or vaginal areas include, but are not limited to mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid or fumaric acid. Examples of excipients that can enhance disintegration and coverage of the affected area include but are not limited to sodium starch glycollate, pregelatinized starch, crospovidone and croscarmellose sodium. Mucoadhesive excipients that are useful in the present invention include but are not limited to microcrystalline cellulose, polycarbophil, hydroxymethyl cellulose, hypromellose, hydroxypropyl cellulose, and PVP. A nonlimiting example of a tablet formulation includes, but is not limited to, microcrystalline cellulose, crospovidone, magnesium stearate, silicon dioxide, polyethylene oxide and mannitol. Another non-limiting example of a tablet formulation has microcrystalline cellulose, magnesium stearate and mannitol. Semi solid dosage forms may include, for example, a mucoadhesive polymer, a solubility/penetration enhancer, a lipophilic solubilizer and a penetration enhancer. The mucoadhesive polymer, for example, may be, but is not limited to, a carbomer, polyethylene glycol, crospovidone, hypromellose, polycarbophil and/or hydroxyethyl cellulose. The solubility/penetration enhancer can be, for example, but not limited to, a mixture of polyoxyl 6 stearate Type I, ethylene glycol stearate and polyoxy 32 stearate Type I, cetyl alcohol, stearyl alcohol, polysorbate 80, sodium lauryl sulphate, mono and di-glycerides, sorbitan monostearate, glyceryl isostearate, polyoxy 15 hydroxystearate, poly15 hydroxystearate, polyoxy 40 hydrogenated castor oil, octyl dodecanol, and/or soybean lecithin. Lipophilic solubilizers include, but are not limited to light mineral oil, mineral oil, white wax and silicone fluid. Penetration enhancers include but are not limited to propylene glycol, transcutol, oleic acid, isopropyl myristate, propylene glycol glycerol monooleate, propylene glycol monocaprylate, PEG-8 Bees wax, cetyl alcohol, stearic acid, cetyl palmitate and/or cetostearyl alcohol. A non-limiting example of a semi-solid formulation includes, for example, one or more of a carbomer, propylene glycol, sorbic acid, EDTA and water. Another non-limiting example of a semi solid formulation includes one or more of a carbomer, mineral oil, a mixture of polyoxy 6 stearate Type I, ethylene glycol stearate, polyoxy 32 stearate Type I, parabens, propylene glycol, EDTA and/or water. Films can be produced, for example, with, but not limited to, hypromellose, polyethylene glycol, polymethacrylates, microcrystalline cellulose, xanthan gum, guar gum and/or polyvinylpyrrolidone. A pessary (vaginal suppository) can be formulated with, for example but not limited to, hard fat (such as Ovucire, Witepsol, Supposi-Base), polyethylene glycol, macrogols, cocoa butter and glycerol. A non-limiting example of a pessary can be made from Witepsol H 15 or Ovucire WL 3264. Therefore, the present invention includes at least the following features: (i) A method to treat a cervical or vaginal HPV-induced infection or an associated condition in a female in need thereof, including but not limited to cervical or vaginal intraepithelial neoplasia, comprising administering to a host in need thereof an effective amount of Compound I or a pharmaceutically acceptable salt thereof such as the monofumarate or hemifumarate, or other solid form, or Compound II or Compound III, in a pharmaceutical composition, in a vaginal tablet or semisolid formulation with a vaginal applicator in combination with the use of a retaining device for sufficient time to collect the post-treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non- diseased tissue. (ii) A method to treat a cervical or vaginal HPV-induced infection, intraepithelial neoplasia, or an associated condition in a human in need thereof, the method comprising (i) administering an effective amount of the anti-HPV therapeutic agent Compound I or a pharmaceutically acceptable salt thereof, such as the monofumarate or hemifumarate, or other solid form or Compound II or Compound III typically in tablet form, to the cervix using a vaginal applicator (which is optionally coated with a lubricant) and then (ii) inserting a retaining device (which is optionally coated with a lubricant) that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post-treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue. (iii) The method of (i) or (ii), wherein the retaining device is selected from a menstrual cup, a menstrual disc, a tampon, diaphragm, cervical cap and a sponge. (iv) The method of (i) or (ii) wherein the retaining device is used as both the applicator and a retaining means in a manner that minimizes toxicity-causing damage to non-diseased tissue. (v) The method of (i) or (ii) wherein the retaining device is a menstrual cup. (vi) The method of (i) or (ii) wherein the retaining device is a menstrual disc. (vii) The method of (i) or (ii) wherein the retaining device is a tampon. (viii) The method of (i), (ii), or (iv) wherein the retaining device is a diaphragm. (ix) The method of (i) or (ii) wherein the retaining device is a cervical cap. (x) The method of (i) or (ii) wherein the retaining device is a sponge. (xi) The method of (v)-(x) wherein the retaining device used as both an applicator and a retaining means. (xii) The method of (i)- (xi) that provides a dosage form containing of from 0.1 mg to 30 mg, from 0.05 to 0.3 mg, from 0.5 mg to 20 mg, from 1 mg to 20 mg, from 1 mg to 15 mg, from 1 mg to 10 mg of a compound of embodiments (i)-(v) and in certain embodiments about or at least 0.03, 0.05, 0.1 mg, 0.3 mg.0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45mg or 50 mg of the active agent. (xiii) The method of (i)-(xi) that provides a dosage form containing from about 0.05 mg to about 1.0 mg, such as 0.1 to 0.5 mg, for example 0.2, 0.3 or 0.4 mg of a compound of embodiments (i)-(v). (xiv) The method of (i)-(xiii) wherein the retaining device is held in place for between 4- 12 hours, 5-10 hours or 6-9 hours and then removed. (xv) The method of (i)-(xiv) wherein the topical formulation with retaining device is administered once a day for one, two, three or four days a week for as long as necessary to achieve the desired results. (xvi) The method of (i)-(xiv) wherein the topical formulation with retaining device is administered three time a week for as long as necessary to achieve the desired results. (xvii) The method of (xv)-(xvi) wherein the topical formulation with retaining device is administered for 2, 3, 4, 5, or 6 weeks. (xviii) The method of (i)-(xvii), wherein the vaginal applicator is first lubricated with a water-based gel such as for example, a glycerol based lubricant, hydroxyethylcellulose-based lubricant, KY jelly or Surgilube. (xix) The method of (i)-(xiii) wherein a water-based gel is inserted into the vaginal area up to the cervix prior to vaginal tablet administration. (xx) The method of (i)-(xix) wherein a lubricant is coated on the retaining device for ease of placement as well as to help maintain the device in position before inserting. (xxi) The method of (i)-(xx) wherein the vaginal applicator and the retaining device are combined into one device for both administration of the vaginal tablet and retaining the post- treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue (xxii) The method of (xxii), wherein the lubricant is selected from water, a glycerol based lubricant and a hydroxyethylcellulose-based lubricant. (xxiii) The methods of any of the above, wherein the active compound is delivered in a morphic form as described herein. (xxiv) A kit that includes a dosage form with a therapeutic compound as described herein and a retaining device. (xxv) The kit of (xxiv) that also includes a vaginal applicator. (xxvi) The kit of (xxiv) or (xxv), wherein the dosage form is a vaginal tablet. (xxvii) The kit of (xxiv) wherein the retaining device is selected from a menstrual cup, a menstrual disc, a tampon, diaphragm, cervical cap and a sponge. (xxviii) The kit of (xxv), wherein the retaining device is used as both the vaginal applicator and a retaining means. BRIEF DESCRIPTION OF THE FIGURES FIG.1A and FIG 1B depict the molecular structure of Compound I monofumarate pattern 1 as determined by the single crystal X-ray diffraction analysis of Example 21. There exists an intermolecular interaction between protonated N5-atom of free base and O7-atom of fumaric acid anion (N(5)–H(5)···O(7)) in the single-crystal form of Compound II. FIG.2 is an in vitro tissue permeation test in vaginal tissue comparing ABI-2280 fumarate salt to ABI-1968. Bar A shows the tissue penetration of a 0.1% ABI-2280 gel in porcine vaginal tissue. Bars B and C show the tissue penetration of a 0.1% ABI-2280 gel in human cervical tissue. Bar D shows the tissue penetration of a 1% formulation of ABI-1968 in 6% NMP into porcine vaginal tissue. Bar E shows the tissue penetration of a 1% nanosuspension of ABI-1968 in porcine vaginal tissue. Bar F shows the tissue penetration of a 3% formulation of ABI-1968 in 6% NMP into porcine vaginal tissue. Bar G shows the tissue penetration of a 3% formulation of ABI-1968 in 20% NMP into porcine vaginal tissue. ABI-1968 penetrates the tissue to a substantially smaller degree, which hinders the ability of the compound to reach the cells which are infected with HPV. This may be a contributing factor to the performance of ABI-1968 in clinical studies. Surprisingly, ABI-2280 displays high tissue penetration in both porcine and human tissues. High tissue penetration may lead to high activity against HPV. This is described in Example 15. FIG. 3 shows a flow diagram for the process of preparing a topical cream formulation described in Example 6. FIG. 4 shows a flow diagram for the process of preparing a topical gel formulation described in Example 6. FIG. 5 shows a flow diagram for the process of preparing a tablet formulation described in Example 7. FIG.6 is a bar graph comparing the tissue penetration of a topical gel and a topical tablet dosage form as described in Example 15. The tablet dosage form produces similar tissue penetration to the topical gel, with an average of 58 ng/mg of compound in the tissue. FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, and 7J depict representative administration procedures to insert a vaginal tablet as described in Example 20. FIG.8 depicts the structure of Compound I monofumarate. The synthesis of Compound I and stereoisomers and pharmaceutically acceptable salts thereof is provided in Examples 1-5. FIG. 9 is a comparison of XRPD diffractograms of Compound I hemifumarate Pattern 1 and Compound I monofumarate Pattern 1 (small scale preparation) obtained in Example 23. FIG.10 is a DSC thermogram of Compound I hemifumarate Pattern 1 obtained in Example 23. FIG.11 is a TGA thermogram of Compound I hemifumarate Pattern 1 obtained in Example 23. FIG. 12A is a DSC thermogram of Compound I monofumarate Pattern 1 (small scale preparation, Example 23), recorder at heating rate of 10°C/min. FIG. 12B is a DSC thermogram of Compound I mono-fumarate Pattern 1 (small scale preparation, Example 23), recorder at heating rate of 2°C/min. FIG. 12C is a DSC cycle of Compound I mono-fumarate Pattern 1 (DSC cycle, 0-150ºC, 150-0ºC, 0-250ºC, 10ºC/min) for the small-scale preparation sample (Example 23). FIG.13 is TGA thermogram of Compound I mono-fumarate Pattern 1 for the small-scale preparation sample (Example 23). FIG.14 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 obtained in stability test experiments at 25°C/84%RH (2 days, open container), 25°C/92%RH (1 week, open container), 40°C/75%RH (1 week, open container), and 60°C (1 week, tight container) as described in Example 24 with original sample of Compound I monofumarate Pattern 1 before the test. FIG. 15 is a Dynamic Vapor Sorption (DVS) plot and DVS change in mass plot for Compound I Pattern 1(Example 26). FIG.16 is a comparison of XRPD diffractograms of Compound I Pattern 1 before and after DVS study (Example 26). FIG. 17 is a Dynamic Vapor Sorption (DVS) plot and DVS change in mass plot for Compound I hemifumarate Pattern 1 (Example 26). FIG.18 is a comparison of XRPD diffractograms of Compound I hemifumarate Pattern 1 before and after DVS study (Example 26). FIG. 19 is a Dynamic Vapor Sorption (DVS) plot and DVS change in mass plot for Compound I monofumarate Pattern 1 (Example 26). FIG.20 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 before and after DVS study (Example 26). FIG.21A is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 obtained in Example 27 (large scale) before and after heating to 106°C. FIG. 21B is a DSC thermogram of Compound I monofumarate Pattern 1 (Example 27), recorded at heating rate of 10°C/min. FIG. 21C is a DSC thermogram of Compound I monofumarate Pattern 1 (large scale preparation, Example 27), recorded at heating rate of 2°C/min. FIG. 21D is a DSC thermogram of Compound I monofumarate Pattern 1 (obtained in Example 27). FIG.22 is TGA thermogram of Compound I monofumarate Pattern 1 (obtained in Example 27). FIG.23 is a comparison of XRPD diffractograms of Compound I mono-fumarate Pattern 1, hemi-fumarate Pattern 2, hemi-fumarate Pattern 3, Pattern 4 obtained from equilibration experiment in water at 25°C for 2 weeks and fumaric acid pattern (obtained in Example 28). FIG.24 is a comparison of XRPD diffractograms of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 2 and 3 weeks (obtained in Example 28). FIG. 25 is a DSC thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 2 weeks (obtained in Example 28). FIG. 26 is a DSC thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 3 weeks (obtained in Example 28). FIG. 27 is a TGA thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 2 weeks (obtained in Example 28). FIG. 28 is a TGA thermogram of Compound I Pattern 4 obtained from equilibration experiment in water at 25°C for 3 weeks (obtained in Example 28). FIG.29 is a comparison of XRPD diffractograms of fumaric acid pattern and Compound I hemifumarate Pattern C obtained in equilibration experiment EQ2 (in acetonitrile) and EQ15 (in 2.9:97.1 v/v water/ acetonitrile) at 25°C for 3 weeks (obtained in Example 28). FIG. 30 is a DSC thermogram of Compound I hemi-fumarate Pattern C obtained from equilibration experiment in acetonitrile at 25°C for 2 weeks (obtained in Example 28). FIG. 31 is a TGA thermogram of Compound I hemi-fumarate Pattern C obtained from equilibration experiment in acetonitrile at 25°C for 2 weeks (obtained in Example 28). FIG.32 is a comparison of XRPD diffractograms of Compound I hemi-fumarate Pattern 2 obtained in equilibration experiments EQ3 (in methyl ethyl ketone), EQ4 (in acetone), and EQ7 (in 1:1 v/v acetone/heptane) at 25°C for 3 weeks (obtained in Example 28). FIG. 33 is a DSC thermogram of Compound I hemi-fumarate Pattern 2 obtained in equilibration experiment in methyl ethyl ketone at 25°C for 2 weeks (obtained in Example 28). FIG. 34 is a TGA thermogram of Compound I hemi-fumarate Pattern 3 obtained in equilibration experiment in methyl ethyl ketone at 25°C for 2 weeks (obtained in Example 28). FIG. 35 is a comparison of XRPD diffractograms of fumaric acid and a mixture of Compound I monofumarate Pattern 1 and unknown pattern obtained in equilibration experiments EQ5 (in isopropanol), EQ8 (in 1:1 v/v isopropanol /heptane), EQ9 (in 1:1 v/v isopropanol/toluene), EQ10 (in 1:3 v/v isopropanol/methyl tertbutyl ether), and EQ12 (in 1:3 v/v ethanol/heptanes) at 25°C for 3 weeks (obtained in Example 28). FIG. 36 is a DSC thermogram of a mixture of Compound I mono-fumarate Pattern 1 and unknown pattern obtained in equilibration experiment EQ5 in isopropanol at 25°C for 2 weeks (obtained in Example 28). FIG.37 is a TGA thermogram of a mixture of Compound I mono-fumarate Pattern 1 and unknown pattern obtained in equilibration experiment EQ5 in isopropanol at 25°C for 2 weeks (obtained in Example 28). FIG.38 is a comparison of XRPD diffractograms of Compound I hemi-fumarate Pattern 5 obtained in equilibration experiment EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 2 and 3 weeks (obtained in Example 28). FIG. 39 is a DSC thermogram of Compound I hemi-fumarate Pattern 5 obtained in equilibration experiment EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 2 weeks (obtained in Example 28). FIG. 40 is a DSC thermogram of Compound I hemi-fumarate Pattern 5 obtained in equilibration experiment EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 3 weeks (obtained in Example 28). FIG. 41 is a TGA thermogram of Compound I hemi-fumarate Pattern 5 obtained in equilibration experiment EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 2 weeks (obtained in Example 28). FIG. 42 is a TGA thermogram of Compound I hemi-fumarate Pattern 5 obtained in equilibration experiment EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 3 weeks (obtained in Example 28). FIG.43 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 obtained in equilibration experiments (procedure in Example 28) EQ11 (in 1:3 v/v tetrahydrofuran/heptane), EQ13 (in 1:3 v/v ethyl acetate/toluene), EQ14 (in 1:3 v/v ethanol/toluene) at 25°C for 2 weeks and Compound I monofumarate Pattern 1 (material obtained in Example 23). FIG.44 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 obtained in equilibration experiments EQ11 (in 1:3 v/v tetrahydrofuran/heptane), EQ13 (in 1:3 v/v EA/toluene), EQ14 (in 1:3 v/v ethanol/toluene) at 25°C for 3 weeks (obtained in Example 28). FIG. 45 is a comparison of XRPD diffractograms of a mixture of Compound I monofumarate Pattern 1 and unknown pattern obtained in equilibration experiments EQ16 (in 1:4 v/v isopropanol/heptane), EQ5 (in isopropanol) (obtained in Example 28) at 25°C for 2 weeks and Compound I monofumarate Pattern 1 obtained in Example 23. FIG. 46 is a comparison of XRPD diffractograms of reference fumaric acid pattern, Compound I monofumarate Pattern 1 (Example 23), Compound I monofumarate Pattern 2 (obtained by precipitation from acetone solution with heptane antisolvent in Experiment AS1, Example 28), Compound I monofumarate Pattern 2 (obtained by precipitation from methyl ethyl ketone solution with heptane antisolvent in Experiment AS4, Example 28), and fumaric acid pattern obtained in Experiment AS2, Example 28. FIG.47 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 (Example 23), Compound I monofumarate Pattern 2 (obtained by precipitation from ethanol solution with heptanes antisolvent in Experiment AS6, Example 28) and Compound I monofumarate Pattern 2 (obtained by precipitation from tetrahydrofuran solution with heptane antisolvent in Experiment AS7, Example 28). FIG.48 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 (Example 23) and Compound I monofumarate Pattern 1 obtained by crystallization at room temperature by slow evaporation from acetone, methylethylketone and ethyl acetate as described in Example 28, Table 41. FIG.49 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 (Example 23) and Compound I monofumarate Pattern 1 obtained by crystallization at room temperature by slow evaporation from methanol, ethanol, isopropanol and tetrahydrofuran as described in Example 28, Table 41. FIG. 50 is a comparison of XRPD diffractograms of reference fumaric acid pattern, Compound I monofumarate Pattern 1 (Example 23) and Compound I monofumarate Pattern 2 obtained by crystallization from hot methyl ethyl ketone saturated solution by slow cooling, Compound I monofumarate Pattern 2 obtained by crystallization from acetone hot saturated solution by slow cooling and Compound I monofumarate Pattern 3 obtained by crystallization from acetonitrile hot saturated solution by slow cooling. FIG.51 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 (Example 23), Compound I monofumarate Pattern 1 obtained by crystallization from hot water saturated solution by slow cooling, and Compound I monofumarate Pattern 1 obtained by crystallization from hot saturated solution in ethanol/toluene (1/1 v/v) by slow cooling (Example 28, Table 42). FIG.52 is a comparison of XRPD diffractograms of Compound I monofumarate Pattern 1 (Example 23), Compound I monofumarate Pattern 2 obtained by crystallization from hot acetone saturated solution by fast cooling, and Compound I monofumarate Pattern 2 obtained by crystallization from hot saturated solution in methyl ethyl ketone by fast cooling (Example 28, Table 43). FIG. 53 is a comparison of XRPD diffractograms of reference fumaric acid pattern, Compound I monofumarate Pattern 1 (Example 23), Compound I monofumarate Pattern 1 obtained by crystallization from hot water saturated solution by fast cooling, Compound I monofumarate Pattern 3 obtained by crystallization from hot saturated solution in acetonitrile by fast cooling, and Compound I monofumarate Pattern 1 obtained by crystallization from hot saturated solution in ethanol/toluene (1/1 v/v) by fast cooling (Example 28). FIG. 54 is a heat-cool-heat DSC thermogram of Compound I monofumarate Pattern 1 (heating to 106°C) (Example 28, Table 44). FIG. 55 is a heat-cool-heat DSC thermogram of Compound I monofumarate Pattern 1 (heating to 130°C) (Example 28, Table 44). FIG.56 is an XRPD diffractogram of Compound II Pattern 1 (Example 29). FIG.57 is a DSC thermogram of Compound II Pattern 1 (Example 29). FIG.58 is a TGA thermogram of Compound II Pattern 1 (Example 29). FIG.59 is an XRPD diffractogram of Compound III Pattern 1 (Example 30). FIG.60 is a DSC thermogram of Compound III Pattern 1 (Example 30). FIG.61 is a TGA thermogram of Compound III Pattern 1 (Example 30). FIG.62 is an XRPD diffractogram of Compound III Pattern 2 (Example 31). FIG.63 is a DSC thermogram of Compound III Pattern 2 (Example 31). FIG.64 is a TGA thermogram of Compound III Pattern 2 (Example 31). DETAILED DESCRIPTION OF THE INVENTION It has been discovered that an effective composition for the treatment of cervical or vaginal HPV infection and cervical or vaginal intraepithelial neoplasia, requires the combination of the selection of a number of aspects that work together to achieve the desired results. It was essential to select the right compound with advantageous lipophilic and tissue penetrating properties combined with a selected pharmaceutically acceptable salt optionally in an advantageous morphic form to achieve the long-sought ability to penetrate the deep epithelial stratified tissues in an effective amount to deliver the active agent. It required years of research to solve this problem, after many failures, to the benefit of patients globally suffering from interepithelial neoplasia that may become cancerous. In addition, an aspect of successful treatment is the use of a retaining device to minimize potential adverse effects on surrounding tissue that can be uncomfortable or even painful for the patient and possibly damaging to the tissue. Thus the invention includes a device system and advantageous therapeutic compound administration to effectively treat HPV cervical or vaginal HPV infection or cervical or vaginal intraepithelial neoplasia. Specifically, it has been discovered that a particularly advantageous treatment for cervical or vaginal HPV infection or cervical or vaginal intraepithelial neoplasia is achieved by the administration of an effective amount of the PMEG prodrug Compound I or a pharmaceutically acceptable salt thereof, such as the hemifumarate or monofumarate salts, including in particular Compound II or Compound III, which may be in a morphic form as described further herein, by steps comprising (i) administering an effective amount of the anti-HPV therapeutic agent Compound I (or a pharmaceutically acceptable salt thereof) or Compound II or III, typically in solid tablet form, to the cervix using a vaginal applicator and then (ii) inserting a retaining device that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post-treatment leakage of vaginal fluids and the remaining therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue. In some embodiments, the retaining device is held in place for 3-10 hours, for example 4-8 hours or 5-7 hours or about or at least around 6 hours. The retaining device is then removed. The retaining device is used after every topical application of the anti-HPV therapeutic agent to prevent undesired PMEG toxicity to surrounding tissue or cells. The retaining device can be, in non- limiting embodiments, a retaining period device, a birth control device, or other type of device that adequately separates the cervix from the vagina, or part of the vagina. Alternatively, it can be any device that forms an adequate barrier between the inserted tablet and surrounding vaginal tissue for a time period of at least 6 hours, and more typically, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, the separate vaginal applicator and retaining device can be combined into one device that can be used to both insert the vaginal tablet and act as a retaining device to protect surrounding tissue. In principal embodiments, the combined device and applicator is similar in structure to a diaphragm with a contoured surface that the vaginal tablet can be placed in for administration, and the tablet can be held in place with a lubricating fluid or other appropriate means. In an alternative embodiment, instead of using a vaginal tablet, a gel or cream formulation with the active therapeutic, as described further below, is administered and then used in combination with the retaining device. While the vaginal tablet is preferred for its solid structure and physical properties, a gel or cream with enough rigidity to deliver the active therapeutic and not unduly escape to surrounding tissue would work. In certain embodiments, the vaginal applicator is first lubricated with a water-based gel such as for example, a glycerol-based lubricant, hydroxyethylcellulose-based lubricant, KY jelly or Surgilube to help maintain the vaginal tablet in place during application and for patient comfort. The lubricant may help the vaginal tablet remain in place for effective disintegration. Alternatively or in addition, a water-based gel can also be inserted into the vaginal area up to the cervix prior to vaginal tablet administration. In addition or alternatively, the lubricant can be coated on the retaining device for ease of placement as well as to help maintain the device in position. Specifically, it was discovered that the key compound for delivery of the active agent is a specific salt of: . Compound I is (ethyl(((2-(2-amino-6-methoxy-9H-purin-9- yl)ethoxy)methyl)(benzyloxy)-phosphoryl)-L-alaninate). U.S. Patent Nos. 9,801,884 and 11,344,555 assigned to the Regents of the University of California claim Compound I and pharmaceutically acceptable salts generally, as well as methods of using the same for treating a papillomavirus infection. Compound I is an acyclic nucleotide phosphonate that metabolizes to a known potent antiviral compound (PMEG; ((9-[2-phosphonomethyoxy)ethyl)guanine])) but has poor cellular permeability and use-limiting systemic toxicity. The assignee has discovered how to improve the prodrug to be delivered topically in a manner that it is rapidly taken up into epithelial cells, a challenging task to date and one that ABI-1968 failed. Compound I (ethyl (((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)- (benzyloxy)phosphoryl)-L-alaninate) has two chiral centers, one at the phosphorus atom and one in the amino acid moiety, either of which can be in the R or S stereoconfiguration. Therefore, Compound I has four stereoisomers. While U.S. Patent Nos.9,801,884 and 11,344,555 describe Compound I generally, the patents do not address the potential stereochemistry of the phosphorus atom. It has been discovered that the stereoisomer of Compound I with R-stereochemistry at the phosphorus and S-stereochemistry at the amino acid carbon has advantageous properties over the other three stereoisomers, as discussed further herein. In a non-limiting embodiment, the advantageous salt (for example fumarate) of Compound I is used as a mixture of (R,S) and (S,S) diastereomers, wherein the first R/S designates the stereochemistry at the phosphorus atom and the second S is the stereochemistry of the carbon in the amino acid moiety (corresponds to the L-alanine residue having S-configuration). While any ratio of the diastereomers can be used that provides the desired results, the (R,S) diastereomer stands out. In other embodiments, the ratio is approximately 1:1 of the R to S enantiomer at the phosphorus atom. In certain aspects, the compound is enantiomerically enriched with the R enantiomer at the phosphorus atom, wherein the amount of R by weight is for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or more. The S-stereoconfiguration at the chiral carbon corresponding to the natural amino acid configuration is advantageous in the present invention. In some aspects, the amount of S by weight is for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or more. In alternative embodiments, the R-stereoconfiguration of the chiral carbon is predominant and is greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or more, In principal aspects, therefore, the invention includes the administration of an effective amount of the fumarate salt of (RP, SC) ethyl (((2-(2-amino-6-methoxy-9H-purin-9- yl)ethoxy)methyl)(benzyloxy)-phosphoryl)-L-alaninate (Compound II) as described herein, optionally in a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical salt of an acyclic nucleotide, methods, compositions, and dosage forms for the treatment of diseases associated with human papilloma virus (HPV).
Figure imgf000031_0001
The compound, compositions, and dosage forms are used in combination with a retaining device to treat conditions related to or occurring as a result of an HPV viral exposure or infection. For example, the active compound can be used to treat precancerous cervical lesions, cervical intraepithelial neoplasia, vaginal intraepithelial neoplasia, cervical cancer and vaginal cancer. The active compounds and compositions with a retaining device can be used to treat an infection caused by the range of HPV types. Most of the cancer-causing HPV types are from the alpha-7 and alpha-9 species including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. The most common cancer-causing HPV types are 16 and 18. HPV-16 and HPV-18 are reported to be the cause of 50% of cervical cancers. And 90% of venereal warts are caused by HPV-6 and HPV-11 (World Health Organization, “Cervical Cancer” https://www.who.int/news- room/fact-sheets/detail/cervical-cancer). Infection with one type of genotype does not preclude a later infection with a different genotype. In some embodiments, Compound I monofumarate, Compound II or Compound III is used to treat HPV-16 in combination with the retaining device. In one embodiment, Compound I monofumarate, Compound II or Compound III is used to treat HPV-18. In one embodiment, Compound I monofumarate, Compound II or Compound III is used to treat a high risk HPV infection. In one embodiment, Compound I monofumarate, Compound II or Compound III is used to treat HPV type 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, or 82. In some embodiments, the compound, formulations, or solid dosage forms that include the compound can also be used prophylactically to prevent or retard the progression of clinical illness in individuals who are HPV positive or who have been exposed to HPV. In particular, it has been discovered that Compound II optionally in a morphic form as described herein, exhibits superior drug-like and pharmacological properties for use in this therapeutic use with retaining device combination. Compound II has R-stereochemistry at the phosphorus atom which has been confirmed with X-ray crystallography (Example 21, see FIG.1A and FIG.1B). In alternative embodiments, Compound II can be used in the form of any desired ratio of phosphorus R- and S-enantiomers, including up to pure enantiomers. In some embodiments, Compound II is used in a form that is at least 90% free of the opposite enantiomer, and can be at least 98%, 99%, or even 100% free of the opposite enantiomer. Unless described otherwise, an enantiomerically pure Compound II is at least 90% free of the opposite enantiomer. In certain embodiments Compound II is used as a racemic mixture of isomers. In addition, in an alternative embodiment, the amino acid of the phosphonamidate can be in the D- or L-configuration, or a mixture thereof, including a racemic mixture. Where a phosphonamidate exhibits chirality, it can be provided as an R or S chiral phosphorus derivative or a mixture thereof, including an enantiomerically enriched form including a racemic mixture. All of the combinations of these stereoconfigurations are alternative embodiments in the invention described herein. In another embodiment, at least one of the hydrogen atoms of Compound I, Compound II, or Compound III can be replaced with deuterium. In certain embodiments, Compound I may be: a pharmaceutically acceptable salt thereof. ay be:
Figure imgf000033_0001
be be
. ethyl (((2-(2-amino-6-methoxy-9H-purin-9- yl)ethoxy)methyl)(benzyloxy) phosphoryl)-L-alaninate (Compound II) In certain embodiments, the active compound of the invention is Compound II, which can be provided in a pharmaceutically acceptable composition or solid dosage form thereof. In an embodiment, Compound II is an amorphous solid. In yet a further embodiment, Compound II is a crystalline solid. II. Salts of Compound I In certain embodiments, the active compound of the invention is Compound I, RP Compound I, or SP Compound I as a monofumarate salt. In certain embodiments, the active compound of the invention is Compound I, RP Compound I, or SP Compound I as a hemifumarate salt. In certain embodiments, the active compound of the invention is Compound I, RP Compound I, or SP Compound I as a sesquifumarate salt. In certain embodiments, the active compound of the invention is RP Compound I or SP Compound I as a sulfate salt. In certain embodiments, the active compound of the invention is RP Compound I or SP Compound I as a hydrochloride salt. In certain embodiments, the active compound of the invention is Compound I, RP Compound I, or SP Compound I as a benzenesulfonate salt. In certain embodiments, the active compound of the invention is RP Compound I or SP Compound I as a tosylate salt. In certain embodiments, the active compound of the invention is RP Compound I or SP Compound I as a succinate salt. or
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
SP Salts of Compound I
Figure imgf000039_0001
The hemifumarate and monosuccinate salts form solids with favorable properties for solid dosage forms including morphic forms for administration in combination with a retaining device to a host such as a human with an HPV infection or an HPV-related disease such as cervical or vaginal intraepithelial neoplasia. However, the monofumarate optionally in morphic form displays superior properties to the hemifumarate and monosuccinate. Thus, the monofumarate salt remains a desired salt form of Compound I for administration in combination with a retaining device.
III. Morphic Forms of Compound I In certain embodiments, the compound used in combination with the retaining device is an isolated morphic form of the compound of the formula: wherein the isolated pattern comprising peaks
Figure imgf000041_0001
independently selected from at least 3, 4, 5, or 6 of the following 2theta values 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the compound used in combination with the retaining device is an isolated morphic form of the compound of the formula: wherein the isolated
Figure imgf000041_0002
pattern comprising peaks independently selected from at least 3, 4, 5, 6, 7, 8 or 9 of the following 2theta values 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°.
In certain embodiments, the compound used in combination with the retaining device is an isolated morphic form of the compound of the formula: wherein the isolated pattern comprising peaks
Figure imgf000042_0001
independently selected from at least 3, 4, 5, 6, 7, 8 or 9 of the following 2theta values 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 20.38+0.2°, 22.94+0.2°, 25.09+0.2°, 26.54+0.2°, 26.90+0.2°, 27.38+0.2°, 28.28+0.2°, 28.95+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the compound used in combination with the retaining device is an isolated morphic form of the compound of the formula: wherein the isolated
Figure imgf000042_0002
pattern comprising peaks independently selected from at least 3, 4, 5, or 6 of the following 2theta values 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. Compound I monofumarate pattern 1 In certain embodiments, Compound I monofumarate pattern 1 characterized by an XRPD pattern in or substantially similar to that in FIG.9 is used in combination with the retaining device. Compound I monofumarate pattern 1 can be produced, for example, by crystallization from isopropyl alcohol and heptane, as described in Example 23. Compound I free base and about 1.0 equivalents of fumaric acid can for example be dissolved in isopropanol at a concentration from about 25% to about 40% w/v and stirred at an elevated temperature, for example about 45°C, about 50°C, about 55°C. The solution is stirred at this temperature until some solids form and is then optionally seeded with Compound I monofumarate Pattern 1 crystalline solids. The mixture is stirred and cooled to a lower temperature to facilitate crystallization, for example less than about 40°C, less than about 30°C, less than about 25°C, less than about 20°C, or less than about 15°C. The mixture is then stirred while heptane is added in an amount ranging from about 1mL per mL of isopropanol to about 5 mL per mL of isopropanol, such as about 4 mL per mL isopropanol. The resulting suspension is stirred while the product crystallizes, for example for at least about 24 hours at 25°C. Next, the suspension is cooled to facilitate crystallization further. The solution can be cooled to less than about 10°C, less than about 5°C, less than about 0°C, or less than about -5°C. The solution is stirred at the decreased temperature to allow time for additional product to crystallize, such as for at least about one day, and then the solids are collected by filtration. The collected solids are dried under reduced pressure and optionally at elevated temperature to provide Compound I monofumarate pattern 1. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least about 3, 4, or 52theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least twelve of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least eleven of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least ten of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least nine of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least eight of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least seven of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least six of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least five of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least four of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. In certain embodiments, the Compound I monofumarate pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least three of the 2theta values selected from 6.0+0.2°, 8.9+0.2°, 9.3+0.2°, 9.7+0.2°, 11.9+0.2°, 14.8+0.2°, 18.0+0.2°, 20.0+0.2°, 23.4+0.2°, 25.2+0.2°, 25.9+0.2°, 26.8+0.2°, and 28.0+0.2°. Compound II pattern 1 In certain embodiments, Compound II Pattern 1 characterized by an XRPD pattern in or substantially similar to that in FIG.56 is used in combination with the retaining device. Compound II Pattern 1 can be produced, for example, by recrystallizing Compound II (Example 28, Table 40), equilibration of Compound II in a suitable solvent, or crystallization by slow evaporation of solvent (Example 28, Table 41). In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least three, four or five 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least seven 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least six 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least five 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least four 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least three 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least two 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least one 2theta values selected from 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. In certain embodiments, the Compound II pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least three, four, or five 2theta values selected from 3.08+0.2°, 9.30+0.2°, 10.66+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 17.45+0.2°, 18.13+0.2°, 19.78+0.2°, 20.14+0.2°, 22.91+0.2°, 23.34+0.2°, 25.14+0.2°, 25.33+0.2°, 25.86+0.2°, 26.78+0.2°, 27.99+0.2°, and 28.82+0.2°. Compound III pattern 1 In certain embodiments, Compound III pattern 1 characterized by an XRPD pattern in or substantially similar to that in FIG.59 is used in combination with the retaining device. Compound III Pattern 1 can be produced, for example, by precipitation from isopropanol and heptanes (Example 30, Table 48). In certain nonlimiting embodiments, Compound III Pattern 1 was prepared by dissolving SP-Compound I free base in isopropanol, for example about 100 mg of SP-Compound I in about 0.25 mL to about 0.5 mL of isopropanol. To this solution was added about 1.0 eq of fumaric acid, and the mixture stirred at ambient or elevated temperature, for example about 25°C to about 60°C. Next, about two to about five times as much heptanes was added as isopropanol. The resulting mixture was stirred at ambient or elevated temperature, for example about 25°C to about 60°C, and then cooled gradually, for example about 0.01°C/min to 1°C/min the solids isolated by filtration and then dried at ambient or reduced pressure. In certain embodiments, the Compound III pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising independently at least 2, 3, 4, 5, or 6 of the 2theta values selected from 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°. In certain embodiments, the Compound III pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least ten 2theta values selected from 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°. In certain embodiments, the Compound III pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least nine 2theta values selected from 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°. In certain embodiments, the Compound III pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least eight 2theta values selected from 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°. In certain embodiments, the Compound III pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least seven 2theta values selected from 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°. In certain embodiments, the Compound III pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least six 2theta values selected from 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°. In certain embodiments, the Compound III pattern 1 used in combination with the retaining device is characterized by an XRPD pattern comprising at least five 2theta values selected from 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°. Compound III pattern 2 In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising independently at least 3, 4, 5, or 6 of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 20.38+0.2°, 22.94+0.2°, 25.09+0.2°, 26.54+0.2°, 26.90+0.2°, 27.38+0.2°, 28.28+0.2°, 28.95+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising independently at least 3, 4, 5, or 6 of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 25.09+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising at least ten of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 25.09+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising at least nine of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 25.09+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising at least eight of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 25.09+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising at least seven of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 25.09+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising at least six of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 25.09+0.2°, 29.64+0.2°, and 38.07+0.2°. In certain embodiments, the Compound III pattern 2 used in combination with the retaining device is characterized by an XRPD pattern comprising at least five of the 2theta values selected from 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 25.09+0.2°, 29.64+0.2°, and 38.07+0.2°. Additional Embodiments of the Present Invention In certain embodiments, the present invention that includes the combined use of the active therapeutic as described herein with a retaining device includes the use of one of the following active agents below which may optionally be used in a morphic form as described herein, and further embodiments that include such active agents. 1. A compound of the formula: or
or A compound of the formula:
Figure imgf000050_0001
ula: or . ula: or . system that includes a pharmaceutical composition comprising the compound of any one of embodiments 1-5, and a pharmaceutically acceptable carrier. 7. The therapeutic use and retaining device system of embodiment 6, wherein the pharmaceutical composition is in a solid dosage form. 8. The therapeutic use and retaining device system of embodiment 6, wherein the pharmaceutical composition is in a semi-solid dosage form. 9. The therapeutic use and retaining device system of embodiment 6, wherein the pharmaceutical composition is in the form of a film. 10. The therapeutic use and retaining device system of embodiment 6, wherein the pharmaceutical composition is in the form of a pessary. 11. The therapeutic use and retaining device system of embodiment 7, wherein the pharmaceutical composition is in the form of a tablet. 12. The therapeutic use and retaining device system of embodiment 8, wherein the pharmaceutical composition is in the form of a cream. 13. The therapeutic use and retaining device system of embodiment 8, wherein the pharmaceutical composition is in the form of a gel. 14. The therapeutic use and retaining device system of any one of embodiments 6-13, wherein the pharmaceutical composition is formulated for topical administration. 15. The therapeutic use and retaining device system of any one of embodiments 6-14, wherein the pharmaceutical composition is formulated for delivery to the cervix. 16. The therapeutic use and retaining device system of any one of embodiments 6-14, wherein the pharmaceutical composition is formulated for delivery to the vagina. 17. The therapeutic use and retaining device system of any one of embodiments 6-14, wherein the pharmaceutical composition is formulated for delivery to the vulva. 18. The therapeutic use and retaining device system of embodiment 11, wherein the tablet is a bilayer tablet. 19. The therapeutic use and retaining device system of embodiment 11, wherein the tablet disintegrates in less than about 250 μL of fluid. 20. The therapeutic use and retaining device system of embodiment 11, wherein the tablet disintegrated in less than about 150 μL of fluid. 21. The therapeutic use and retaining device system of any one of embodiments 6-20, wherein the pharmaceutical composition comprises from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, or from about 0.05 mg to about 0.3 mg of the compound. 22. The therapeutic use and retaining device system of any one of embodiments 6-20, wherein the pharmaceutical composition comprises from about 0.05 mg to about 5 mg of the compound. 23. The therapeutic use and retaining device system of embodiment 22, wherein the pharmaceutical composition comprises from about 0.05 mg to about 0.5 mg of the compound. 24. The pharmaceutical composition of embodiment 23, wherein the pharmaceutical composition comprises from about 0.1 mg to about 0.3 mg of the compound. 25. The therapeutic use and retaining device system of embodiment 22, wherein the pharmaceutical composition comprises at least about 0.1 mg of the compound. 26. The therapeutic use and retaining device system of embodiment 22, wherein the pharmaceutical composition comprises at least about 0.3 mg of the compound. 27. The therapeutic use and retaining device system of embodiment 22, wherein the pharmaceutical composition comprises at least about 1 mg of the compound. 28. The therapeutic use and retaining device system of embodiment 22, wherein the pharmaceutical composition comprises at least 1.5 mg of the compound. 29. The therapeutic use and retaining device system of embodiment 22, wherein the pharmaceutical composition comprises at least about 2 mg of the compound. 30. The therapeutic use and retaining device system of embodiments 12-13, wherein the pharmaceutical composition comprises from about 0.01% to about 10% of the compound. 31. The therapeutic use and retaining device system of embodiments 12-13, wherein the pharmaceutical composition comprises from about 0.01% to 0.5% of the compound. 32. The therapeutic use and retaining device system of embodiments 12-13, wherein the pharmaceutical composition comprises from about 0.1% to 5% of the compound. 33. The therapeutic use and retaining device system of any one of embodiments 6-32, wherein the pharmaceutical composition comprises mucoadhesive polymer. 34. The therapeutic use and retaining device system of any one of embodiments 6-20, wherein the pharmaceutical composition comprises from about 5% to about 20% mucoadhesive polymer. 35. The therapeutic use and retaining device system of any one of embodiments 6-20, wherein the pharmaceutical composition comprises from about 10% to about 50% mucoadhesive polymer. 36. The therapeutic use and retaining device system of any one of embodiments 6-20, wherein the pharmaceutical composition comprises from about 50% to about 90% mucoadhesive polymer. 37. The therapeutic use and retaining device system of any one of embodiments 6-36, wherein the pharmaceutical composition comprises a disintegration enhancing excipient. 38. The therapeutic use and retaining device system of any one of embodiments 6-37, wherein the pharmaceutical composition comprises a penetration enhancing excipient. 39. The therapeutic use and retaining device system of any one of embodiments 6-38, wherein the pharmaceutical composition comprises an excipient which allows for the controlled release of the active compound. 40. The therapeutic use and retaining device system of embodiment 12, wherein the pharmaceutically acceptable carrier includes at least one of light mineral oil, propylparaben, Tefose 63, water, EDTA, methylparaben and Carbopol 974P. 41. The therapeutic use and retaining device system of embodiment 13, wherein the pharmaceutically acceptable carrier includes at least one of EDTA, methyl paraben, Carbopol 974P, propylene glycol and sorbic acid. 42. The therapeutic use and retaining device system of embodiment 11, wherein the tablet includes at least of mannitol, polycrystalline cellulose and magnesium stearate. 43. A method to treat a cervical or vaginal HPV-induced infection or an associated condition caused by a human papillomavirus in a human in need thereof, including but not limited to cervical or vaginal intraepithelial neoplasia, comprising administering to a host in need thereof an effective amount of the compound or pharmaceutical composition for use as described in any one of embodiments 1-42, with a vaginal applicator in combination with the use of a retaining device for sufficient time to collect the post-treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non- diseased tissue. 44. A method to treat a cervical or vaginal HPV-induced infection, intraepithelial neoplasia, or an associated condition in a human in need thereof, the method comprising: (i) administering an effective amount of the compound or pharmaceutical composition for use as described in any one of embodiments 1-42, to the cervix using a vaginal applicator (which is optionally coated with a lubricant); and then (ii) inserting a retaining device (which is optionally coated with a lubricant) that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post- treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue. 45. The method of embodiments 43 or 44, wherein the retaining device is selected from a menstrual cup, a menstrual disc, a tampon, diaphragm, cervical cap and a sponge. 46. The method of embodiments 43 or 44, wherein the retaining device is used as both the applicator and a retaining means to collect the post-treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue. 47. The method of embodiments 43 or 44, wherein the retaining device is a menstrual cup. 48. The method of embodiments 43 or 44, wherein the retaining device is a menstrual disc. 49. The method of embodiments 43 or 44, wherein the retaining device is a tampon. 50. The method of embodiments 43, 44, or 46, wherein the retaining device is a diaphragm. 51. The method of embodiments 43 or 44, wherein the retaining device is a cervical cap. 52. The method of embodiments 43 or 44, wherein the retaining device is a sponge. 53. The method of embodiment 46, wherein the retaining device used as both an applicator and a retaining means is a diaphragm. 54. The method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is atypical squamous cells of undetermined significance (ASC-US). 55. The method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is atypical glandular cells (AGC). 56. The method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is low-grade squamous intraepithelial lesions (LSIL). 57. The method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is atypical squamous cells, cannot exclude high grade squamous intraepithelial lesion (ASC-H). 58. The method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is high grade squamous intraepithelial lesions (HSIL). 59. The method of any one of embodiments 43-53, wherein the condition caused by a human papillomavirus is adenocarcinoma in situ (AIS). 60. The method of any one of embodiments 43-53, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia. 61. The method of embodiment 60, wherein the cervical intraepithelial neoplasia is Grade 1 cervical intraepithelial neoplasia. 62. The method of embodiment 60, wherein the cervical intraepithelial neoplasia is Grade 2 cervical intraepithelial neoplasia. 63. The method embodiment 60, wherein the cervical intraepithelial neoplasia is Grade 3 cervical intraepithelial neoplasia. 64. The method of any one of embodiments 43-53, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia. 65. The method of any one of embodiments 43-64, wherein from about 0.05 milligrams to about 40 milligrams of compound or pharmaceutical composition is administered. 66. The method of any one of embodiments 43-64, wherein from about 0.1 milligrams to about 30 milligrams is administered. 67. The method of any one of embodiments 43-64, wherein from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, from about 0.05 mg to about 0.3 mg, from about 0.03 mg to about 0.07 mg, from about 0.05 mg to about 0.15 mg, or from about 0.15 mg to about 0.45 mg of the compound is administered. 68. The method of any one of embodiments 43-64, wherein from about 0.05 mg to about 0.3 mg of the compound is administered. 69. The method of any one of embodiments 43-68, further comprising applying a lubrication means to the epithelial tissue before inserting the dosage form in the affected area. 70. The method of any one of embodiments 43-68, further comprising applying a lubrication means to the dosage form before inserting the dosage form in the affected area. 71. The method of embodiment 69 or 70 wherein the lubrication means is selected from water, a glycerol based lubricant and a hydroxyethylcellulose-based lubricant. 72. The method of any one of embodiments 43-71, wherein the compound or pharmaceutical composition is administered once per day. 73. The method of any one of embodiments 43-71, wherein the compound or pharmaceutical composition is administered twice per day. 74. The method of any one of embodiments 43-71, wherein the compound or pharmaceutical composition is administered twice per week. 75. The method of any one of embodiments 43-71, wherein the compound or pharmaceutical composition is administered three times or more per week. 76. The method of any one of embodiments 43-75, wherein the compound or pharmaceutical composition is administered for about one week. 77. The method of any one of embodiments 43-75, wherein the compound or pharmaceutical composition is administered for about two weeks. 78. The method of any one of embodiments 43-75, wherein the compound or pharmaceutical composition is administered for about three weeks. 79. The method of any one of embodiments 43-75, wherein the compound or pharmaceutical composition is administered for about four weeks. 80. The method of any one of embodiments 43-75, wherein the compound or pharmaceutical composition is administered for about five weeks. 81. The method of any one of embodiments 43-75, wherein the compound or pharmaceutical composition is administered for about six weeks. 82. The method of any one of embodiments 43-81, wherein the compound or pharmaceutical composition is wherein the compound is administered in a therapeutic cycle comprising: a. a treatment cycle comprising administering the compound, and b. a rest cycle, comprising a period of no treatment. 83. The method of embodiment 82, wherein the rest cycle is about one week. 84. The method of embodiment 82, wherein the rest cycle is about two weeks. 85. The method of embodiment 82, wherein the rest cycle is about three weeks. Isotopic Substitution The present invention includes but is not limited to compounds, pharmaceutical compositions, and the use of any of the active compounds described herein in combination with the retaining device, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as fumarate, Compound II or Compound III, with desired isotopic substitutions of atoms at amounts above the natural abundance of the isotope, i.e., enriched. Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons. By way of general example and without limitation, isotopes of hydrogen, for example, deuterium (2H) and tritium (3H) may be used anywhere in described structures. Alternatively, or in addition, isotopes of carbon, e.g., 13C and 14C, may be used. A preferred isotopic substitution is deuterium for hydrogen at one or more locations on the molecule to improve the performance of the drug. The deuterium can be bound in a location of bond breakage during metabolism (an α-deuterium kinetic isotope effect) or next to or near the site of bond breakage (a β-deuterium kinetic isotope effect). Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280) describes deuteration of nucleotides to improve their pharmacokinetic or pharmacodynamic, including at the 5-position of the molecule. Substitution with isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. Substitution of deuterium for hydrogen at a site of metabolic break- down can reduce the rate of or eliminate the metabolism at that bond. At any position of the compound that a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including protium (1H), deuterium (2H) and tritium (3H). Thus, reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise. The term "isotopically-labeled" analog refers to an analog that is a "deuterated analog", a "13C-labeled analog," or a "deuterated/13C-labeled analog." The term "deuterated analog" means a compound described herein, whereby a H-isotope, i.e., hydrogen/protium (1H), is substituted by a H-isotope, i.e., deuterium (2H). Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium. In certain embodiments, the isotope is 90, 95 or 99% or more enriched in an isotope at any location of interest. In some embodiments it is deuterium that is 90, 95 or 99% enriched at a desired location. Unless indicated to the contrary, the deuteration is at least 80% at the selected location. Deuteration of the nucleoside can occur at any replaceable hydrogen that provides the desired results. V. Treatment or Prophylaxis of HPV-induced Intraepithelial Neoplasia In exemplary non-limiting embodiments, a method for the treatment of HPV infection or HPV-induced intraepithelial neoplasia is provided that includes (i) administering an effective amount of the anti-HPV therapeutic agent such as ABI-2280 or a pharmaceutically acceptable salt thereof, or other compound or its salt described herein, optionally in morphic form, and typically in tablet form, to the cervix using a vaginal applicator and then (ii) inserting a retaining device that is maintained at the base of the cervix or in the vaginal canal for sufficient time to collect the post- treatment leakage of vaginal fluids and the therapeutic agent or its metabolite such as PMEG in a manner that minimizes toxicity-causing damage to non-diseased tissue as described in detail above. Types of HPV-induced intraepithelial neoplasia which can be treated include but are not limited to cervical and vaginal intraepithelial neoplasia. Patients can be instructed in the procedure for self-administration. The females can optionally administer the first dose of one of the active compounds disclosed herein in a vaginal tablet to the cervix using the vaginal applicator followed by the retaining device in the clinic. The single-use tablet applicator can be pre-moistened with a water-based lubricating gel. The patient or healthcare provider can then place the retaining device in its proper location. Following placement of the retaining device, the patient can be instructed to remain supine for several hours, for example, 3, 4, 5, 6, 7 or 8 hours with minimal activities and may resume normal activities after that time-period. The patient should repeat this procedure once daily for a total of 2, 3 or 4 applications in a week as instructed. It is preferred to carry out the two step administration process in the evening. Patients typically administer three doses on alternate days over a 1-week period. Patients can optionally be instructed to use the following preventative measures to protect against leakage of drug or metabolite-containing fluid to healthy tissue: • Minimize amount of water-based lubricant (e.g.,KY gel) used for dosing. • Do not bathe the genital area until after the instructed administration dosage time period. An example is to bathe in the morning after dosing the night before. • After bathing, a non-water based barrier gel (for example zinc oxide) can be applied to the external genitalia and perineum for additional protection. • Peripads can be used and can be changed frequently to avoid continued exposure to any leaked drug or metabolite containing fluid. • It is preferred to avoid dosing during the patient’s period. Dosings are preferably initiated and completed prior to anticipated starting date of the period. • Examples of suitable schedules include days 1, 3, and 5 followed by days 8, 10 and 12, followed by days 15, 17 and 19. Patients can combine this protocol with other standard of care procedures who have screening biopsy results of HSIL (CIN2 or CIN3). For example, 10-14 weeks after the first administration of the active compound in a vaginal tablet with retainer device, the patient may opt to undergo a LLETZ procedure or another procedure described in the Background section above. In certain embodiments, the Compound I, or a pharmaceutically acceptable salt thereof such as Compound II or Compound III, vaginal tablet is self-administered. In certain embodiments, one or more doses of the compound are administered by a physician. In certain embodiments, the first dose is administered by a physician or medical provider and the remaining doses, for example doses on days 3, 5, 8, and 10, are administered at home. In certain embodiments, a formulation for the treatment of intraepithelial neoplasia is a dosage form that contains from about 0.01 mg to about 10 mg, from about 0.05 to about 5 mg, from about 0.05 to about 0.15 mg, from about 0.15 mg to about 0.45 mg, or from about 0.5 to about 1.5 mg of any of the active compounds described herein including but not limited to Compound I or a pharmaceutically acceptable sale such as a monofumarate, hemifumarate, or other solid form, or Compound II or Compound III. In certain embodiments, a formulation for the treatment of intraepithelial neoplasia is a dosage form that contains about or at least 0.01, 0.03, 0.05, 0.1 mg, 0.3 mg.0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg or 20 mg, of Compound I monofumarate, Compound II or Compound III, optionally in a selected morphic form. The dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form. In certain embodiments, the topical formulation is administered once a day, or several days a week (such as 2 or 3 days a week) in combination with the retaining device, as long as necessary to achieve the desired results. In certain embodiments, the topical formulation is administered on a weekly schedule for one, two, three, four, five, six or more weeks. In certain aspects, the topical formulation is administered on a schedule of three dosages a week for two, three, four, five, or six weeks. In certain embodiments, the compound can be administered in one or more therapeutic cycles comprising a treatment cycle and a rest cycle, wherein the treatment cycle comprises administering the compound as described herein in combination with the retaining device, followed by a rest cycle (comprising a period of no treatment) before the next treatment cycle. In certain embodiments, the rest cycle is from about one day to about six months. In certain embodiments the rest cycle is one, two, three, four, five, six, seven, eight or more weeks before the next treatment cycle. In certain embodiments, multiple therapeutic cycles are administered, for example one, two, three, four, five, or six therapeutic cycles. Dosage forms which do not adhere well to the target site may be dislodged, interfering with treatment. Dosage forms have been discovered that adhere to the target site and dissolve rapidly in low fluid volumes. Adhesion to the target site also prevents exposure to healthy tissues, which may limit toxicity and side effects. Dosage forms which soften, break down, and/or disintegrate quickly in low fluid volumes are advantageous to cause a rapid release of the active compound to the target tissue. Dosage forms that disintegrate in, for example, less than about 50 µL, less than about 100 µL, less than about 125 µL, less than about 150 µL, less than about 175 µL, less than about 200 µL, or less than about 250 µL fluid particularly facilitate drug penetration into the target site. In certain embodiments, the dosage form is a gel. In certain embodiments, the dosage form is a cream. In certain embodiments, the dosage form is a tablet. In certain embodiments, the dosage form disintegrates in about one to about ten seconds. In certain embodiments, the dosage form disintegrates in about ten seconds to one minute. In certain embodiments, the dosage form disintegrates in about one minute to about one hour. In certain embodiments, the dosage form disintegrates in about one to six hours. The physical dimensions of the dosage form can impact the effectiveness of the dosage form. A tablet that is thinner provides a greater surface area to volume ratio and may degrade quicker and cover the target area better. In certain embodiments the dosage form is less than about 6, 5, 4, 3, or 2 millimeters thick in its smallest dimension. The formulation of the dosage form is important for adequate administration of the active agent into the intraepithelial tissue. The formulation for example, can be prepared for use as a tablet, a reconstituted powder, a dry powder, a semi solid dosage form, a film or a pessary (i.e., a vaginal suppository). Some embodiments disclosed herein include the use of an effective amount of Compound I monofumarate, Compound II or Compound III, in the manufacture of a medicament for ameliorating or treating a human papillomavirus infection, wherein the infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA. Other embodiments disclosed herein include the use of an effective amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II or Compound III, for ameliorating or treating a human papillomavirus infection, wherein the human papillomavirus infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA. Certain nonlimiting embodiments disclosed herein include a method for ameliorating or treating a human papillomavirus infection that can include contacting a cell infected with the human papillomavirus in a subject with an effective amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound I hemifumarate, or other solid form of Compound I, Compound II or Compound III, wherein the infection is ameliorated or treated by inhibiting the synthesis of viral DNA. Yet still other embodiments disclosed herein include a method for ameliorating or treating a human papillomavirus infection that can include administering to a subject infected with the human papillomavirus an effective amount of Compound I monofumarate, Compound II or Compound III, wherein the human papillomavirus infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA. Some embodiments disclosed herein relate Compound I monofumarate, Compound II or Compound III, for use in ameliorating or treating a human papillomavirus infection, wherein the human papillomavirus infection can be ameliorated or treated by inhibiting viral replication by inhibiting the synthesis of viral DNA. In some embodiments, the human papillomavirus can be a high-risk human papillomavirus, such as those described herein. For example, the high-risk human papillomavirus can be selected from HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV- 56, HPV-58, HPV-59, HPV-68, HPV-73 and HPV-82. In some embodiments, the human papillomavirus can be HPV-16. In some embodiments, the human papillomavirus can be HPV-11. In some embodiments, the human papillomavirus can be HPV-18. In some embodiments, the human papillomavirus can be one or more of the following high-risk types: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73 and HPV-82. As described herein, the presence of an HPV infection can be detected using the Papanicolaou test (Pap smear) and/or DNA probe testing (for example, HPV DNA probe testing for one or more high-risk HPV types). Therefore, in some embodiments, an effective amount of Compound I or a pharmaceutically acceptable salt thereof, such as a monofumarate, Compound II or Compound III, can be provided to a subject diagnosed with an HPV infection in combination with the retaining device, for example a high-risk HPV infection, by a DNA test. In some embodiments, an effective amount of Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III can be provided to a subject diagnosed with an HPV infection, or a disease associated with HPV infection, as identified by a Papanicolaou test. In certain embodiments, an effective amount of Compound I monofumarate, Compound II, or Compound III may be provided to a subject with a Papanicolaou test result that does not indicate the disease has progressed to cervical cancer. The Bethesda system is a standardized scoring system for reporting pap smear test results and assigns a grade of 1-3 based on severity. Grade 1 CIN (CIN 1) indicates mild dysplasia. Grades 2 and 3 CIN (CIN 2, CIN 3) are more serious and typically require intervention. In certain embodiments, Compound I monofumarate, Compound II or Compound III is used to treat CIN 1 (Grade 1 cervical intraepithelial neoplasia). In certain embodiments, Compound I monofumarate, Compound II or Compound III is used to treat CIN 2 (Grade 2 cervical intraepithelial neoplasia) in combination with the retaining device. In certain embodiments, Compound I monofumarate, Compound II or Compound III is used to treat CIN 3 (Grade 3 cervical intraepithelial neoplasia) in combination with the retaining device. In certain embodiments, a pharmaceutical composition comprising Compound I monofumarate, Compound II or Compound III, is used in the manufacture of a system for the treatment of CIN 1 (Grade 1 cervical intraepithelial neoplasia) that includes the use of a retaining device, as described further herein. In certain embodiments, a pharmaceutical composition comprising Compound I monofumarate, Compound II or Compound III, is used in the manufacture of a system for the treatment of CIN 2 (Grade 2 cervical intraepithelial neoplasia) that includes the use of a retaining device as described further herein. In certain embodiments, a pharmaceutical composition comprising Compound I monofumarate, Compound II or Compound III, is used in the manufacture of a medicament for the treatment of CIN 3 (Grade 3 cervical intraepithelial neoplasia). In certain embodiments, Compound I monofumarate, Compound II or Compound III, optionally in a pharmaceutically acceptable carrier, is used to treat a condition selected from the group consisting of atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells (AGC), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cells (cannot exclude high grade squamous intraepithelial lesion) (ASC-H), high grade squamous intraepithelial lesions (HSIL), adenocarcinoma in situ (AIS), and cervical cancer (e.g. squamous cell carcinoma or adenocarcinoma). In certain embodiments, Compound II optionally in a pharmaceutically acceptable carrier, is used in the therapeutic use and retaining device system to treat a condition selected from the group consisting of atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells (AGC), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cells (cannot exclude high grade squamous intraepithelial lesion) (ASC-H), high grade squamous intraepithelial lesions (HSIL), adenocarcinoma in situ (AIS), and cervical cancer (e.g. squamous cell carcinoma or adenocarcinoma). In some embodiments, the human papillomavirus can be a low-risk human papillomavirus, including those described herein. In some embodiments, the human papillomavirus can be HPV- 6. In some embodiments, the human papillomavirus can be HPV-11. Compound I monofumarate, Compound II or Compound III, can be used in the manufacture of a system that includes a retaining device for use to ameliorate and/or treat an infection caused by one or more types of human papillomaviruses. For example, Compound I monofumarate, Compound II or Compound III, can be used in the manufacture of a system that includes a retaining device for use to ameliorate and/or treat an infection of HPV-16 and/or HPV- 18. In certain embodiments, Compound I monofumarate, Compound II or Compound III can be used in the manufacture of a system that includes a retaining device for use to treat a high-risk HPV infection. In certain embodiments, Compound I monofumarate, Compound II or Compound III can be used in the manufacture of a system that includes a retaining device for use to treat a related disease or condition occurring as a result of a high-risk HPV infection. In some embodiments, Compound I monofumarate, Compound II or Compound III, can be used in the manufacture of a system that includes a retaining device for use to ameliorate and/or treat an infection comprising both high-risk and low-risk HPV. As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials and in vitro studies. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the therapeutic system that includes a retaining device to treat conditions related to or occurring as a result of exposure to or an infection of HPV. In certain embodiments, a pharmaceutical composition comprising Compound II is used to treat precancerous cervical lesions. In certain embodiments, a pharmaceutical composition comprising Compound II is used to treat cervical intraepithelial neoplasia. In certain embodiments, a pharmaceutical composition comprising Compound II is used to treat vaginal intraepithelial neoplasia. In certain embodiments, a pharmaceutical composition comprising Compound II is used to treat cervical cancer. In certain embodiments, a pharmaceutical composition comprising Compound II is used to treat vaginal cancer. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the manufacture of a system that includes a retaining device for the treatment of conditions related to or occurring as a result of exposure to or an infection of HPV. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the manufacture of a system that includes a retaining device for the treatment of precancerous cervical lesions. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the manufacture of a system that includes a retaining device for the treatment of cervical intraepithelial neoplasia. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the manufacture of a system that includes a retaining device for the treatment of vaginal intraepithelial neoplasia. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the manufacture of a system that includes a retaining device for the treatment of cervical cancer. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the manufacture of a system that includes a retaining device for the treatment of vaginal cancer. In certain embodiments, a pharmaceutical composition comprising Compound II is used in the manufacture of a system that includes a retaining device for the treatment of oropharyngeal cancer. It is advantageous for the dosage form to be easily applied to the target site. Direct application to the target site prevents systemic exposure and toxicity. To place the dosage form on the target site, the dosage form may be applied with an applicator. In certain embodiments the dosage form is applied with a vaginal applicator. In certain embodiments, additional fluid (such as a lubricant) is delivered along with the dosage form, applied to the dosage form, or applied to the target site or surrounding tissues. In certain embodiments, a lubricating fluid is administered in combination with the dosage form to enhance the coverage of the cervix, vagina or vulva. In certain embodiments, water is used as the fluid administered with the dosage form. In certain embodiments, a lubricating glycerol- or hydroxyethylcellulose-based, water soluble fluid is used in combination with the dosage form. In certain embodiments the dosage form is administered without additional fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 5 milliliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 4 milliliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 3 milliliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 2 milliliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 1 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.75 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.5 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.25 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.2 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.15 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.125 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.1 milliliter of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 10 microliters to about 100 microliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 75 microliters to about 250 microliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 200 microliters to about 500 microliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 400 microliters to about 750 microliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 700 microliters to about 1,000 microliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 1 milliliter to about 2 milliliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 2 milliliters to about 3 milliliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 3 milliliters to about 4 milliliters of fluid. In certain embodiments, the dosage form will soften, disintegrate, and/or dissolve in from about 4 milliliters to about 5 milliliters of fluid. In certain embodiments, Compound II is administered in combination with the use of a retaining device for at least 1, 2, 3, 4, 5, or 6 consecutive or nonconsecutive days. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device for up to 12 weeks. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device for up to 10 weeks. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device for up to 8 weeks. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device for up to 6 weeks. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device for up to 4 weeks. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device for up to 2 weeks. In certain embodiments, Compound II is administered once a week in combination with the use of a retaining device for up to 1 week. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 12 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 10 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 8 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 6 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 4 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 2 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device twice a week for up to 1 week. In certain embodiments, Compound II is administered in combination with the use of a retaining device three times a week. In certain embodiments, Compound II is administered in combination with the use of a retaining device three times a week for up to 12 weeks. In certain embodiments, Compound II is administered in combination with the use of a retaining device three times a week for up to 10 weeks. In certain embodiments, Compound II is administered in combination with a retaining device three times a week for up to 8 weeks. In certain embodiments, Compound II is administered in combination with a retaining device three times a week for up to 6 weeks. In certain embodiments, Compound II is administered in combination with a retaining device three times a week for up to 4 weeks. In certain embodiments, Compound II is administered in combination with a retaining device three times a week for up to 2 weeks. In certain embodiments, Compound II is administered in combination with a retaining device three times a week for up to 1 week. In certain embodiments, Compound II is administered in combination with a retaining device daily. In certain embodiments, Compound II is administered in combination with a retaining device daily for up to 12 weeks or indefinitely as instructed by a healthcare provider. In certain embodiments, Compound II is administered in combination with a retaining device daily for up to 10 weeks. In certain embodiments, Compound II is administered in combination with a retaining device daily for up to 8 weeks. In certain embodiments, Compound II is administered in combination with a retaining device daily for up to 6 weeks. In certain embodiments, Compound II is administered in combination with a retaining device daily for up to 4 weeks. In certain embodiments, Compound II is administered in combination with a retaining device daily for up to 2 weeks. In certain embodiments, Compound II is administered in combination with a retaining device daily for up to 1 week. In certain embodiments, from about 0.05 mg to about 0.3 mg of Compound II is administered in combination with a retaining device daily for one, two, three, four, five, six, or more weeks, as instructed by a healthcare provider. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 12 weeks. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 10 weeks. In certain embodiments Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 8 weeks. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 6 weeks. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 4 weeks. In certain embodiments, Compound I monofumarate or hemifumarate or other solid form is administered in combination with a retaining device three times a week for up to 2 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device three times a week for up to 1 week. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 12 weeks or indefinitely as instructed by a healthcare provider. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 10 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 8 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 6 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 4 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 2 weeks. In certain embodiments, Compound I monofumarate is administered in combination with a retaining device daily for up to 1 week. In certain embodiments, Compound I monofumarate, or other solid form Compound II or Compound III may be administered in combination with a retaining device three, four, five or six times a week. In certain embodiments, Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device once per day. In certain embodiments, Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device twice per day. In certain embodiments, Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device three, four, or more times per day. In certain embodiments, Compound I monofumarate, Compound II or Compound III may be administered in combination with a retaining device daily. In certain embodiments, the compound can be administered in combination with a retaining device in one or more therapeutic cycles comprising a treatment cycle and a rest cycle, wherein the treatment cycle comprises administering the compound as described herein, followed by a rest cycle (comprising a period of no treatment) before the next treatment cycle. In certain embodiments, the rest cycle is from about one day to about six months. In certain embodiments the rest cycle is one, two, three, four, five, six, seven, eight or more weeks before the next treatment cycle. In certain embodiments, multiple therapeutic cycles are administered, for example one, two, three, four, five, or six therapeutic cycles. As described above, a number of compounds have been investigated for the treatment of HPV-induced neoplasia, however none has been approved yet. For non-limiting examples of investigated approaches, see Ahn W.S., et al. Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions. Eur. J. Cancer Prev.2003;12:383–390; Ashrafian L,et al. Double-blind randomized placebo-controlled multicenter clinical trial (phase IIa) on diindolymethane’s efficacy and safety in the treatment of CIN: implications for cervical cancer prevention. EPMA J.2015;6:doi: 10.1186/s13167-13015-10048-13169; Bossens M., et al. Safety and tolerance of cidofovir as a 2% gel for local application in high-grade cervical intraepithelial neoplasia: A phase 1 investigation. Int. J. Clin. Pharmacol.2018;56:134–141; Chen F.P. Efficacy of imiquimod 5% cream for persistent human papillomavirus in genital intraepithelial neoplasm. Taiwanese J. Obstetrics Gynecol.2013;52(4):475–478; Choo Y., et al. Intravaginal application of leukocyte interferon gel in the treatment of cervical intraepithelial neoplasia (CIN) Arch Gynecol. 1985;237:51–54; de Witte C.J et al. Imiquimod in cervical, vaginal and vulvar intraepithelial neoplasia: a review. Gynecol. Oncol. 2015;139:377–384; Desravines N, et al. Low dose 5- fluorouracil intravaginal therapy for the treatment of cervical intraepithelial neoplasia 2/3: A case series. J. Gynecol. Surg. 2020;36; DiSilvestro P.A., et al. Treatment of cervical intraepithelial neoplasia levels 2 and 3 with adapalene, a retinoid-related molecule. J. Low Genit Tract. Dis. 2001;5:33–37; Graham V., et al. Phase II trial of beta-all-transretinoic acid for cervical intraepithelial neoplasia via a collagen sponge and cervical cap. West. J. Med.1986;145:192–195; Grimm C., et al. Treatment of cervical intraepithelial neoplasia with topical imiquimod: a randomized controlled trial. Obstet. Gynecol.2012;120(1):152–159; Hampson L., et al. A single- arm, proof-of-concept trial of lopimune (lopinavir/ritonavir) as treatment for HPV-related pre- invasive cervical disease. PLoS ONE. 2016;11; Helm C.W. et al. Retinoids for preventing the progression of cervical intra-epithelial neoplasia. Cochrane Systematic Review. 2013; Hubert P., et al. Local applications of GM-CSF induce the recruitment of immune cells in cervical low-grade squamous intraepithelial lesions. Am. J. Reprod. Immunol.2010;64:126–136; Koeneman MM, et al. Topical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC trial): study protocol for a randomized controlled trial. BMC Cancer.2016:doi: 10.1186/s12885-12016- 12187-12883; Krause S., et al. Interferon and cervical dysplasia: CIN III treated with local interferon application. Colposcopy Gynecologic Laser Surgery. 1987;3:195–198; Krebs H.B., et al. Chronic ulcerations following topical therapy with 5-fluorouracil for vaginal human papillomavirus-associated lesions. Obstet. Gynecol.1991;78(2):205–208; Laccetta G. et al. Effect of the treatment with beta-glucan in women with cervical cytologic report of atypical squamous cells of undetermined significance (ASCUS) and low-grade intraepithelial lesions (L-SIL) Minerva Ginecol.2015;67:113–120; Meyskens F.L., et al. A phase I trial of beta-all-transretinoic acid delivered via a collagen sponge and a cervical cap for mild or moderate intraepithelial cervical neoplasia. J. Natl Cancer Inst. 1983;71:921–925; Niwa K., et al. Topical vidarabine of 5- fluoruracil treatment against persistent HPV in genital (pre)cancerous lesions. Oncol Reports. 2003;10:1437–1441; Pachman DR, et al. Randomized clinical trial of imiquimod: an adjunct to treating cervical dysplasia. Am. J. Obstet. Gynecol. 2012;206(1):42 e41-47; Rahangdale L et al. Topical 5-fluorouracil for treatment of Cervical Intraepithelial Neoplasia 2: a randomized controlled trial. Am. J. Obstet. Gynecol. 2014;210:e1–e8; Schneider A., et al. Efficacy trial of topically administered Interferon gamma-1beta gel in comparison to laser treatment in cervical intraepithelial neoplasia. Arch. Gynecol Obste. 1995;256:75–83; Silman F.H., et al. 5- fluorouracil/chemosurgery for intraepithelial neoplasia of the lower genital tract. Obstet. Gynecol. 1981;58:356–360; Snoeck R., Noel J.C., Muller C., Clercq De, Bossens M. Cidofovir, a new approach for the treatment of cervix intraepithelial neoplasia III (CIN III) J. Med. Virol. 2000;60:205–209; Stentella P., Biamonti A., Carraro C. Efficacy of carboxymethyl beta-glucan in cervical intraepithelial neoplasia: a retrospective, case-control study. Minerva Ginecol. 2017;69:425–430; Suh-Burgmann E., Sivret J., Duska L.R., Del Carmen M., Seiden M.V. Long- term administration of intravaginal dehydroepiandrosterone on regression of low-grade cervical dysplasia - a pilot study. Gynecol. Obstet. Invest.2003;55:25–31; Valencia M.H., Pacheco A.C., Quijano T.H., Giron A.V., Lopez C.V. Clinical response to glycyrrhizinic acid in genital infection due to human papillomavirus and low-grade squamous intraepithelial lesion. Clin. Pract. 2011 1(e93); van de Sande A., Koeneman M., Gerestein C., Kruse A., van Kemenade F., van Beekhuizen H. Topical Imiquimod treatment of residual or recurrent cervical intraepithelial neoplasia (TOPIC-2 trial): a study protocol for a randomized controlled trial. BMC Cancer. 2018;18:4510–4517; and Van Pachterbeke C., Bucella D., Rozenberg S. Topical treatment of CIN 2+ by cidofovir: Results of a phase II, double-blind, prospective, placebo-controlled study. Gynecol Onc.2009;115:69–74. VI. Pharmaceutical Compositions and Dosage Forms In an aspect of the invention, pharmaceutical compositions according to the present invention comprise an anti-HPV effective amount of any of the active compounds described herein optionally in a morphic form as described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof, including the monofumarate, Compound II or Compound III as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive, or excipient, and/or in combination or alternation with at least one other active compound and used in combination with the retaining device as described herein. In one embodiment, the invention includes a solid dosage form of Compound II in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is administered directly to the cervix, vagina or vulva. Table 1. A nonlimiting example of a formulation for ABI-2280 vaginal tablet Ingredient Function Amount per 175 mg Tablet AP
Figure imgf000073_0001
ABI-2280 vaginal tablet products are formulated to deliver drug to the cervical epithelium following topical administration onto the cervix. The excipients in the tablet products should be preferably compendial (USP/NF), included in the US FDA’s List of Inactive Ingredients, or appear on the US FDA’s Food Additive Mannitol is a Generally Recognized As Safe (GRAS) listed naturally occurring sugar alcohol, widely used in pharmaceutical dosage forms and food products. It has been extensively studied including by vaginal route, and is currently in oral, parenteral, nasal and topical products. The tablet has good adherence to mucosal tissue. PMEG prodrug, for example, ABI-2280, vaginal tablets can be packaged for example in bottles with a desiccant. Nonlimiting examples are high density polyethylene bottles with a silica gel desiccant canister and rayon, induction sealed and capped with a child-resistant closure. The vaginal tablet can be administered using the single-use vaginal tablet applicator or retaining device. It is preferred that tablets are stored refrigerated (2-8℃) in bottles with desiccant. Prior to administration, the bottle should be brought to room temperature for at least 30 minutes before opening and removing the tablet. Tablets are typically stored refrigerated (2-8℃) in bottles with desiccant. Prior to clinical use, the tablet should be removed from refrigeration (2-8℃) and brought to room temperature for at least 30 minutes before opening and removing the tablet. The tablets have been designed to deliver the required dose with a single tablet at each application using a tablet applicator or retaining device. In an aspect of the invention, pharmaceutical compositions used in the present invention comprise an anti-HPV effective amount of Compound II described herein, optionally in a morphic form, in combination with a pharmaceutically acceptable carrier, additive, or excipient, further optionally in combination with at least one other antineoplastic agent or antiviral agent, such as an anti-HPV agent in combination with the retaining device. In certain embodiments the pharmaceutical composition includes Compound II in combination with a second antiviral drug in combination with the retaining device. In certain embodiments the pharmaceutical composition includes Compound II in combination with an anticancer drug in combination with the retaining device. One of ordinary skill in the art will recognize that a therapeutically effective amount will vary with the infection or condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetic of the agent used, as well as the patient or subject (animal or human) to be treated, and such therapeutic amount can be determined by the attending physician or specialist. In certain pharmaceutical dosage forms, the prodrug form of the compounds, especially including acylated (acetylated or other), and ether (alkyl and related) derivatives, phosphate esters, thiophosphonamidates, phosphonamidates, and various salt forms of the present compounds, may be used to achieve the desired effect. One of ordinary skill in the art will recognize how to readily modify the present compounds to prodrug forms to facilitate delivery of active compounds to a targeted site within the host organism or patient. The person of ordinary skill in the art also will take advantage of favorable pharmacokinetic parameters of the prodrug forms, where applicable, in delivering the present compounds to a targeted site within the host organism or patient to maximize the intended effect of the compound. The amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II, or Compound III included within the therapeutically active formulation according to the present invention is an effective amount to achieve the desired outcome according to the present invention in combination with the retaining device, for example, for treating the HPV infection, reducing the likelihood of a HPV infection or the inhibition, reduction, and/or abolition of HPV or its secondary effects, including disease states, conditions, and/or complications which occur secondary to HPV infection. In certain embodiments, Compound I monofumarate, Compound II, or Compound III may be administered in a gel in combination with the use of a retaining device. In certain embodiments the gel contains from about 0.001% to about 10%, from about 0.01% to about 10%, from about 0.05% to about 5%, from about 0.1 to about 3% from about 0.1 to about 2% Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II, or Compound III (weight/weight). In certain embodiments the gel contains from about 0.001% to about 0.05% Compound I monofumarate, Compound II, or Compound III. In certain embodiments, the gel contains from about 0.01% to about 0.5% Compound I monofumarate, Compound II, or Compound III. In certain embodiments, the gel contains from about 0.1% to about 5% Compound I monofumarate, Compound II, or Compound III. In certain non-limiting embodiments, any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II, or Compound III is administered topically via a tablet, capsule, suspension, liquid, emulsion, implant, particle, sphere, cream, ointment, suppository, pessary, transdermal form, gel, mucosal, and the like in combination with the retaining device. The dosage form may also be a bilayer tablet, in which the full dose of active compound is released one direction (for example towards the target tissue). In certain embodiments, the dosage form can soften, disintegrate, and/or release the drug in low fluid volumes. In certain embodiments, the dosage form softens and begins to release the drug immediately. In certain embodiments, the dosage form softens and begins to release the drug gradually. In certain embodiments, the dosage form softens and begins to release the drug within one hour. In certain embodiments, the dosage form softens and begins to release the drug within two hours. The dosage form may be prepared to maximize surface area, facilitating disintegration. In certain embodiments, the dosage form is a round tablet. In certain embodiments, the dosage form is an oval tablet. In certain embodiments, the dosage form is a caplet. The tablet width is the largest dimension, and the tablet thickness is the smaller dimension. In certain embodiments, the dosage form is twice as wide as it is thick. In certain embodiments, the dosage form is three times as wide as it is thick. In certain embodiments, the dosage form is four or more times as wide as it is thick. In certain embodiments the dosage form is from about 0.1 mm thick to about 5 mm thick. In certain embodiments, the dosage form is from about 1 mm to about 2 mm thick. In certain embodiments, the dosage form is from about 2 mm to about 3 mm thick. In certain embodiments, the dosage form is from about 3 mm to about 4 mm thick. In certain embodiments the dosage form is from about 4 mm to about 5 mm thick. In certain embodiments the tablet is from about 5 mm to about 15 mm thick. In certain embodiments, the dosage form is less than 5 grams. In certain embodiments, the dosage form is from about 0.05 gram to about 0.15 gram. In certain embodiments, the dosage form is from about 0.1 to about 1 gram. In certain embodiments, the dosage from about 0.75 grams to about 2 grams. In certain embodiments, the dosage form is from about 1 gram to about 5 grams. The dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form. In certain embodiments, the dose form is not easily removed, dislodged, or moved from the target site. These desirable properties may be achieved by inclusion of a mucoadhesive polymer into the pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises a mucoadhesive polymer or mucoadhesive excipient. Nonlimiting examples of mucoadhesive polymers and excipients include: Hypromellose, lectin, thiolated polymers (e.g. chitosan–iminothiolane, poly(acrylic acid)–cysteine, poly(acrylic acid)–homocysteine, chitosan– thioglycolic acid, chitosan–thioethylamidine, alginate– cysteine, poly(methacrylic acid)–cysteine and sodium carboxymethylcellulose–cysteine), polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidinone, polyacrylic acid (Carbopol®), polyheroxyethyl methacrylate, chitosan, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, sodium carboxymethyl cellulose, aminated corn starch, cellulose derivatives, poly (acrylic acid) polymers, poly (hydroxyethyl methylacrylate), poly (ethylene oxide), poly (vinyl pyrrolidone), poly (vinyl alcohol), tragacanth, sodium alginate, karaya gum, guar gum, xanthan gum, soluble starch, gelatin, pectin, chitosan, methyl cellulose, hyaluronic acid, hydroxy propyl methylcellulose, hydroxy propyl cellulose, gellan gum, carrageenan, cationic hydroxyethyl celluloses, hydrogel, dihydroxyphenylalanine, and alginate-polyethylene glycol acrylate. In certain embodiments, the pharmaceutical composition comprises from about 0 to about 10% mucoadhesive polymer excipients selected from the list consisting of carbomer, polyethylene glycol, crospovidone, polycarbophil, hypromellose, and hydroxyethyl cellulose. In certain embodiments, the pharmaceutical composition comprises from at least about 0.1% to about 90, about 92%, about 93%, about 95%, about 98% about 97%, about 98%, or about 99% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 0.1% to about 1% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 0.5% to about 5% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 1% to about 10% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 5% to about 20% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 10% to about 50% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 20% to about 75% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 50% to about 90% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 75% to about 99% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises about or at least about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 percent mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises no more than about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 percent mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises 0% mucoadhesive polymer. In this instance, the adhesion to the target site is achieved by use of other pharmaceutically acceptable excipients. To prepare the pharmaceutical compositions used in the present invention, a therapeutically effective amount of any of the active compounds described herein including but not limited to Compound I monofumarate, Compound II, or Compound III optionally in morphic form according to the present invention is often admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. In preparing pharmaceutical compositions in topical dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid or semi-solid topical preparations such as gels, creams, ointments, suspensions, elixirs, and solutions, suitable carriers and additives including water, glycols, oils, alcohols, preservatives, and the like may be used. In certain embodiments, the pharmaceutical composition comprises propylene glycol. In certain embodiments, the pharmaceutical composition comprises carboxypolymethylene. In certain embodiments, the pharmaceutical composition comprises ethylenediaminetetraacetic acid (EDTA). In certain embodiments, the pharmaceutical composition comprises sorbic acid. In certain embodiments, the pharmaceutical composition comprises carbomer. In certain embodiments, the pharmaceutical composition comprises hydroxyethyl cellulose. In certain embodiments, the pharmaceutical composition comprises polyethylene glycol. For solid topical preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose, and related carriers, diluents, granulating agents, lubricants, binders, mucoadhesive polymer, disintegrating agents, and the like may be used. If desired, the tablets or capsules may be coated or sustained release by standard techniques. The use of these dosage forms may significantly enhance the bioavailability of the compounds in the patient. In certain embodiments, the pharmaceutical composition comprises mannitol. In certain embodiments, the pharmaceutical composition comprises magnesium stearate. In certain embodiments, the pharmaceutical composition comprises microcrystalline cellulose. In certain embodiments, the pharmaceutical composition comprises polycarbophil. In certain embodiments, the pharmaceutical composition comprises polyethylene oxide. In certain embodiments, the pharmaceutical composition comprises colloidal silicon dioxide. In certain embodiments, the pharmaceutical composition comprises povidone. In certain embodiments, the pharmaceutical composition comprises isopropyl alcohol. In certain embodiments, the pharmaceutical composition comprises sodium starch glycolate. In certain embodiments, the pharmaceutical composition comprises croscarmellose sodium. In certain embodiments, the pharmaceutical composition comprises crospovidone. In certain embodiments, the pharmaceutical composition comprises hydroxypropylmethylcellulose. In certain embodiments, the pharmaceutical composition comprises lactose. In certain embodiments, a powder pharmaceutical composition comprises one or more excipient from the group consisting of xanthan gum, microcrystalline cellulose, polyethylene oxide, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose sodium, povidone, mannitol, colloidal silicon dioxide, sodium benzoate, sodium starch glycolate, sodium lauryl sulfate, poloxamer 407, polyoxypropylene- polyoxyethylene copolymers, and the like. In certain embodiments, the pharmaceutical composition comprising an effective amount of a fumarate salt of any of the active compounds described herein including but not limited to Compound I, further comprises a pharmaceutically acceptable excipient selected from the list consisting of Acacia, agar, alginic acid, ascorbyl palmitate, bentonite, benzoic acid, butylated hydroxyanisole, butylated hydroxytoluene, butylene glycol, calcium acetate, calcium hydroxide, canola oil, carob bean gum, carrageenan, castor oil, cellulose, corn starch, disodium edetate, erythorbic acid, ethyl lactate, ethylcellulose, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, hypromellose, lactic acid, lauric acid, lecithin, linoleic acid, medium chain triglycerides, methyl paraben, methylcellulose, microcrystalline cellulose, microcrystalline wax, myristic acid, oleic acid, palmitic acid, peanut oil, pectin, phosphoric acid, polycarbophil, potassium alginate, propionic acid, propyl gallate, propyl paraben, propylene glycol, propylene glycol alginate, silicon dioxide, simethicone, sodium alginate, sodium benzoate, sodium bicarbonate, sodium carboxymethylcellulose, sodium chloride, sodium citrate, sodium lactate, sodium lauryl sulfate, sodium metabisulfate, sodium phosphate, sodium sulfite, sodium thiosulfate, sorbic acid, stearic acid, talc, tapioca starch, tartaric acid, thymol, urea, vitamin E polyethylene succinate, beeswax, xanthan gum, and zinc acetate. In certain embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients for use as a pessary. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III further comprises up to 99.9% pessary excipient selected from the group consisting of hard fat, PEG, macrogols, cocoa butter, and glycerol. Non limiting examples of hard fat include Ovucire® (mono-, di- and triglyceride esters of fatty acids (C10 to C18), the triester fraction being predominant and ethoxylated fatty alcohols), Witepsol® (glycerol esters of vegetable saturated fatty acids, such as lauric acid), and Supposi-base™ (a blend of saturated polyglycolysed glycerides). In certain embodiments, the pharmaceutical composition comprising an effective amount of any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III further comprises a pharmaceutically acceptable excipient that enhances the penetration, disintegration, film forming and/or controlled release properties of the composition. In certain embodiment, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate, Compound II or Compound III further comprises a penetration enhancing excipient. In certain embodiments, the penetration enhancing excipient is selected from the group consisting of oleic acid, eucalyptol, Caprylol, Labrafil, Labrasol, Lauroglycol, diethylene glycol monomethyl ether (Transcutol), propylene glycol, sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, poloxamer (231, 182, 184), Tween 20, 40, 60, 80, fatty acids and fatty acid esters, isostearic acid, glycerin, and chitosan. In certain embodiments, the pharmaceutical composition comprising Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III contains from 0% to about 20% penetration enhancing excipients selected from the group consisting of cetyl alcohol, propylene glycol, transcutol P, oleic acid, isopropyl myristate, propylene glycol dicaprylate, glyceryl monooleate, propylene glycol monocaprylate, PEG-8 bees wax, cetyl alcohol, stearic acid, cetyl palmitate, and cetosteryl alcohol. In certain embodiments, the pharmaceutical composition comprises from about 0 to about 25% penetration enhancing excipients selected from the list consisting of stearyl alcohol, polysorbate 80, sodium lauryl sulfate, mono and diglycerides, sorbitan monostearate, glyceryl isostearate, polyoxyl 15 hydroxystearate, polyoxyl 40 hydrogenated castor oil, octyl dodecanol, and soybean lecithin. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises a film forming excipient. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III contains from 0% to about 99% film forming excipients selected from the group consisting of hypromellose, polyethylene glycol, polymethacrylates, microcrystalline cellulose, guar gum, xanthan gum, and polyvinylpyrrolidone. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises and excipient which allows for controlled release of the active compound. In certain embodiments, the controlled release pharmaceutical composition comprises ethylcellulose, hypromellose, microcrystalline wax, polycarbophil, beeswax. Percentage ranges of excipients and other components of the pharmaceutical composition are given as a percent by weight, unless otherwise specified. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises a disintegration enhancing excipient. In certain embodiments, the disintegration enhancing excipient is selected from the group consisting of cellulose, guar gum, crospovidone, polyplasdone, soy polysaccharides, calcium silicate, gelatin, cation exchange resins, bentonite, citrus pulp, alginic acid, calcium alginate, methylcellulose, microcrystalline cellulose, sodium carboxymethylcellulose, croscarmellose, solka floc, corn starch, sodium starch glycolate (Explotab, Primojel), glycine, hydroxypropyl starch, and starch 1500. In certain embodiments, the pharmaceutical composition comprises up to about 99% disintegration enhancing excipient such as mannitol and/or microcrystalline cellulose. In certain embodiments, the pharmaceutical composition comprises from about 0 to about 70% disintegration enhancing excipients selected from the list consisting of lactose, sucrose, and calcium phosphate. In certain embodiments, the pharmaceutical composition comprises from about 0 to about 50% disintegration enhancing excipients selected from the list consisting of sodium bicarbonate, citric acid, maleic acid, adipic acid, and fumaric acid. In certain embodiments, the pharmaceutical composition comprises from about 0 to about 20% disintegration enhancing excipients selected from the list consisting of sodium starch glycollate, pregelatinized starch, crospovidone, and croscarmellose sodium. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 70% mannitol, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%. In certain embodiments, the pharmaceutical composition comprising Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 70% lactose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 70% sucrose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 70% microcrystalline cellulose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 20%, about 30% about 40%, about 50%, about 60% or about 70%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 20% sodium starch glycolate, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 20% pregelatinized starch, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 20% crospovidone, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from about 0 to about 20% croscarmellose sodium, including but not limited to any amount that achieves the desired results, for example up to about 1%, about 2%, about 3%, about 5% about 7%, about 10%, about 12%, about 15% or about 20%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% xanthan gum, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% polycarbophil, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% polyethylene oxide, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% hydroxyethylmethyl cellulose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% hydroxyethyl cellulose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% hypromellose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% hydroxypropyl cellulose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% PVP, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In certain embodiments, the pharmaceutical composition comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises from 0 to about 50% microcrystalline cellulose, including but not limited to any amount that achieves the desired results, for example up to about 5%, about 10%, about 15%, about 20% about 25%, about 30%, about 35%, about 40% or about 45%. In typical embodiments according to the present invention, any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III and the compositions described are used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV. In certain embodiments, a tablet used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 250 mg of microcrystalline cellulose, about 20 mg of crospovidone, about 5 mg of magnesium stearate, about 5 mg of silicon dioxide, about 5 mg of polyethylene oxide, and about 100 mg of mannitol. In certain embodiments, a tablet used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV further comprises about 155 mg microcrystalline cellulose, about 1.75 mg of magnesium stearate, and about 17.5 mg of mannitol. In certain embodiments, a semi-solid formulation used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 15 mg of carbomer, about 50 mg of propylene glycol, about 10 mg of sorbic acid, about 5 mg of EDTA, and about 920 mg of water. In certain embodiments, a semi-solid formulation used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 20 mg of carbomer; about 70 mg of mineral oil; about 80 mg of a mixture of polyoxyl 6 stearate Type I, ethylene glycol stearates and polyoxyl 32 stearate type 1; about 5 mg parabens; about 60 mg propylene glycol; about 5 mg EDTA; and about 760 mg water. In certain embodiments, a dry powder for reconstitution is used to treat, prevent, or delay an HPV infection or a secondary disease state, condition, or complication of HPV comprising any of the active compounds described herein, including but not limited to Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III further comprises about 15.5 mg xanthan gum, about 19.8 mg mannitol, about 5 mg silicon dioxide, and about 0.5 mg sodium benzoate. VII. Combination and Alternation Therapy The treatments described herein for cervical or vaginal intraepithelial neoplasia can be combined with conventional approaches such as, but not limited to, excision or ablation of the transformed zone. Techniques include cryotherapy, laser therapy, loop electrosurgical procedure (LEEP) and cone biopsy. All of these surgical procedures damage the affected areas and can lead to scarring. The most common intervention for cervical intraepithelial neoplasia, LEEP, is effective in 60-90% of cases, however, it has been associated with a significantly increased risk of miscarriage, ectopic pregnancies, and negative psychological outcomes. In certain embodiments, the treatments described herein in combination with the retaining device are used to lessen, ameliorate or substitute for the use of these conventional practices. In certain embodiments, the treatments described herein can be used in combination with a surgical technique. In certain embodiments, a patient in need thereof can receive surgery before, during and/or after administration of an effective amount of a compound described herein. In certain embodiments, the compound is administered in combination with the retaining device about or less than about 8 months, 6 months, 4 months, 3 months, 2 months, or 1 month prior to surgery. In certain embodiments, the compound is administered in combination with the retaining device about or at least about 1 month, 2 months, 3 months, 4 months, 6 months, or 8 months after surgery. In certain embodiments, the surgical procedure can be an excision of the target and/or diseased tissue, including but not limited to loop electrosurgical excision procedure (LEEP), large loop excision of the transformation zone (LLETZ), knife conization, cold knife conization, knife cone biopsy, or laser conization. In certain embodiments, the surgical procedure can be ablation, including but not limited to laser ablation or cryoablation. The efficacy of a drug against an HPV infection or neoplasia, may be prolonged, augmented, or restored by administering the compound in combination or alternation with another, and perhaps even two or three other, antiviral compounds that induce a different mutation or act through a different pathway, from that of the principal drug. Alternatively, the pharmacokinetic, biodistribution, half-life, or other parameter of the drug can be altered by such combination therapy (which may include alternation therapy if considered concerted). Furthermore, since HPV is associated with several types of cancer, combination therapy with anticancer therapeutics can provide better outcomes for patients. Since the disclosed Compound I or a pharmaceutically acceptable salt thereof such as a monofumarate or Compound II or Compound III are DNA Polymerase inhibitors, it may be useful to administer the compound to a host in need thereof in combination with, for example: a) a protease inhibitor; b) another DNA polymerase inhibitor; c) an inhibitor of E6 or E6AP such as MEDI0457, luteolin, CAF-24 or gossypetin; d) an inhibitor of E7; e) an inhibitor of E1 or E2, including inhibitors of the E1-E2 protein interaction; f) L2 lipopeptides; g) an inhibitor or degrader of L1 or L2; h) an HDAC inhibitor such as vorinostat; i) degraders of tetraspanins such as CD9, CD63 or CD151; j) immunotherapeutics such as T-cell therapies (including adoptive T-cell therapies) and checkpoint inhibitors; k) anti-proliferative drugs; l) a therapeutic vaccine; m) a prophylactic vaccine; n) trichloroacetic acid; o) salicylic acid; p) imiquimod; q) podofilox; r) Gardasil® 9; s) Gardasil® 4; t) Cervarix; u) VGX-3100; v) GGX-188E; and/or w) ADXS11-001. VIII. Variations of Solid-State Forms Any of the materials described herein that include Compound I in any stereochemical designation, or mixtures thereof, along with one or more additional compounds in any selected ratio, such as but not limited to an acid as described herein or water (or other solvent) in a solid form, can be used for HPV anti-viral or intraepithelial neoplasia therapy. Such solid forms, when they contain Compound I so described and one or more additional compounds, may be considered multicomponent solids and, when crystalline, multicomponent crystalline forms. For example, “crystalline A B” would be considered a multicomponent crystalline form of compounds A and B. A salt is an example of a multicomponent solid, and a crystalline salt is a multicomponent crystalline form. Salts typically exhibit proton transfer between an acid and a base. Other multicomponent crystalline forms include cocrystals. A cocrystal is typically a crystalline solid of two or more neutral species. Because charge balance is not necessary for neutral species, the stoichiometry of cocrystals cannot be determined based on charge balance. In pharmaceutically relevant cocrystals, one component is typically an active drug or prodrug and the other (or others) is usually termed a coformer, and it interacts nonionically with the active drug or prodrug in the crystal lattice. In some embodiments, the solid form may be both a salt and a cocrystal. For example, the active drug or prodrug may be in the form of a salt with a counterion and interact nonionically with a coformer. Such a solid form would be a multicomponent solid and, when crystalline, a multicomponent crystalline form. Examples of such cocrystals when the coformer is volatile includes solvates. A solid form may exist in a single crystalline solid form or be polymorphic where more than one crystalline form of the same solid form exists. Polymorphism is typically determined by use of x-ray powder diffraction. Other examples of multicomponent solids are hydrates and solvates. A hydrate is a solid form that includes water in the solid form. In crystalline hydrates, the water may form part of the unit cell of the crystal in which case it is often a stoichiometric hydrate. A solvate is a solid form that includes one or more solvent molecules and, when crystalline, may form part of the unit cell of the crystal in which case it is often a stoichiometric solvate. A hydrate or a solvate can also be used in the therapy described generally herein. In some embodiments, the solid form may be partially crystalline, disordered, or amorphous.. In certain embodiments, any of the solid forms described above that comprise Compound I can be used in an effective amount in the treatment of HPV infection or cervical or vaginal intraepithelial neoplasia in a patient in need thereof, as generally taught herein. A multicomponent crystal comprising Compound I can include a salt form of Compound I, a cocrystal form of Compound I and/or a mixture of a salt and cocrystal form of Compound I. The solid form formed can be a function of the synthesis and crystallization or solidification conditions used, among other factors. In certain embodiments, an effective amount of a multicomponent crystal comprising Compound I and a pharmaceutically acceptable coformer is used in the treatment of cervical or vaginal intraepithelial neoplasia in a patient in need thereof. In certain embodiments, an effective amount of a multicomponent crystal comprising Compound I and a pharmaceutically acceptable coformer is used in combination with a retaining device to treat cervical or vaginal intraepithelial neoplasia in a patient in need thereof. In certain embodiments, the crystalline form formed by addition of about 1 equivalent of fumaric acid to a solution of Compound I optionally in isopropyl alcohol and heptane, optionally in a pharmaceutically acceptable carrier, is used in the treatment of cervical or vaginal intraepithelial neoplasia, optionally in combination with a retaining device. Any solid form based on desired properties that includes Compound I can be used in an effective amount with or without a retaining device to deliver therapy to the HPV-infected patient in need thereof. EXAMPLES The dose strengths in mg used herein refer to the weight of the active acyclic phosphonamidate and do not include the salt in the molecular weight, and thus the total weight in the dosage form. Example 1: Preparation of Fumarate Salts Preparation of Compound I Hemifumarate About 200 mg of Compound I free base was added to 0.5 mL of EtOH. While stirring, 0.5 molar amount of fumaric acid was added at 50°C and the mixture was stirred for 2 hours. A clear solution was obtained. The solution was then cooled to 25°C within 1 hour. Hemi-fumarate seeds of Sample RC13 (Example 5, Table 7) were added, followed by addition of 2.5 mL of heptanes to induce precipitation. An oil was obtained and stirred at 25°C for about 4 days. After 4 days, the resulting suspension was cooled to 5°C. After stirred at 5°C for about 4 days, the precipitated solids were collected by filtration and dried at 40°C under vacuum for about 3 hours. As a result, 116 mg of light-orange hemi-fumarate were obtained in yield of 52%. Preparation of Compound I Monofumarate Pattern 1 (small scale preparation) About 244 mg of Compound I free base was added into 0.8 mL of IPA. Then, 1.0 eq. of fumaric acid was added with stirring at 50°C for about 1.5 hours. The yellow clear solution obtained was cooled to 25°C and stirred for about 5 minutes. Mono-fumarate seeds of Sample RC 18 (Table 7) were added to the mixture, followed by addition of 4 mL of heptanes as an antisolvent. The mixture was stirred at 25°C for 4 days. The precipitated material was collected by filtration and dried at 40°C under vacuum for about 2 hours. As a result, 208 mg of mono- fumarate solids were obtained in yield of 69%. Example 2: Large Scale Preparation of Mono-Fumarate About 4.16 g of Compound I free base was dissolved in 11 mL of IPA. Then, 1.0 eq. of fumaric acid was added to the yellow clear solution under stirring at 50°C. After about 1 hour, some solids precipitated out. Then, 10 mg seeds of mono-fumarate (from the above ‘Small scale preparation’) were added. The mixture was stirred at 50°C for about 1.5 hours and cooled to 25°C, then it was stirred at 25°C for about 10 min. Then, 40 mL of heptanes was added as antisolvent. Obtained suspension was stirred at 25°C for about 24 hours, then cooled to 5°C at a rate of 0.1°C/min and stirred at 5°C for about 1 day. The solids were collected by filtration and dried in the oven at 40°C for about 2 hours under vacuum. About 4.1 g of light pink solids were obtained in a yield of 81.2%. Example 3: Synthesis of Mixture of (R, S) and (S, S) ethyl-2-((((2-(2-amino-6-methoxy-9H- purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (Compound I)
Step 1: Preparation of diethyl-((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)- phosphonate (3)
Figure imgf000091_0001
g, 0.296 mol, 1 equiv.), Cs2CO3 (96.37 g, 0.296 mol, 1 equiv.) and DMF (250 mL) under N2 atmosphere at room temperature. To this at room temperature and under stirring was added diethyl 2- chloroethoxymethyl phosphonate 2 (74.85 g, 0.325 mol, 1.1 equiv.) in a drop-wise manner. The reaction was stirred at 40-50°C for 0.5 to 1.5 hours, heated to 60-70°C and stirred for 0.5-1.5 hours, and then stirred at 75 to 85°C for 18-24 h. After bringing the reaction temperature to 20- 30°C, the reaction mixture was filtered and the resulting cake was washed with DMF (100 mL x 2). The combined filtrate was concentrated to a half volume below 70°C, diluted with n-heptane (250 mL) and again concentrated to a half volume below 75°C. The resulting solution was poured into DCM (1 L), stirred at 20 to 30°C for 20-40 min., then aqueous 10% Na2SO4 solution (~100 mL) was added. The resulting bi-phasic solution was stirred for 20-40 minutes then filtered through diatomite and the wet cake was washed with DCM (~100 mL). From the filtrate, the aqueous phase was separated and the organic phase was again washed with aqueous 10% Na2SO4 solution (~100 mL). The combined aqueous phases upon washing (back extraction) with DCM (200-300 mL), the organic phases were combined and concentrated. The resulting crude product 3 was then purified by silica gel column chromatography using DCM to 1% MeOH in DCM. The fractions containing products were combined and the solvent was evaporated below 40°C. The solid product 3 was treated with the repeated dilution with MTBE and concentration (up to 1/3rd volume). The resulting slurry was then diluted with MTBE (400-500 mL) and agitated at 40-50°C for 4-6 h and at 15-25°C for 8-15 h. The suspension was filter and washed with MTBE and dried at 35-40°C for 15-20 h to afford the desired product, diethyl-((2-(2-amino- 6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonate 3 in 43.4% (48.66 g) isolated yield with 91.8 % purity by HPLC.1H NMR (400 MHz, DMSO-d6), δppm: 8.08 (s, 1H), 6.91 (s, 2H), 4.24 (d, 2H, J= 8 Hz), 3.92 (m, 4H), 3.86 (q, 4H, J= 8Hz), 1.14 (t, 6H, J= 8 Hz). LCMS (m/z): 364.2 (MH+) and 366.2 (MH+). Step 2: Preparation of ((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonic acid (4)
Figure imgf000092_0001
with diethyl-((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonate 3 (100 g, 0.275 mol) followed by 2,6-lutidine (147.33 g, 1.375 mol, 5 equiv.) and the temperature was adjusted to 0- 5°C. To this was added TMSBr (167.47 g, 1.102 mol, 4.0 equiv.) in a drop-wise manner and stirred further for 0.5-1 h at 0-5°C and 15-20 h at 20-25°C. After adjusting the reaction temperature at 0-5°C, a drop-wise addition of 1144 g aqueous 1N NaOH was performed. After maintaining the temperature at 20-30°C for 1-2 h, the aqueous alkaline layer was separated and repeatedly washed with MTBE. An aqueous solution was acidified with a drop-wise addition of aqueous 2N HCl to pH= 6-7 at 15-25°C and charged with MeOH (10 Vol.). This resulting methanolic solution was further acidified with a drop-wise addition of aqueous 2N HCl to pH= 3-4 at 35-45°C After addition of seeds of product 4, the methanolic acidic solution was stirred at 35-45°C for 3-5 h and acidified furthermore using a drop-wise addition of aqueous 2N HCl to pH= 1.5-2.5 and stirred for 11-20 h at 15-20°C. The resulting solid was isolated by filtration, washing with MeOH (2 x 100 mL) and drying at 45-55°C for 20-30 h to yield the desired product, ((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonic acid 4 in 96.5 % (84.4 g) isolated yield with 99.8 % purity by HPLC.1H NMR (400 MHz, DMSO-d6), δ ppm: 8.1 (s, 1H), 6.92 (bs, 2H), 4.5-5.5 (bs, 2H), 4.22 (dd= t, 2H, J= 8 Hz), 3.84 (t, 2H, J= 8 Hz), 3.58 (t, 2H, J= 8 Hz). LCMS (m/z): 308 (MH+) and 310 (MH+). Step 3: Preparation of ((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-phosphonic acid (5)
Figure imgf000093_0001
6-chloro-9H-purin-9- yl)-ethoxy)-methyl)-phosphonic acid 4 (50 g, 0.162 mol) at 20-30°C, and stirred for 10-30 min. To this solution was added a 30 wt% NaOMe solution in MeOH (1.62 mol, 10 equiv.) in a drop- wise manner and then stirred at 50-60°C for 15-24 h. The reaction was maintained at 20-30°C for 20-40 min and then filtered. The filtrate was then acidified at 20-30°C by a drop-wise addition of conc. HCl to adjust pH= 6-7 and concentrated below 40°C to one third of the volume. The temperature of the concentrated solution was raised to 35-45°C and acidified to adjust the pH= 3- 4 by means of a drop-wise addition of conc. HCl. The resulting acidic solution was charged with the seeds of product 5, and stirred at 35-45°C for 1.5-2.5 h. At this temperature, the addition of conc. HCl to adjust the pH= 2-3 was done in a drop-wise rate, stirred for 3-5 h, cooled to -3°C to 3°C range and stirred for 8-15 h. The resulting solid was filtered, washed with MeOH (~100 mL) and n-heptane (~100 mL). The resulting cake was dried at 50-60°C for 16-24 h under vacuum to afford the desired product, ((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-phosphonic acid 5 in 89.3 % (48.22 g) isolated yield with 99.5 % purity by HPLC.1H NMR (400 MHz, DMSO-d6), δ, ppm: 7.88 (s, 1H), 6.47 (bs, 4H), 4.18 (t, 2H, J= 8 Hz), 3.96 (t, 2H, J= 8 Hz), 3.60 (d, 2H, J= 12 Hz). LCMS (m/z): 304.20 (MH+). Step 4a: Preparation of a mixture of (R,S)- and (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H- purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (8)
Figure imgf000094_0001
To a solution of ((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-phosphonic acid 5 (40 g, 0.132 mol, 1 equiv.) in DCM (560 mL) at 20-30°C under stirring was charged (S)- ethyl 2-aminopropionate hydrogen chloride salt 6 (20.19 g, 0.132 mol, 1 equiv.), benzyl alcohol 7 (71.28 g, 0.66 mol, 5 equiv.) and TEA (159.98 g, 1.58 mol, 12 equiv.), and the solution was stirred for 10-30 min. To this was added a solution prepared from Ph3P (207.5 g, 0.792 mol, 6 equiv.) and 2,2’-dithiopyridine (Aldrithiol-2) (174.24 g, 0.792 mol, 6 equiv.) in DCM (320 mL) at 20-30°C over 60 min. The resulting reaction mixture was stirred at 35-45°C for 15-20 h and concentrated to remove 3/4th of solvent under vacuum below 40°C. To the resulting residue were added MeOH (~120 mL), distilled water (~400 mL), toluene (~400 mL) and n-heptane (~400 mL) and stirred at 20-30°C for 0.5-1 h. After allowing the reaction mixture to stand for 0.5 to 1 h at 20-30°C, the organic phase was separated and the aqueous phase was extracted few more times with a mixture of toluene (~400 mL) and n-heptane (~400 ml) to remove maximum amount of remaining reagents and by-products. The remaining aqueous phase was then extracted with DCM (2 x 400 mL) and upon concentration of DCM under vacuum below 40°C, the crude product was purified by silica gel column chromatography with DCM to 2% MeOH in DCM as a mobile phase. The eluting fractions containing product were combined and solvent was removed under vacuum below 40°C to give the desired product as a mixture of diastereoisomers namely, (R, S) and (S, S); (+) (2S)-ethyl-2-((((2-(2-amino-6-methoxy-9H- purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate 8 (Compound I) in 45.8 % (29.74 g) isolated yield with 98.8 % purity by HPLC.1H NMR (400 MHz, DMSO-d6), δ, ppm: 7.85 (s, 1H), 7.34 (m, 5H), 6.44 (s, 2H), 5.36 (m, 1H), 4.90 (m, 2H), 4.17 (m, 2H), 4.07 (m, 2H), 3.95 (s, 3H), 3.82 (m, 5H), 1.18-1.24 (m, 6H). LCMS (m/z): 493.3 (MH+). Step 4b: Preparation of a mixture of (R,R)- and (S,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H- purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate
Figure imgf000095_0001
To synthesize the (R,R) and (S,R) mixture, the procedure of Step 4a can be performed substituting D-alanine ethyl ester ((R)-ethyl 2-aminopropionate hydrogen chloride salt) for the L- alanine ethyl ester ((S)-ethyl 2-aminopropionate hydrogen chloride salt). Step 4c: Preparation of ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)-phosphoryl)-amino)-propionate
Figure imgf000095_0002
can aminopropionate hydrogen chloride salt) for the L-alanine ethyl ester ((S)-ethyl 2- aminopropionate hydrogen chloride salt). Example 4: Preparation of (+)-Compound I Monofumarate ((+)-(2S)-Ethyl-2-((((2-(2- amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate monofumarate) (9)
Figure imgf000096_0001
To a solution of (+)-(2S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (Compound I) 8 (29.72 g, 0.06 mol, 1 equiv.) in IPA was added a solution of fumaric acid (7.66 mol, 1.1 equiv.) in IPA through a filter at 45-55°C and stirring was continued for 1-2 h. The seeds of the compound 9 were added to the reaction mixture and stirring was continued for 1-2 h at 45-55°C. After allowing the reaction mixture to settle at 20-30°C for 4-6 h, a drop-wise addition of n-heptane (~300 mL) was performed and stirring was continued for another 8-15 h at 20-30°C and 0-5°C for 8-15 h. The solid observed was filtered and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, ~50-60 mL). The solid cake was dried at 35-45°C for 16-24 h under vacuum to afford the desired product, (+)-(2S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)-phosphoryl)-amino)-propionate monofumarate 9 (Compound I monofumarate) in 87.9 % (32.74 g) isolated yield with 99.1 % purity by HPLC.1H NMR (DMSO-d6), δ, ppm: 1.14 (t, 3H, J= 7.2 Hz), 1.22 (d, 3H, J= 7.2 Hz), 3.82 (m, 2H; dd, 1H, J= 4.0 Hz; bs, 2H), 3.95 (s, 3H), 4.06 (m, 2H), 4.17 (m, 2H), 4.87 (m, 2H), 5.38 (q, 1H, J= 4 Hz), 6.44 (s, 2H), 6.64 (s, 2H), 7.33 (m, 5H), 7.82 (s, 1H), 13.18 (bs, 2H). LCMS (m/z): 493.20 (MH+).
Example 5: Chiral Separation of (R,S)- and (S,S)-Isomers of Compound I and preparation of their monofumarate salts, Compound II and Compound III
Figure imgf000097_0002
Figure imgf000097_0003
Figure imgf000097_0001
ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (S,S)-ethyl-2-((((2-(2- amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate Compound I (a diastereoisomeric mixture of Isomers I and II) (22.50 g) was subjected to a chiral chromatography separation under SFC separation conditions as shown below to separate and obtain the 11.7 g of (R,S)-Isomer I (10) with 98.6% purity by HPLC and 9.1 g of (S,S)- Isomer II (11) with 95.6% purity by HPLC. SFC Conditions: Column: ChiralPak AD, 250×30mm I.D., 10µm; Mobile phases: A: CO2 and B: Ethanol (0.1% NH3H2O); Gradient: B 45% isocratic; Flow rate: 200 mL /min; Wavelength: 310 nm; Cycle time: ~6 min; Back pressure: 100 bar; Injection amount: ~1g. Characterization of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (Isomer I) as a free base: Purity by HPLC: 98.6%; 1H NMR (DMSO-d6), δ, ppm: 7.82 (s, 1H), 7.30 (m, 5H), 6.38 (s, 2H), 5.30 (t, 1H), 4.83 (d, 2H), 4.18 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.84 (m, 2H), 3.60 (m, 5H), 1.20 (d, 3H), 1.15 (t, 3H); LCMS (m/z): 493 (MH+). Characterization of (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (Isomer II) as a free base: Purity by HPLC: 95.6%; 1H NMR (DMSO-d6): δppm 7.82 (s, 1H), 7.35 (m, 5H), 6.45 (s, 2H), 5.30 (t, 1H), 4.80 (d, 2H), 4.18 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.80 (m, 3H), 3.70 (m, 2H), 1.20 (d, 3H), 1.15 (t, 3H); LCMS (m/z): 493 (MH+). In certain nonlimiting embodiments, the stereoisomers are separated using HPLC or SFC with achiral or chiral stationary phases. Non limiting examples of chiral stationary phases which may be used include Chiralpak AD, Chiralpak AS, Chiralcel OG, and Chiralcel OJ. In alternative non limiting embodiments, the individual isomers can be synthesized asymmetrically. For nonlimiting examples of asymmetric synthesis of phosphonamidates see Numan, A et al. “Asymmetric Synthesis of Stereogenic Phosphorus P(V) Centers Using Chiral Nucleophilic Catalysis”, Molecules 2021, 26, 3661 and Ambrosi, A. et al. “Synthesis of Rovafovir Etalafenamide (Part III): Evolution of the Synthetic Process to the Phosphonamidate Fragment” 2021, Org. Process Res. Dev.25, 5, 1247-1262. Step 1b: Chiral Separation of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)- ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (S,S)-ethyl-2-((((2-(2- amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate
Figure imgf000099_0001
Separation of (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)-phosphoryl)-amino)-propionate and (S,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H- purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (synthesis described in Step 4b of Example 3) can be performed using same techniques for the (R,S) and (S,S) mixture as described above.
Step 1c: Chiral Separation of (RP)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)- ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (SP)-ethyl-2-((((2-(2- amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate
Figure imgf000100_0001
Separation of (RP)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)-phosphoryl)-amino)-propionate and (SP)-ethyl-2-((((2-(2-amino-6-methoxy-9H- purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (synthesis described in Step 4c of Example 3) can be performed using same techniques for the (R,S) and (S,S) mixture as described above. Step 2a: Preparation of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate (Compound II)
To a solution of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate 10 (3 g, 6 mmol, 1 equiv.) in IPA was added a solution of fumaric acid ( 0.765 g, 6.6 mmol, 1.1 equiv.) in IPA through a filter at 45- 55°C and stirring was continued for 1-2. The seeds of the compound 12 were added to the reaction mixture and stirring was continued for 1-2 h at 45-55°C. After allowing the reaction mixture to settle at 20-30°C for 4-6 h, a drop-wise addition of n-heptane (~30 mL) was performed and stirring was continued for another 8-15h at 20-30°C and 0-5°C for 8-15h. The solid observed was filtered and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, ~5 mL). The solid cake was dried at 35-45°C for 16-24 h under vacuum to afford the desired product, (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)- phosphoryl)-amino)-propionate monofumarate 12 (Isomer I monofumarate or Compound II) in 85 % (3.1 g) isolated yield with 98.6 % purity by HPLC.1H NMR (DMSO-d6), δ, ppm:δ7.80 (s, 1H), 7.35 (m, 5H), 6.63 (s, 2H), 6.40 (s, 2H), 5.53 (t,
Figure imgf000101_0001
2H), 4.15 (t, 2H), 4.00 (m, 2H), 3.92 (s, 3H), 3.80 (m, 3H), 3.75 (m, 2H), 1.20 (d, 3H), 1.13 (t, 3H); Base (10): Fumaric acid ratio: 1: 1.00 (by 1H NMR). Step 2b: Preparation of (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate (13), also referred as Compound III
Figure imgf000101_0002
To a solution of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate 11 (3 g, 6 mmol, 1 equiv.) in IPA was added a solution of fumaric acid ( 0.765 g, 6.6 mmol, 1.1 equiv.) in IPA through a filter at 45- 55°C and stirring was continued for 1-2. The seeds of the compound 13 were added to the reaction mixture and stirring was continued for 1-2 h at 45-55°C. After allowing the reaction mixture to settle at 20-30°C for 4-6 h, a drop-wise addition of n-heptane (~30 mL) was performed and stirring was continued for another 8-15h at 20-30°C and 0-5°C for 8-15h. The solid observed was filtered and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, ~5 mL). The solid cake was dried at 35-45°C for 16-24 h under vacuum to afford the desired product, (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)- phosphoryl)-amino)-propionate monofumarate 13 (Isomer II monofumarate or Compound III) in 80 % (2.9 g) isolated yield with 95.6 % purity by HPLC.1H NMR (DMSO-d6), δ, ppm: δ7.82 (s, 1H), 7.35 (m, 5H), 6.62 (s, 2H), 6.35 (s, 2H), 5.30 (t, 1H), 4.90 (d, 2H), 4.15 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.80 (m, 3H), 3.70 (m, 2H), 1.20 (d, 3H), 1.15 (t, 3H); Base (11): Fumaric acid ratio: 1: 1.2 (by 1H NMR). Step 2c: Preparation of (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (S,R)-ethyl-2-((((2- (2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate monofumarate
Figure imgf000102_0001
Figure imgf000102_0002
Synthesis of (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)- (benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (S,R)-ethyl-2-((((2-(2-amino-6- methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate can be carried out as in Steps 2b and 2c for the (R,S) and (S,S) stereosiomers, substituting the starting materials in Steps 2b and 2c with the product of the chiral separation in Step 1b. Step 2d: Preparation of (RP)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (SP)-ethyl-2-((((2- (2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)- propionate monofumarate
Figure imgf000103_0001
Figure imgf000103_0002
- - - - (benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (SP)-ethyl-2-((((2-(2-amino-6- methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate can be carried out as in Steps 2b and 2c for the (R,S) and (S,S) stereosiomers, substituting the starting materials in Steps 2b and 2c with the product of the chiral separation in Step 1c. Example 6. Nonlimiting examples of preparation of semisolid formulations A topical cream formulation can be prepared, for example, by emulsifying an oil phase and an aqueous phase along with an active pharmaceutical ingredient. In a non-limiting embodiment, the oil phase of the cream was prepared by mixing light mineral oil, propylparaben and Tefose® 63. Next, the aqueous phase of the cream was prepared by mixing water, EDTA, methylparaben, and Carbopol®974P. The oil and water phases were then emulsified. To the emulsified mixture was added the active pharmaceutical ingredient and propylene glycol. The mixture was pH adjusted and then filled into tubes. A topical gel formulation can be prepared, for example, by mixing an aqueous gel carrier with an active pharmaceutical ingredient. In a non-limiting embodiment, the aqueous phase of the topical gel was prepared by mixing water, EDTA, methylparaben (or sorbic acid) and Carbopol®974P. The active pharmaceutical ingredient and propylene glycol was added to this solution, mixed and pH adjusted, then filled into tubes. In certain nonlimiting embodiments, from about 0.001% w/w to about 10% w/w active pharmaceutical ingredient is added to the semisolid formulation. For example, from about 0.0025% w/w to about 2.5% w/w, such as 0.003%, 0.01%, 0.03%, 0.1%, 0.3% or 1%. Example 7. Preparation of Compound I Monofumarate vaginal tablets A nonlimiting example of the preparation of vaginal tablets of Compound I monofumarate is provided below (See FIG.5 for a flow diagram). Two or more of the excipients are combined, blended, and screened to make the excipient blend. Then the active pharmaceutical ingredient (such as Compound I monofumarate) is screened and added to a portion of the excipient blend. The resulting mixture is then blended and then more excipient blend is added. The mixture is thus gradually diluted with the excipient blend, with thorough mixing after each addition of excipient blend. Once the Excipient blend has been used up, the magnesium stearate is added and the mixture blended once more. The mixture is then compressed into tablets and packaged. Table 2. Batch Formula for Compound I monofumarate vaginal tablets, 0.3 mg free base dose for batch size of 1.0 Kg Ingredient % w/w Qty/batch (g)
Figure imgf000104_0001
Ingredient % w/w Qty/batch (g)
Figure imgf000105_0001
Compound I monofumarate vaginal tablets, 0.3 mg A non- example of a process to prepare the vaginal tablet of Compound I monofumarate is provided Dispensing 1. Weigh the materials as per the batch manufacturing formula and dispense in separate poly bags. Screening 1. Sieve all excipients through a screen. Blending of Active and Screening 1. Blend the screened excipients: microcrystalline cellulose and mannitol in a diffusion blender. 2. Take 49.5 grams of the excipients blend and add 2.12 grams of Compound I monofumarate 3. Blend the active and the excipients, and screen to remove chunks 4. To this blend add 148.5 grams of the excipients blend 5. Blend the active blend and the excipients and screen to remove chunks 6. To this blend add 247.5 grams of the excipients blend 7. Blend the active blend and the excipients and screen to remove chunks 8. To this blend add the remaining 495 grams of the excipients blend 9. Blend the active blend and the excipients and screen to remove chunks Final Blending 10. Add magnesium stearate to the diffusion blender and mix the contents. 11. Discharge and reconcile blend. Compression 1. Compress the blend on a rotary tablet press using appropriate tooling (punches and die), to target weight. Check friability and disintegration at the beginning of the compression run, and check periodically for individual tablet weights, thickness and hardness. Packaging 1. Package bulk tablets into double lined re-closable clear PE bags with desiccants between the bags and further into an aluminum foil pouch with desiccant and heat sealed. Illustrative excipients for a vaginal tablet formulation Tablet formulations are optionally selected to display the properties of mucoadhesion and substantivity and include excipients that have solubilizing, erosion-generating (for disintegration), porosity (for water uptake) and viscosity enhancing (to keep the drug at the target site) properties. Examples of excipients that will cause rapid disintegration to cover the cervix or vaginal areas include, but are not limited to mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid or fumaric acid. Examples of excipients that can enhance disintegration and coverage of the affected area include but are not limited to sodium starch glycollate, pregelatinized starch, crospovidone and croscarmellose sodium. Mucoadhesive excipients that are useful in the present invention include but are not limited to microcrystalline cellulose, polycarbophil, hydroxymethyl cellulose, hypromellose, hydroxypropyl cellulose, and PVP. The table below lists non-limiting examples of combinations of excipients which have the desired properties for a tablet formulation. A tablet formulation may for example comprises the active pharmaceutical ingredient, microcrystalline cellulose and may contain mannitol. In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the rapid disintegrant category (left column of Table 3). In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the disintegration enhancement category (middle column of Table 3). In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the mucoadhesive excipient category (right column of Table 3). Table 3. Excipients for Tablets Rapidly disintegrating To Enhance Mucoadhesive polymers d i t t Dii t ti d e o
Figure imgf000107_0001
Example 8. Illustrative excipients for a reconstitution powder or dry powder formulation A reconstitution powder or dry powder formulation may improve the shelf stability of a pharmaceutical agent or formulation. In certain nonlimiting embodiments, the dry powder formulation may be mixed with saline, propylene glycol or other aqueous carrier shortly before it is administered, minimizing the time for degradation. In certain nonlimiting embodiments, the dry powder formulation is mixed with an oil, cream, or other nonaqueous carrier shortly before it is administered. In certain embodiments, the reconstitution powder or dry powder formulation rapidly covers the infected or diseased tissue in the cervix, vulva or vagina. Excipients which enhance the rapid coverage of the cervix, vulva or vagina, include but are not limited to mannitol, lactose, sucrose, calcium phosphate, and microcrystalline cellulose. In certain embodiments the excipient for rapid coverage of the cervix, vulva or vagina is mannitol. In certain embodiments, the reconstitution powder or dry powder formulation has good coverage of the cervix, vulva or vagina. Nonlimiting examples of excipients which enhance the coverage of the cervix, vulva or vagina, include sodium starch glycollate, pregelatinized starch, crospovidone, and croscarmellose sodium. In certain embodiments the reconstitution powder or dry powder formulation contains mucoadhesive properties once it has been reconstituted. This prevents smearing of the dosage form or otherwise exposing healthy tissues to the active pharmaceutical ingredient. Excipients which improve the mucoadhesive properties of the reconstituted powder or dry powder formulation include but are not limited to xanthan gum, polycarbophil, polyethylene oxide, hydroxyethylmethyl cellulose, hydroxyethyl cellulose, Hypromellose, hydroxypropyl cellulose, PVP, and microcrystalline cellulose. In certain embodiments, the excipient which improves mucoadhesion is xanthan gum. The table below lists combinations of excipients which have the desired properties for a reconstitution powder or dry powder formulation. A dry powder or reconstitution powder formulation comprises the active pharmaceutical ingredient and may contain mannitol and/or xanthan gum. In certain non-limiting embodiments, the dry powder or reconstitution powder formulation comprises one or more excipients selected from the rapid coverage category (left column of Table 4). In certain non-limiting embodiments, the dry powder or reconstitution powder formulation comprises one or more excipients selected from the coverage enhancement category (middle column of Table 4). In certain non-limiting embodiments, the dry powder or reconstitution powder formulation comprises one or more excipients selected from the mucoadhesive excipient category (right column of Table 4). Table 4. Excipients for Reconstitution Powders of Dry Powder Dosage Forms Rapidly covering To Enhance coverage Mucoadhesive polymers PVP (0 to 50%) e
Figure imgf000110_0001
Example 9. Illustrative excipients for a semisolid formulation Semisolid formulations are selected to display the properties of mucoadhesion and assist in the drug penetration into the tissue. Semisolid formulations may include excipients that have solubilizing, lipophilic (to assist in solubilizing lipophilic compound), penetration enhancing (for higher activity) and mucoadhesive (to keep the drug at the target site) properties. In certain embodiments, the semisolid formulation is mucoadhesive. Excipients which contribute to the mucoadhesive properties include but are not limited to carbomer, polyethylene glycol, crospovidone, polycarbophil, Hypromellose, and hyroxyethyl cellulose. In certain embodiments, the semisolid formulation enhances the penetration and/or solubility of the active pharmaceutical ingredient. Excipients which enhance the penetration and/or solubility of the active pharmaceutical ingredient include but are not limited to polyoxyl 6 stearate type I, ethylene glycol stearate, polyoxyl 32 stearate type I, and propylene glycol. The table below lists combinations of excipients which have the desired properties for a semisolid formulation. A semisolid formulation comprises the active pharmaceutical ingredient and one or more excipients from each column of Table 5. In certain non-limiting embodiments, the semisolid formulation comprises one or more excipients selected from the mucoadhesive polymer category (left column of Table 5). In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the solubility and penetration enhancers category (second column of Table 5). In certain non-limiting embodiments, the semisolid formulation comprises one or more excipients selected from the lipophilic solubilizer category (third column of Table 5). In certain non-limiting embodiments, the semisolid formulation comprises one or more excipients selected from the penetration enhancer category (right column of Table 5). Table 5. Excipients for semisolid dosage forms Mucoadhesive Solubility and Lipophilic Penetration l t ti l bili E h
Figure imgf000111_0001
Sorbitan Propylene glycol monostearate (0 to monoca r late (0 to
Figure imgf000112_0001
Example 10. Illustrative excipients for a semisolid formulation Pessary and film forming formulations are selected to be solid at room temperature but soften to release the active pharmaceutical ingredient at body temperature. This allows for easy handling and storage of the formulation as well as achieving desired tissue coverage at the cervix. In a non-limiting embodiment of a film forming formulation, one or more excipients from the left column of Table 6 provide the desired properties. In a non-limiting embodiment of a pessary formulation, one or more excipients from the right column of Table 6 provide the desired properties. Table 6. Excipients for films and pessaries Films (Film Formers) Pessaries (Vaginal Suppositories)
Figure imgf000113_0001
Example 11. Illustrative vaginal tablet formulations In certain non-limiting embodiments, the formulation for a vaginal tablet dosage form comprises the ingredients in Table 7. In certain non-limiting embodiments, the formulation for a tablet dosage form comprises the ingredients in Table 7. An illustrative process for combining these ingredients into a tablet dosage form can be found in Example 7. Table 7. Example Tablet Formulation Material Amount in tablet
Figure imgf000114_0001
Table 8. Example Tablet Formulation Material Amount in tablet
Figure imgf000114_0002
Example 12. Illustrative semisolid formulations In certain non-limiting embodiments, the formulation for a gel semisolid dosage form comprises the ingredients in Table 9. In certain non-limiting embodiments, the formulation for a cream semisolid dosage form comprises the ingredients in Table 10. An illustrative process for combining these ingredients into a cream or gel semisolid dosage form can be found in Example 6 above. Table 9. Example semisolid formulation (gel) Material Amount per Gram h
Figure imgf000115_0001
Table 10. Example semisolid formulation (cream) Material Amount per Gram
Figure imgf000115_0002
Mineral oil 70 mg
Figure imgf000116_0001
Example 13. Illustrative pessary formulation In certain non-limiting embodiments, the pessary formulation comprises the ingredients listed in Table 11 or Table 12. The pessary dosage form can be prepared, for example, by mixing the active pharmaceutical ingredient with the excipient. In one non-limiting embodiment, the excipient is heated in a mixing apparatus while stirring until it has softened or melted, then the active pharmaceutical ingredient is added portionwise. The temperature, stirring speed, and rate of addition are controlled to ensure an even distribution of active pharmaceutical ingredient. The mixture is then mixed until homogeneous and placed into pessary or suppository molds to solidify.
Table 11. Example formulation for a pessary Pessary Excipients Amount per Gram h
Figure imgf000117_0001
Table 12. Example formulation for a pessary Pessary Excipients Amount per Gram h
Figure imgf000117_0002
Example 14. In-vitro cytotoxicity testing Compounds: Three compounds (Compound I, Compound II and Compound III) were solubilized at 40 mM in DMSO and stored at -20°C. The test compounds were evaluated using a high test concentration of 50 µM. Serial half-logarithmic dilutions were performed for the in vitro cytotoxicity assays. Tamoxifen citrate was purchased from Sigma-Aldrich (St. Louis, MO). Tamoxifen citrate was solubilized in DMSO at 40 mM and used as a positive control compound at a high test concentration of 100 µM for the cytotoxicity assays. In Vitro Cytotoxicity Evaluations: Cells listed in Table 13 were enumerated by Trypan Blue Dye exclusion method and seeded in the interior wells of a 96 well flat bottom microtiter plate at 100 µL/well. Proliferating cells were incubated overnight at 37°C/5% CO2 to allow the cells to adhere to the plates at approximately 70% confluency. Tissue culture medium was removed and replaced with 100 µL/well of fresh medium. One-hundred microliters (100 µL) of each compound at six concentrations was transferred to the 96-well plate containing the cells in triplicate. Table 70 lists the cell line, type of cell, source of cell stock, base tissue culture medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, and microtiter plate seeding density. Table 13: Cell Culture for in vitro Cytotoxicity Cell Seeding Cell Line Cell Type Cell Source C lt D ity l) 4
Figure imgf000118_0001
Cytotoxicity XTT: Following incubation at 37°C in a 5% CO2 incubator for five days, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5- sulfopheny1)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product. XTT solution was prepared daily as a stock of 1 mg/ml in RPMl1640. Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/ml in PBS and stored in the dark at - 20°C. XTT/PMS stock was prepared immediately before use by adding 40 µL of PMS per ml of XTT solution. Fifty microliters of XTT/PMS were added to each well of the plate and the plate was reincubated for 4 hours at 37°C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader. Data Analysis and Evaluation: Microsoft Excel 2010 was used to analyze and graph the raw data. CCso (50% reduction in cell viability) values are tabulated and provided. Raw data for cytotoxicity with a graphic representation of the data are provided in a printout summarizing the compound effect on cell viability. In Vitro Cytotoxicity Evaluations: Compounds I, II, and III were evaluated for cytotoxicity to proliferating Hs27, HeLa, C33A and HEK293 cells by measuring cell viability using XTI tetrazolium dye following five days in culture (Table 14). The CC50 values calculated from these assays are summarized in the below tables. Tamoxifen citrate was evaluated in parallel as a control compound. The CC50 value for tamoxifen citrate in proliferating C33A, HeLa, Hs27, and HEK293 cells was 17.2, 19.9, 21.2 and 21.3 µM, respectively. Compound I and its two isomers were similarly cytotoxic when evaluated in parallel against each of the four cell lines. CC50 values for the three test compounds ranged from approximately 0.1 to 0.28 µM in C33A cells. In HeLa cells, CC50 values for the three test compounds ranged from 15.1 to 18.6 µM. The CC50 values for the three test compounds ranged from approximately 7.62 to 23.2 µM in Hs27 cells. In HEK293 cells, CC50 values for the three test compounds was approximately 0.1 µM. Table 14: In vitro Cytotoxicity Data Compound C33A HeLa Hs27 HEK293 )
Figure imgf000119_0001
Example 15. Ex Vivo permeation and penetration of antiviral drugs across porcine vaginal tissue Preparation of Porcine Vaginal Tissue Freshly harvested porcine vaginal tissue was procured from local slaughterhouse in an ice box. The vaginal tissue was cut open to expose the mucosal surface and tissue was cleaned by gentle flow of PBS pH 7.4. The porcine vaginal tissue was cut into circular portions (approximately 2 cm2) with help of a telemetric punch. Mounting Porcine vaginal tissue on Franz diffusion cells The circular portion of tissue was sandwiched between two chambers of a Franz diffusion cell with an active diffusion area of 1 cm2, and the mucosal layer was exposed to the donor chambers. The resistance across porcine vaginal tissue was measured using a wave form generator to ensure the integrity of the tissue segment used for the permeation study. Porcine vaginal tissue with resistance of ≥3 KΩ.cm2 was used for study. The receiver chamber was filled with 8 ml of 5% solutol PBS 7.4 pH, which was stirred at 600 rpm with a 3 mm magnetic stir bar and the temperature was maintained at 37 °C with a circulating water bath. Loading of formulation in the donor chamber The ~200mg of gel was filled into tared 1mL syringe and gel was dispensed to donor chamber. The gel was spread on to mucosal surface with pre weighed applicator. After loading and spreading of the gel onto surface of mucosal surface, the weight of 1mL syringe and applicator was noted to determine the exact amount of gel loaded into donor chamber. Permeation and penetration study Time course (2h, 4h and 8h; Table 15) porcine vaginal permeation studies were performed. After loading gel, 500 μL samples were withdrawn from the receiver compartment at different time intervals and each time an equal volume of fresh receiver media was used to replace withdrawn samples. The sample withdrawn at each time interval were stored immediately in -20 ̊C until analysis. After 2, 4 and 8h, the formulation was removed from the donor chamber with help of syringe and cleaned with cotton swab. The tissue was removed and washed gently with wash solution (50% methanol in water) 5 times and alternatively cleaning with cotton swab. Table 15: Time course IVPT study design Study Receptor fluid sampling time points Study Period 2h IVPT 0 and 2h 2h
Figure imgf000121_0001
An 8 mm punch of active diffusion area of washed porcine vaginal tissue was removed, weighed and transferred into a tube. This tube was immediately placed into dry ice for ~15 min. After specified time, tissue was removed and placed in precooled dish. The tissue was minced into smaller pieces on a dish with a precooled surgical blade. The minced tissue was transferred to sample tubes and dish was rinsed with 1 ml of 5% solutol in PBS 7.4 pH and transferred to same tissue sample tube. These tubes were stored in -70 ̊C until analysis. Preparation of receptor fluid for analysis The samples stored at -20 °C was removed and thawed at room temperature for 30 min. The drug from receptor fluid was centrifuged at 13000 rpm for 5 min and to 200μL of supernatant equal volume of extraction solvent was added. These samples were centrifuged at 13000 rpm for 5 min and supernatant was transferred into vials for analysis. Extraction of Drug from Porcine Vaginal tissue The minced tissue samples stored at -70 ̊C were removed and thawed at room temperature for ~90 min. The samples were kept for shaking at room temperature for 4 h in BioShaker. After 4 h, samples were centrifuged at 13000 rpm for 5 min. To 100 μL of supernatant, 400 μL of extraction solvent was added and vortexed for 2 min. These samples were centrifuged at 13000 rpm for 5 min and supernatant was transferred into vials for analysis. Example 16. Nonclinical Studies in Non-human Animals Two in vivo studies evaluated the local toxicity and toxicokinetics of Compound I vaginal tablets following repeated intravaginal administration to rabbits for 2 weeks (6 doses, 3x/week) at doses ranging from 0.003 mg to 2.9 mg Compound I per tablet. Rabbits (approximately 3 kg in size) were administered tablets with the same excipient profile as those proposed for use in clinical studies, but smaller in size (50 mg rabbit tablets vs.175 mg clinical tablets). Tablets were administered through a lubricated cannula inserted into the vagina, followed by a small amount of normal saline to aid in tablet disintegration. Rabbits were dosed 6 times over 2 weeks (dose administration on Study Days 1, 3, 5, 8, 10, and 12) and necropsied on Day 15. In general, Compound I administration was well tolerated. Findings from the two rabbit studies in the locally exposed tissues of vulva, vagina and cervix are summarized below. Vulva The most significant findings in both tablet studies were in the vulvar tissues, with dose- dependent erythema and edema observed after about the third dose (i.e., the second week when administered 3x/week) that was dose-strength related. While some transient increases in erythema and edema were seen across all groups (including placebo control tablets and sham controls in which the cannula was inserted into the vagina) which was attributed to the dosing procedure, in both studies there were individual erythema and edema scores of ≥ 3 (scored out of 4) seen in the 2.9 mg Compound I animals (high dose group) starting at Day 8. In the first of the two studies, there were a few instances of more substantial vulvar irritation seen at necropsy in the 2.9 mg dose group (abrasion, scab and/or thickened vulva) and microscopic findings of moderate vulvar ulceration and inflammation; these vulvar findings were not seen in the second study where animals were also dosed at 2.9 mg and may be due in part to more familiarity with the dosing procedure by the technical staff. The vulvar findings are presumed to be related to direct contact with Compound I, possibly due to leakage given the position of the urethra in the vagina of rabbits, resulting in increased vulvar exposure to Compound I during urination. These results support the need for the use of a retaining device in combination with the therapeutic administration. Vagina and Cervix Many animals, including sham and placebo controls, had microscopic infiltrates of a mixed cell population noted within the lamina propria of sections of vulva and vagina after repeated administration of Compound I vaginal tablets. Within the vagina, microscopic findings in the two studies included minimal to mild epithelial degeneration, mixed cell inflammation and luminal exudate in animals administered tablets ≥ 0.03 mg Compound I. In the first study, two cases of minimal arterial degeneration/necrosis were seen at the 2.9 mg dose, which was characterized by disruption of the arterial wall with karyorrhectic (nuclear) debris and/or fibrinous change. In addition, one animal treated at 2.9 mg had moderate ulceration in the middle portion of the vagina. These findings were not observed in the second, larger GLP study. In general, the lower portion of the vagina near the vaginal opening was less affected than the upper, or cervical/cranial, vagina nearer the site of administration. Systemic Exposure Compound I vaginal tablets are formulated for topical administration at the site of action with the goal of producing minimal systemic exposure. In rabbits, dose-dependent increases in systemic exposure (Cmax and AUC) were observed following intravaginal administration of Compound I vaginal tablets at doses of 0.03, 0.29 and 2.9 mg Compound I. No accumulation was observed after repeated (i.e., 3x/week for 2 weeks) administration. The extent of systemic exposure in humans is expected to be lower than observed in rabbits due to the differences in body mass. In addition, physiologic/anatomic differences between rabbits and humans are expected to result in decreased bioavailability in humans compared with rabbits due to a smaller relative surface area and increased thickness of vaginal and cervical epithelial tissue. Toxicity Studies in Rabbits In addition, three in vivo studies of up to 2-week duration were conducted in rabbits using Compound I Vaginal Gel. Those studies were designed to examine both the local and systemic toxicity associated with intravaginal administration of Compound I at dose strengths ranging from 0.01% to 1% (in a 1 mL dose, this correlates to administering 0.1 mg to 10 mg of Compound I). In general, the local findings were similar in nature to those described following administration of the vaginal tablet, although more vulvar irritation was seen, presumably due to the greater extent of direct tissue exposure with gel leakage and urination. One of the 2-week studies included 4-week recovery groups to understand the reversibility of any toxicity findings. By Day 45 (Recovery), there was partial recovery of Compound I-related microscopic findings in the vulva and lower vagina, and complete recovery of the findings seen in the middle and upper vagina. Systemically, Compound I Vaginal Gel was well tolerated, with no significant findings. Although systemic exposure in patients is intended to be low, the safety evaluation of Compound I included studies with parenteral administration to ensure 100% systemic exposure to the dose, to characterize the potential systemic toxicity. Compound I administered by the IV route in safety pharmacology studies evaluated the potential for adverse effects on the respiratory, cardiovascular, and central nervous systems. No adverse effects were observed. Single-dose IV toxicity studies were also conducted to describe potential systemic toxicity. In rabbits, single doses of 0 (vehicle control), 0.03, 0.3 and 3.0 mg/kg Compound I were given intravenously and animals were monitored for 7 or 23 days before sacrifice for necropsy and microscopic examination of tissues. Compound I was well tolerated with no signs of toxicity at any dose and all animals survived to the scheduled termination. There were no changes in chemistry or coagulation parameters. Decreased bone marrow cellularity, likely due to inhibition of DNA polymerase, was observed at the high dose only (3 mg/kg) in animals sacrificed on Day 7. Although there were no cytologic abnormalities observed in bone marrow smears, there was evidence in bone marrow smears of increased myeloid and erythroid precursors. The bone marrow changes completely reversed by Day 23. Hence, the NOAEL was 3 mg/kg. Systemic exposures (AUC) at a dose of 3 mg/kg after IV dosing were more than 20-fold higher than those observed in intravaginal toxicology studies at maximally tolerated dose strengths and are, on that basis, also expected to be much higher than potential human exposure by intravaginal application. Toxicity Studies in Dogs A similarly designed single-dose IV toxicity study was also completed in female dogs given doses of 0, 0.02, 0.06 and 0.3 mg/kg, again with 7- and 23-day post-treatment observation periods. As in rabbits, Compound I was well tolerated with no clinical signs and all animals survived to their scheduled necropsy. There were no changes in chemistry, hematology, urinalysis or coagulation parameters. The only treatment-related finding was microscopic renal tubular karyocytomegaly at the high dose (0.3 mg/kg) on both Days 7 and 23. Although the change was not considered adverse, it was considered to be related to treatment with Compound I. Karyomegaly is an enlargement of the cell nucleus that has been proposed to be a response to chemical/drug exposure or insult (Hard, 2018). As indicated, the NOAEL for this study was 0.3 mg/kg which is approximately 12-fold greater exposure (by AUC) than the plasma exposure in rabbits dosed intravaginally at the 0.29 mg dose strength. Genotoxicity A standard battery of genotoxicity studies to assess the potential mutagenicity of Compound I was completed. A bacterial reverse mutation (Ames) study was negative. A mammalian chromosomal aberrations study was positive in the presence of metabolic activation at the highest concentration tested; however, there was no evidence of mutagenicity in an in vivo micronucleus assay. These data suggest the risk of genotoxicity following administration of Compound I is low. Taken together, data from 5 intravaginal safety studies (2 with tablet, 3 with gel) suggest that if adverse effects following administration of Compound I vaginal tablet were to occur, they would be expected to be minor and limited to the site of application. Example 17. Human Clinical Studies Compound I monofumarate salt was investigated in a single and multiple-dose, ascending, randomized, placebo-controlled study in healthy human volunteers. The study was conducted in 2 parts. Part A consisted of single ascending dose cohorts administering Compound I Vaginal Gel 0.01% or placebo, and Compound I vaginal tablet up to 0.3 mg or placebo. Part B consisted of 2 multiple ascending dose cohorts administering Compound I vaginal tablet at 0.1 mg and 0.3 mg or placebo (3 doses over 1-week period). The study completed in June 2023. A total of 35 subjects were enrolled in the study: 19 subjects in Part A (13 received Compound I; 6 received placebo) and 16 subjects in Part B (12 received Compound I; 4 received placebo). In Part A and the 0.1 mg dose cohort of Part B, Compound I was safe and well tolerated. No dose limiting toxicities (DTLs) were reached or serious adverse events (SAEs) reported. All AEs were mild or moderate in severity. After review of safety data, it was determined that DLT at the 0.3 mg dose in Part B was reached. Although no SAEs were reported in this cohort, 3 out of 6 subjects receiving Compound I experienced 2 or more moderate genitourinary AEs (i.e. vaginal discomfort, vulvovaginal erythema, vulvovaginal pain, vulvovaginal swelling, urinary tract infection, and bacterial vaginosis), thereby meeting the protocol definition of DLT. As such, the SMC confirmed the maximum tolerated dose (MTD) at 0.1 mg administered 3 times over 1-week period. There was no detectable Compound I in plasma from any subjects in any cohort at any time-points (up to Day 8; LLOQ = 0.1 ng/mL). This study supports the use of a retaining device as an integral part of the therapeutic protocol. Example 18. Human Patient Self-Administration Patients should keep the bottles of vaginal tablets from extreme high temperatures and preferably store the tablets in the refrigerator. Prior to administration, the vaginal tablet is removed from the refrigerator and allowed to come to room temperature for at least 30 minutes. Compound I vaginal tablets can be administered with a vaginal applicator, followed by application of a retaining device. In certain embodiments, Compound I vaginal tablets can be administered with a diaphragm or menstrual cup. Example 19. Exemplary Instructions For Self-Administration Of Compound I vaginal tablets With Vaginal Applicator Tablet administration is preferably carried out at bedtime to reduce displacing medication that may occur when standing or walking. 1. Place all supplies on a clean flat surface 2. Optionally, gloves for administration, vaginal tablet applicator, water-based lubricant (such as KY Jelly, Surgilube or similar), and bottle of Compound I monofumarate vaginal tablets are assembled. 3. Wash and dry hands, and put on gloves. 4. Remove the applicator from its packaging. 5. Remove the Compound I monofumarate vaginal tablet from the bottle and place it into the end of the applicator. 6. To load the tablet into the applicator, retract the plunger slightly and insert the tablet into the barrel of the applicator until it is securely seated. Illustrations of the tablet placed in the barrel of the applicator are shown in FIG 7A. If loaded properly, the tablet will be firmly in the barrel and will not drop even when the applicator is held with tablet facing down. In this position the tablet can be released by pushing in the plunger. 7. Prior to self-administration, add a small amount (about a teaspoon, which should be enough to cover/coat the tablet and applicator) of water-based lubricant onto the tablet and applicator.The lubricant may be provided in individual packets if available; in that case, open the packet and dispense the entire amount of lubricant onto the Compound I monofumarate vaginal tablet. 8. For insertion, the female can position her body in one of two ways: a. Stand upright (standing with her feet apart and her knees bent). It may also be helpful to raise one leg up on a chair or the bed. b. Lie on her back with her knees bent and legs slightly apart. 9. When in a comfortable position, spread the lips at the entrance to the vagina and gently insert the applicator into vagina (as she would for inserting a tampon). Gently advance the applicator until resistance is met. When in position, deliver the Compound I monofumarate tablet onto the cervix by pushing the plunger until it stops. 10. Remove the applicator from the vagina and place in the disposal bag. 11. It is preferred to not engage in any vigorous activities such as jogging, working out, or swimming for 24 hours after applying the vaginal tablet. Example 20. Participant Instructions for Self-Administration of Compound I monofumarate vaginal tablets with Vaginal Retaining Device 1. The diaphragm should be a flexible device that is created to be inserted through the vagina and placed to cover the cervix. The retaining device is used to help prevent leakage from the dissolved Compound I monofumarate vaginal tablet. The device will form a seal against the cervix. The diaphragm with the vaginal tablet is inserted, and then removed approximately 5-10 hours after insertion, and more typically 5-8 hours, for example around or at least 6 hours. 2. vaginal tablet administration should be preferably done at bedtime to reduce displacing medication that may occur when standing or walking. 3. Remove the diaphragm from its packaging. 4. Prior to self-administration, remove the Compound I monofumarate vaginal tablet from the bottle and place it inside the diaphragm first (FIG.7B) 5. Place a small amount (about a teaspoon or 5 mL) of water-based lubricant into the diaphragm to cover the Compound I monofumarate vaginal tablet. The lubricant may be provided in individual packets if available; in that case, open the packet and dispense the amount of lubricant onto the Compound I monofumarate vaginal tablet (FIG.7C). 6. Hold the diaphragm with one hand by placing the thumb and index finger on the grip dimples along the rim. The arrow should point towards her body. See FIG.7D. 7. Proceed to fold the diaphragm. The vaginal tablet will be located on either the left or right side of the fold (See FIG.7E). 8. Alternatively, if the lubricant and vaginal tablet rise up too close to the rim when folding, and appear to be coming out, the patient can push down on the diaphragm dome while folding to allow for one big fold (See FIG.7F). 9. For insertion of the diaphragm the patient can position her body in one of three ways: kneeling down in a sitting position; lying down with legs bent; or standing with one leg elevated. 10. Using her free hand, spread the labia. Push the diaphragm with her other hand into the vagina, and push down gently towards the back, advancing the diaphragm until the cervix rests inside the diaphragm (FIG.7G). 11. Optionally use a finger to verify that the cervix is covered by the diaphragm. If you can feel the cervix through the diaphragm (it should feel like the tip of the nose), then it is positioned correctly (See FIGS.7H, 7I, and 7J). 12. The edge of the removal dome should be located securely and safely behind the upper part. 13. It is preferred not to engage in any vigorous activities such as jogging, working out, or swimming for 24 hours after applying the vaginal tablet. Example 21: Single-Crystal X-Ray Diffraction (SC-XRD) study of Compound II Pattern 1 Single crystals of Compound II Pattern 1 suitable for SC-XRD study were obtained in a temperature cycling experiment in MeOH. X-ray diffraction data were collected on a D8 Venture diffractometer equipped with a CMOS area detector at 170(2) K using Cu-Kα radiation (λ = 1.5418 Å); X-ray generator power: 50 kV, 1.4 mA; Distance from sample to area detector: 40 mm; Exposure time 150 seconds; Resolution: 0.81. Structure refinement: on F2. Hydrogen site location: mixed. H atoms were treated by a mixture of independent and constrained refinement. X-ray diffraction data and crystal data are presented in Table 16. Table 16. Crystallographic parameters and x-ray diffraction data Chemical Formula C21H30N6O6P·C4H3O4 Molecular weight 608.54 of
Figure imgf000129_0001
Crystalline form of Compound II Pattern 1 crystallized in monoclinic system, P21 space group with Rint = 5.7% and the final R1 [I>2σ(I)] = 7.4%. The crystalline form did not contain solvent molecule. This crystalline form of Compound II Pattern 1 was found to have a free base to fumaric acid ratio of 1:1 and corresponds to monofumarate salt of Isomer I with Flack parameter (absolute structure parameter) of 0.16(10). As shown in FIG.1A and 1B, protonated free base and fumaric acid anion are linked through a N+(5)-H(5)···O(7) ionic bond in the single crystal structure. Proton transfer was observed from fumaric acid to the N(5)-nitrogen atom of purine. Example 22: Synthesis of Salts of Compound I Compound I Sulfate Two methods were used to synthesize the sulfate salt of Compound I. Method A To a solution of Compound I (0.049 g, 0.1 mmol) in dry THF (1 mL) at 0-10°C was added a solution of sulfuric acid (1N in THF) slowly. During the addition of sulfuric acid solution, the pink colored, clear THF solution of Compound I free base was changed to an off-white semisolid. The reaction mixture was brought to room temperature over 20-30 minutes and shaken. After allowing the solid material to settle, the supernatant was decanted carefully. The resulting semi- solids were washed with an additional amount of dry THF (2 x 2 mL), and the resulting solids were dried under high vacuum to yield 0.053 g of Compound I Sulfate Salt-1 as an off-white solid. Method B To a solution of Compound I (0.024 g, 0.05 mmol) in dry ethyl acetate (EtOAc, 0.5 mL) at 0-10°C was added a solution of sulfuric acid (1N in EtOAc) slowly. During the addition of sulfuric acid solution, the pink colored EtOAc clear solution of Compound I free base was changed to an off-white colored solid. The reaction mixture was brought to room temperature over 20-30 min and shaken well. After allowing the solid material to settle, the supernatant was decanted carefully. The off-white solids were washed with an additional amount of EtOAc (2 x 2 mL). The resulting off-white solids were dried under high vacuum to yield 0.023 g of Compound I Sulfate Salt-2. Compound I Methanesulfonate
Figure imgf000130_0001
to synthesize the methanesulfonate salt of Compound I. Method A To a solution of Compound I (0.049 g, 0.1 mmol) in dry EtOAc (1mL) at 0-10°C was added neat methanesulfonic acid (MSA; MW=96.11; d= 1.47; 0.007 mL; 0.11 mmol) dropwise. During the addition of MSA solution, the pink colored EtOAc clear solution of Compound I free base was changed in to an off-white semi-solid (glue like). The heterogeneous mixture was brought to room temperature in 20-30 min and shaken well. After allowing semi-solid material to settle down, the supernatant was decanted carefully. The resulting semi-solid (glue like) was washed with methyl tert-butyl ether (MTBE; 2 x 2 mL) and was dried under high vacuum to yield 0.054 g of Compound I Methanesulfonate Salt-1. Method B To a solution of Compound I (0.049 g, 0.1 mmol) in dry IPA (1mL) at 0-10°C was added methanesulfonic acid solution (1N in THF; 0.11 mmol; 0.110 mL) dropwise. During the addition of MSA solution, the pink colored EtOAc clear solution of Compound I free base was changed in to an off-white solid. The heterogeneous mixture was brought to room temperature in 20-30 min and shaken well. After allowing solid material to settle down, the supernatant was decanted carefully. The resulting solid was washed with methyl tert-butyl ether (MTBE; 2 x 2 mL) and was dried under high vacuum to yield 0.049 g of Compound I Methanesulfonate Salt-2. Compound I Hydrochloride Salt
Figure imgf000131_0001
I (MW=492; 0.049 g, 0.1 mmol) in dry EtOAc (1mL) at 0- 10°C was added HCl solution (4N in dioxane; 0.11mmole; 0.027 mL) dropwise. During the addition of HCl solution, the pink colored EtOAc clear solution of Compound I free base was changed in to an off-white solid. This heterogeneous mixture was brought to room temperature in 20-30 min and shaken well. After allowing solid material to settle down, the supernatant was decanted carefully. The resulting solid was washed with methyl tert-butyl ether (MTBE; 2 x 2 mL) and was dried under high vacuum to yield 0.045 g of Compound I HCl Salt. Compound I monofumarate
Figure imgf000131_0002
I (0.035 g, 0.071 mmol) in dry iso-propanol (0.1 mL) at 0- 10°C was added a fumaric acid (MW= 116; 12.3 mg; 0.106 mmol; 1.5 equivalents). The reaction mixture was brought to the room temperature then heated at 60°C for 30 min and stirred at room temperature for 12 h. The reaction mixture was filtered, and the filtrate was concentrated under a reduced pressure at 40°C. The mixture was diluted with MTBE (2 mL), shaken well and the MTBE was decanted carefully. The colorless solid was washed with an additional amount of MTBE (2 mL) and dried under high vacuum to yield 0.022 g of Compound I sesquifumarate salt. Compound I monofumarate salt can be synthesized by washing Compound I sesquifumarate salt with additional MTBE and drying under high vacuum. Compound I Benzenesulfonic Acid Salt g, 0.115 mmol) in dry EtOAc (1.0 mL) at 0-10°C was
Figure imgf000132_0001
a (BsOH, MW=158; 0.020 g; 0.127 mmol in 0.2 mL of EtOAc) dropwise. During the addition of BsOH solution, the pink colored EtOAc clear solution of Compound I free base precipitated as a colorless solid. The reaction mixture was brought to room temperature in 20-30 min, shaken well. After allowing solid material to settle, the supernatant was decanted carefully. The colorless solid was washed with an additional amount of MTBE (2 x 2 mL). The resulting solid was dried under high vacuum to yield 0.064 g of Compound I Besylate Salt. Compound I Tosylate Salt
Figure imgf000132_0002
I (0.024 g, 0.05 mmol) in dry EtOAc (0.5 mL) at 0-10°C was added a solution of p-toluenesulfonic acid (p-TsOH, 0.055 mL; 0.055 mmol; 1N in EtOAc) dropwise. During the addition of p-TsOH solution, the pink colored EtOAc clear solution of Compound I free base precipitated as a colorless solid. The reaction mixture was brought to room temperature in 20-30 min and shaken well. After allowing solid material to settle, the supernatant was decanted carefully. The colorless solid was washed with an additional amount of MTBE (2 x 2 mL). The resulting solid was dried under high vacuum to yield 0.027 g of Compound I Tosylate Salt. Melting points of salts of Compound I obtained in this example were determined by differential scanning calorimetry. The results are presented in Table 4. The monofumarate salt, produced by washing of the sesquifumarate salt has the highest melting point of the salts tested. Table 17. Melting points of salts of Compound I Compound I salt Onset of melting (°C) Sulfate-1 85
Figure imgf000133_0001
Example 23: Preparation of Fumarate Salts Preparation of Compound I Hemifumarate Pattern 1 About 200 mg of Compound I free base was added to 0.5 mL of EtOH. While stirring, 0.5 molar amount of fumaric acid was added at 50°C and the mixture was stirred for 2 hours. A clear solution was obtained. The solution was then cooled to 25°C within 1 hour. Hemi-fumarate seeds of Sample RC13 were added, followed by addition of 2.5 mL of heptanes to induce precipitation. An oil was obtained and stirred at 25°C for about 4 days. After 4 days, the resulting suspension was cooled to 5°C. After stirred at 5°C for about 4 days, the precipitated solids were collected by filtration and dried at 40°C under vacuum for about 3 hours. As a result, 116 mg of light-orange hemi-fumarate Pattern 1 were obtained in yield of 52%. XRPD is shown in FIG.9; DSC is shown in FIG.10; and TGA is shown in FIG.11. Preparation of Compound I Monofumarate Pattern 1 (small scale preparation) About 244 mg of Compound I free base was added into 0.8 mL of IPA. Then, 1.0 eq. of fumaric acid was added with stirring at 50°C for about 1.5 hours. The yellow clear solution obtained was cooled to 25°C and stirred for about 5 minutes. Mono-fumarate seeds of Sample RC 18 were added to the mixture, followed by addition of 4 mL of heptanes as an antisolvent. The mixture was stirred at 25°C for 4 days. The precipitated material was collected by filtration and dried at 40°C under vacuum for about 2 hours. As a result, 208 mg of mono-fumarate Pattern 1 solids were obtained in yield of 69%. XRPD is shown in FIG. 9. DSC at 10°C/min is shown in FIG.12A, DSC at 2°C/min is shown in FIG.12B. DSC cycle results are shown in FIG.12C. TGA is shown in FIG.13. Table 18. Characterization of Compound I free base pattern and its mono- and hemifumarate patterns Compound I Compound I Compound I Monofumarate Pattern %
Figure imgf000134_0001
Compound I Pattern 1 is in high crystallinity. The hemifumarate and monofumarate Patterns are in medium crystallinity. Compound I Pattern 1 is an anhydrate and has a melting peak at Tonset of 75.0°C with an enthalpy of about 64 J/g. It shows about 0.3% weight loss at about 70°C. KF shows it contains about 1.7% of water. About 0.7% of MTBE (by weight) residue was detected by 1H NMR. Compound I hemifumarate Pattern 1 is an anhydrate. The stoichiometry of free form: fumaric acid is about 1:0.5 based on 1H NMR result. It has a melting peak at Tonset of 85.2°C with an enthalpy of about 37 J/g. It shows about 1.0% weight loss at about 85°C. KF shows it contains about 1.7% of water. About 0.7% of EtOH and 0.7% of heptanes (by weight residual) was detected by 1H NMR. Compound I monofumarate Pattern 1 is an anhydrate. The stoichiometry of free form: fumaric acid is about 1:1.0 based on 1H NMR result. It has a melting peak at Tonset of 107.2°C with split peaks and with an enthalpy of about 78 J/g. The two thermal events cannot be resolved with 2 K/min and 0.5 K/min heating rate by DSC as well. It shows about 0.3% weight loss at about 107°C. KF shows it contains about 1.2% of water. About 0.7% of IPA and 2.2% of heptanes (by weight) residue was detected by 1H NMR. Example 24. Stability of Compound I free base Pattern 1, Compound I Monofumarate Pattern 1 and Compound I hemifumarate Pattern 1 Initial chemical purity: Initial purities of Compound I free base Pattern 1, Compound I monofumarate Pattern 1 and Compound I hemifumarate Pattern 1 are 98.7%, 98.6% and 97.8%, respectively. Bulk stability: Accelerated stability experiments were conducted at 25°C/92%RH and 40°C/75%RH in an open container, 60°C in a tight container for one week. Results are presented in Table 19. Results for Compound I monofumarate Pattern 1 are also presented in FIG.14. All the three candidates showed good physical stability after exposure to the three conditions. They all show some degradation after exposure to 40°C/75% RH for one week, and two are not stable at high temperature (60°C for one week). Compound I hemifumarate Pattern 1 tends to degrade more than Compound I monofumarate Pattern 1 in the two conditions mentioned above. Table 19. Bulk stability of Compound I free base Pattern 1, Compound I monofumarate Pattern 1 and Compound I hemifumarate Pattern 1 Compound I Compound I Compound I Storage Conditions free base Hemifumarate Monofumarate
Figure imgf000135_0001
Example 25. Solubility of Compound I free base Pattern 1, Compound I Monofumarate Pattern 1 and Compound I Hemifumarate Pattern 1 About 4 mg of Compound I Pattern 1 was added into 2 mL of buffer solution. For Compound I hemifumarate Pattern 1 and Compound I monofumarate Pattern 1, about 4 mg was added into 1.8 mL and 1.6 mL buffer solution, respectively. pH value was adjusted in simulated vaginal fluid with 0.2N NaOH. After stirring at 37℃ for 0.5 hour and 2 hours, clear solution was obtained for all the three candidates. Table 20. Solubility of Compound I free base Pattern 1, Compound I monofumarate Pattern 1 and Compound I hemifumarate Pattern 1 Compound I free base Compound I Compound I Pattern 1 Hemifumarate Monofumarate
Figure imgf000136_0001
Solubility was tested in five media, pH 3.0 citrate buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, water, simulated vaginal fluid (pH 4.2) at 37°C for 0.5 h and 2 h. All the three candidates are highly soluble in the media (>2 mg/mL). Example 26: Hygroscopicity of Compound I free base Pattern 1, Compound I Hemifumarate Pattern 1, and Compound I Monofumarate Pattern 1 Hygroscopicity was investigated by DVS at 25°C, using the following method: Method: 40-0-95-0-40%RH, dm/dt=0.002 The results are presented in Table 21 and in FIGS.15 to 20. Table 21. Hygroscopicity of Compound I free base Pattern 1, Compound I hemifumarate Pattern 1, and Compound I monofumarate Pattern 1 Compound I free base Compound I Compound I Pattern 1 Hemifumarate Pattern 1 Monofumarate Pattern 1 le 9 8 7 7 7 No
Figure imgf000137_0001
Hygroscopicity was investigated by DVS at 25°C. All three Patterns are moderately hygroscopic. Compound I Pattern 1 shows about 4.3% water uptake up to 90% RH but absorbs about 14.3% water in 95% RH. For Compound I monofumarate Pattern 1 and Compound I hemifumarate Pattern 1, they show about 2.9% and 6.9% water uptake in up to 95% RH. There is no form change after the DVS tests. Example 27: Large Scale Preparation of Mono-Fumarate Pattern 1 About 4.16 g of Compound I free base was dissolved in 11 mL of IPA. Then, 1.0 eq. of fumaric acid was added to the yellow clear solution with stirring at 50°C. After about 1 hour, some solids precipitated out. Then, 10 mg seeds of mono-fumarate (from the above ‘Small scale preparation’) were added. The mixture was stirred at 50°C for about 1.5 hours and cooled to 25°C, then it was stirred at 25°C for about 10 min. Then, 40 mL of heptanes was added as antisolvent. Obtained suspension was stirred at 25°C for about 24 hours, then cooled to 5°C at a rate of 0.1°C/min and stirred at 5°C for about 1 day. The solids were collected by filtration and dried in the oven at 40°C for about 2 hours under vacuum. About 4.1 g of light pink solids were obtained in a yield of 81.2%. XRPD is shown in FIG.21A; DSC at 10°C/min rate is shown in FIG.21B; DSC at 2°C/min rate is shown in FIG. 21C; mDSC thermogram is shown in FIG. 21D; TGA is shown in FIG.22. Table 22. Properties of Compound I Monofumarate Pattern 1 Parameter Method Result Purity HPLC 99.3% y
Figure imgf000138_0002
Example 28: Polymorph Screening Study of Compound I fumarate salts The polymorph screening study was performed for fumarate salts of Compound I (mixture of diastereomers). Their polymorphic behaviors were investigated by equilibration, precipitation by addition of antisolvent, slow cooling, fast cooling and slow evaporation experiments. Approximate solubility of the Compound I Monofumarate Pattern 1 at 25°C and 50°C
Figure imgf000138_0001
vial, and a 20 μL aliquot of each solvent (as shown in Table 23) was added to determine solubility at 25°C. About 10 mg of Compound I monofumarate Pattern 1 (Example 27) was weighed to a 2 mL glass vial and aliquot of 20 μL of each solvent (as shown in Table 23 below) was added to determine solubility at 50°C. Maximum volume of each solvent added was 1 mL. Approximate solubility was determined by visual observation. Table 23. Approximate solubility of Compound I monofumarate Pattern 1 at 25°C and 50°C Experiment Solvent Solubility (mg/mL) 25°C 50°C
Figure imgf000139_0001
Equilibration with solvents at 25°C for 2 weeks and 3 weeks About 30 mg of Compound I monofumarate Pattern 1 (obtained in Example 27) was equilibrated in suitable amount of solvent at 25°C for 2 weeks and 3 weeks with a stirring plate. Obtained suspension was filtered. The solid part (wet cake) was investigated by XRPD. When differences were observed, additional studies were performed (e.g., NMR, DSC, TGA, HPLC, and PLM). The results are presented in Tables 24-39 and in FIGS.23-45. Table 24. Results of equilibration with water for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional l t 2 k t t 2 k k t t k
Figure imgf000139_0002
For Pattern D, XRPD diffractograms are shown in FIG. 23 and FIG. 24; DSC thermograms are shown in FIG.25 and FIG.26; TGA thermograms are shown in FIG.27 and FIG.28. Table 25. Results of equilibration with acetonitrile for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000140_0001
, . .30; and TGA thermogram is shown in FIG.31. Table 26. Results of equilibration with MEK for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000140_0002
For Pattern B, XRPD diffractogram is shown in FIG.32; DSC thermogram is shown in FIG.33; and TGA thermogram is shown in FIG.34. Table 27. Results of equilibration with acetone for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000141_0001
XRPD diffractogram is shown in FIG.32 (3 weeks). Table 28. Results of equilibration with isopropanol for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000141_0002
or a ern , racogram s sown n . ; ermogram s sown n .36; and TGA thermogram is shown in FIG.37. Table 29. Results of equilibration with acetone/toluene (1:1 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional Sl t 2 t t 2 k 3 k t t 3 k
Figure imgf000141_0003
~1.8% at 55-100ºC PLM: Irregular particles HPLC Purity: 98.9% F P tt E XRPD diff t i h i FIG 38 DSC th h i FIG 39
Figure imgf000142_0001
Table 30. Results of equilibration with acetone/heptane (1:1 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks) ne ne
Figure imgf000142_0002
XRPD diffractogram is shown in FIG.32 (3 weeks). Table 31. Results of equilibration with IPA/heptane (1:1 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000142_0003
XRPD diffractogram is shown in FIG.35.
Table 32. Results of equilibration with IPA/toluene (1:1 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000143_0001
XRPD diffractogram is shown in FIG.35. Table 33. Results of equilibration with IPA/MTBE (1:3 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000143_0002
XRPD diffractogram is shown in FIG.35. Table 34. Results of equilibration with THF/heptanes (1:3 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional
Figure imgf000143_0003
Note: Salt ratio is free base form:fumaric acid ratio XRPD is shown in FIG.43 and FIG.44. Table 35. Results of equilibration with EtOH/heptanes (1:3 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks) by
Figure imgf000144_0001
. . Table 36. Results of equilibration with EA/toluene(1:3 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000144_0002
XRPD is shown in FIG.43 and FIG.44 Table 37. Results of equilibration with EtOH/toluene (1:3 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional
Figure imgf000144_0003
Note: Salt ratio is free base form:fumaric acid ratio XRPD is shown in FIG.43 and FIG.44. Table 38. Results of equilibration with water/ACN (2.9:97.1 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000145_0001
Table 39. Results of equilibration with IPA/heptanes (1:4 v/v) for Compound I monofumarate Pattern 1 at 25°C for 2 weeks and 3 weeks Exp. XRPD Additional XRPD Additional (Solvent) (2 weeks) test (2 weeks) (3 weeks) test (3 weeks)
Figure imgf000145_0002
XRPD diffractogram is shown in FIG.45. Precipitation by addition of antisolvent About 30 mg of Compound I monofumarate Pattern 1 (Example 7) was dissolved in a good solvent. Antisolvent was added into the obtained solutions slowly. Precipitates were collected by filtration. The solid part (wet cake) was investigated by XRPD. When differences were observed, additional investigations were performed (e.g., NMR, DSC, TGA). If no precipitation was obtained, the solutions were cooled to 5°C for crystallization. After stirred at 5°C for about 23 days, no precipitation was obtained, the solutions were put in a -20°C freezer for crystallization. Results are presented in Table 40. XRPD diffractograms are shown in FIG. 46 and FIG. 47. Table 40. Precipitation of Compound I fumarate salts by addition of antisolvent Exp. Solvent Antisolvent XRPD Comments AS1 Acetone Heptanes Pattern B + Salt ratio =1:0.89 C 9- he nd C 7- le es C 0-
Figure imgf000146_0001
Crystallization at room temperature by slow evaporation Combined with approximate solubility experiment, solubility samples were filtered through a 0.45 μm nylon filter. Obtained solutions were slow evaporated at ambient condition. Solid residues were examined for their polymorphic form. Results are presented in Table 41. XRPD diffractograms are shown in FIG. 48 and FIG. 49. Table 41. Crystallization at room temperature by slow evaporation Exp. Solvent XRPD 1 Acetone Pattern 1 with medium crystallinity
Figure imgf000147_0001
Crystallization from hot saturated solutions by slow cooling About 30 mg of Compound I monofumarate Pattern 1 was dissolved in the minimal amount of selected solvents at 50°C. Obtained solutions were cooled to 5°C at the rate of 0.1°C/min. Precipitates were collected by filtration. The solid part (wet cake) was investigated by XRPD. When differences were observed, additional investigations were performed (e.g., NMR, DSC, TGA). If no precipitation was obtained, the solutions were put in a -20°C freezer for crystallization. After storing in -20°C freezer for about 5 days, no precipitation was obtained, heptane was added as antisolvent. Precipitates were collected by filtration. The solid part (wet cake) was investigated by XRPD. Results are presented in Table 42. XRPD diffractograms are shown in FIG. 50 and FIG. 51. Table 42. Crystallization from hot saturated solutions by slow cooling Exp. Solvent XRPD Comments
Figure imgf000147_0002
Residual solvent: 2.0% (by weight) ACN solvent DSC onset (enthal ): 740°C (89 J/ ) at N ar ar
Figure imgf000148_0001
Crystallization from hot saturated solutions by fast cooling About 30 mg of Compound I monofumarate Pattern 1 was dissolved in the minimal amount of selected solvents at 50°C. Obtained solutions were put into an ice bath and agitated. Precipitates were collected by filtration. The solid part (wet cake) was investigated by XRPD. When differences were observed, additional investigations were performed (e.g., NMR, DSC, TGA). If no precipitation was obtained, the solutions were put in -20°C freezer for crystallization. After storing in -20°C freezer for about 7 days, no precipitation was obtained, heptanes was added as antisolvent. Precipitates were collected by filtration. The solid part (wet cake) was investigated by XRPD. Results are presented in Table 43. XRPD diffractograms are shown in FIG. 52 and FIG. 53. Table 43. Crystallization from hot saturated solutions by fast cooling Exp. Solvent XRPD Comments
Figure imgf000148_0002
5 MEK Pattern B with medium Free base form:fumaric acid=1:0.64; crystallinity 3.0% (by weight) MEK solvent residue 6 EtOH/t l n P tt rn 1 with m di m Aft r t r t -20°C f r b t 5 d l ar
Figure imgf000149_0002
Behavior of Compound I Monofumarate Pattern 1 under heating and cooling by two
Figure imgf000149_0001
Cycle 1: 0°C to 106°C at 10°C/min; 106°C to 0°C at 10°C /min; reheat from 0°C to 250°C at 10°C/min. Cycle 2: 0°C to 130°C at 10°C /min; 130°C to 0°C at 10°C /min; reheat from 0°C to 250°C at 10°C /min. The results are presented in Table 44and in FIG.54 and FIG.55. Table 44. Behavior of Compound I monofumarate Pattern 1 under heating and cooling Exp. Heating rate Thermal events 1 Cycle 1: 0°C to 106°C at 10°C/min; 106°C Heating:
Figure imgf000149_0003
Behavior under compression About 10 mg of Compound I monofumarate Pattern 1 was compressed for 5 minutes at 10 MPa with a hydraulic press. XRPD characterization was performed to investigate the polymorphic behavior under compression. According to the XRPD, no form change was observed. Grinding simulation experiments About 10 mg of Compound I monofumarate Pattern 1 was ground manually with a mortar and pestle for 5 min. Form transformation and degree of crystallinity was evaluated by XRPD. According to the XRPD, no form change was observed; crystallinity slightly decreased. Granulation simulation experiments Water or ethanol was added drop wise to about 10 mg of Compound I monofumarate Pattern 1 until solids were wetted sufficiently. The samples were ground manually with a mortar and pestle for 3 minutes. Samples are dried under ambient condition for 10 min. Form transformation and degree of crystallinity were evaluated by XRPD. According to the XRPD, neither in ethanol, nor in water form change was observed. Summary of identified mono- and hemifumarate polymorphs Compound I monofumarate Pattern 1 used in the study was prepared from Compound I free base according to Example 7. The initial form of monofumarate used in the polymorph study described below (Pattern 1) is an anhydrate of monofumarate with HPLC purity of about 99.3%. The ratio of free form and fumaric acid is about 1: 0.96 by 1H NMR. It has two melting peaks with Tonset of about 98.5°C with an enthalpy of about 14 J/g and 109.6°C with an enthalpy of about 25 J/g, as measured by differential scanning calorimetry (DSC). By thermogravimetric analysis (TGA), Pattern 1 shows about 0.5% weight loss at about 98°C and 0.6 % weight loss from 98°C to 140°C. About 1.0% (by weight) heptanes and 0.2% (by weight) IPA residue were detected by 1HNMR. Karl Fischer titration shows it contains about 1.3% water. During the polymorph study, four new patterns were identified. Although monofumarate was used as initial physical form obtained new patterns showed different stoichiometric ratio. Pattern B, Pattern C and Pattern E are hemifumarate salts, and Pattern D is a degradation product. Pattern B is an anhydrate of hemifumarate with HPLC purity of about 99.6%. With MEK, acetone, acetone/heptanes, MEK/heptanes and EtOH/MTBE as solvents, it can be obtained from equilibration experiment, addition of antisolvent, slow cooling and fast cooling experiments. The ratio of free base form to fumaric acid is about 1: 0.52 by 1H-NMR. It has two thermal events with Tonset of about 77.4°C with an enthalpy of about 71 J/g and 88.4°C with an enthalpy of about 18 J/g. It shows about 0.7% weight loss at about 77°C and 4.2% weight loss from 77°C to 130°C. About 4.6% (by weight) MEK residue was detected by 1H NMR. With ACN and ACN/water as solvents, a
Figure imgf000151_0001
Pattern C and fumaric acid was obtained by equilibration, slow cooling and fast cooling experiments. The ratio of free base form to fumaric acid of the mixture is about 1: 0.95 by 1H NMR. After washing by water, the ratio decreased to about 1:0.76. This indicates that Pattern C is not a monofumarate. It has two thermal events with Tonset of about 74.0°C with an enthalpy of about 89 J/g and Tonset of about 90.6°C with an enthalpy of about 15 J/g. It shows about 0.4% weight loss at about 73°C and 2.1% weight loss from 73°C to 144°C. About 2.0% (by weight) ACN residue was detected by 1H NMR. Pattern D is a degradation product with HPLC purity of about 0.2%. It was obtained in water by equilibration experiment experiments. It has two thermal events with Tonset of about 41.4°C with an enthalpy of about 67 J/g and Tonset of about 72.1°C with an enthalpy of about 29 J/g. It shows about 0.6% weight loss at about 41°C and 8.5% weight loss from 41°C to 178°C. Pattern E is an anhydrate of hemifumarate with HPLC purity of about 98.9%. It was obtained in acetone/toluene by equilibration experiment. The ratio of free base form to fumaric acid of the mixture is about 1: 0.69 by 1H NMR. It has two thermal events with Tonset of about 53.1°C with an enthalpy of about 33 J/g and Tonset of about 96.5°C with an enthalpy of about 34 J/g. It shows about 1.0% weight loss at about 53°C and 3.6% weight loss from 53°C to 96°C. About 0.6% (by weight) acetone residue was detected by 1H NMR. Results of the study on Compound I mono- and hemifumarate polymorphs are summarized in Table 45 below. The salt ratio is the ratio between Compound I free base and the salt counterion. “AS” indicates the form can be prepared by antisolvent addition, using the solvent/antisolvent pairs listed in the table. “EQ” indicates the form can be prepared through equilibration in the listed solvent. “SC” indicates the form can be prepared by slowly cooling a solution of Compound I monofumarate in the listed solvent. “FC” indicates t the form can be prepared by quickly cooling a solution of Compound I monofumarate in the listed solvent. Table 45. Summary of identified Compound I fumarate salt Patterns A, B, C, and E Melting Polymorph Salt Temperatures Salt ratio Preparation E th l s,
Figure imgf000152_0001
Example 29: Preparation of Compound II Pattern 1 Experiment 1. Small-scale synthesis and preparation of seeds One hundred milligrams of RP Compound I free base and 0.3 mL of IPA were added into a glass vial. To it was added 1.0 equiv. of fumaric acid and the resulting mixture was stirred at 50°C for 2 min, most of the material was precipitated out. To it 1.0 mL of heptanes was added. The sample was stirred at 50°C for 1 h, then cooled to 3°C with 0.1°C/min. After stirring at 3°C for about 8 h, 0.4 mL of heptanes was added. Solids obtained was isolated by filtration and drying in vacuum oven at 40°C for about 2 h to obtain Compound II Pattern 1. The characterization results are reported in Table 46. XRPD diffractogram of Compound II Pattern 1 is shown in FIG.56. Table 46. Properties of Compound II Pattern 1 (small scale preparation) Parameter Method Result
Figure imgf000152_0002
Stoichiometry and 1H NMR Free base : fumaric acid=1:0.98; residual solvent 0.2% (by weight) IPA residue
Figure imgf000153_0003
Experiment 2. Large-scale preparation Three grams of RP Compound I free base and 9 mL of IPA were added into a glass vial. To it was added 1.0 equiv. of fumaric acid and the resulting mixture was stirred at 50°C for 5 min. About 21 mg of Compound II Pattern 1 seeds from Experiment 1 were added into the mixture.30 mL of heptanes was added into the mixture. The sample was stirred at 50°C for 1 h then cooled to 3°C with 0.1°C/min. After stirring at 3°C for about 16 h, the solid obtained was isolated by filtration and drying in vacuum oven at 40°C for about 4 h and at 50°C for about 3 h. This resulted in 2.9 g of white solid Compound II Pattern 1 in a yield of 78.3%. XRPD diffractogram of Compound II Pattern 1 is shown in FIG.56. DSC thermogram of Compound II Pattern 1 is shown in FIG.57. TGA thermogram of Compound II Pattern 1 is shown in FIG.58. Table 47. Properties of Compound II Pattern 1 (large scale preparation) Parameter Method Result Yield 783% of
Figure imgf000153_0002
Compound II Pattern 1 is an anhydrate. The stoichiometry of free base:fumaric acid is about 1:1.0 based on 1H NMR result. It has a melting peak at Tonset of 141.5°C combined with decomposition.
Figure imgf000153_0001
about 0.3% weight loss at about 130°C. No residual solvent was detected by 1H NMR. Example 30: Preparation of Compound III Pattern 1 SP Compound I free base (100 mg) and 0.3 mL of IPA were added into a glass vial. To it was added 1.0 equiv. of fumaric acid and the mixture was stirred at 50°C for 15 min, most of the material was precipitated out. After addition of 1.0 mL heptanes, the sample was stirred at 50°C for 1 h, then cooled to 3°C with 0.1°C/min. After stirring at 3°C for about 8 h, 0.4 mL of heptanes was added into the mixture to get better suspension. White solid was isolated by filtration and drying in vacuum oven at 40°C for about 2 h to yield Compound III Pattern 1. The characterization results are reported in Table 48. XRPD diffractogram of Compound III Pattern 1 is shown in FIG. 59. DSC thermogram of Compound III Pattern 1 is shown in FIG. 60. TGA thermogram of Compound III Pattern 1 is shown in FIG.61. Table 48. Properties of Compound III Pattern 1 Parameter Method Result X-ray diffraction 3-40° (2 theta) Compound III Pattern 1
Figure imgf000154_0001
Example 31: Preparation of Compound III Pattern 2 SP Compound I free base (3 g) and 9 mL of IPA were added into a glass vial. After the addition of 1.0 equiv. of fumaric acid, a lot of solid precipitated out immediately. 30 mL of heptanes was added into the mixture, then about 20 mg of Compound III Pattern 1 seeds were added into the mixture. The sample was stirred at 50°C for 1 h, then cooled to 3°C with 0.1°C/min. After stirring at 3°C for about 20 h, the sample was heated from 3°C to 50°C within 20 mins, then 0.2 equiv. of fumaric acid and 1.5 ml of heptanes were added into the mixture. The resulting mixture was stirred at 50°C for about 2 h then cooled to 3°C with 0.1°C/min. After stirring at 3°C for about 13 h, it was reheated to 50°C within 20 mins and kept stirred at 50°C for about 6 h. It was cooled to 3°C with 0.1°C/min and stirred at 3°C for about 2 days. The resulting solid was isolated by filtration and drying in vacuum oven at 50°C for about 3 h to obtain 3.1 g of white solid in a yield of 83.6%. The results are reported in Table 49. XRPD diffractogram of Compound III Pattern 2 is shown in FIG.62. DSC thermogram of Compound III Pattern 2 is shown in FIG.63. TGA thermogram of Compound III Pattern 2 is shown in FIG.64. Table 49. Properties of Compound III Pattern 2 Parameter Method Result Yield 83.6%
Figure imgf000155_0001
Compound III Pattern 2 is an anhydrate. The stoichiometry of free base: fumaric acid is about 1:1.2 based on 1H NMR result. It has two melting peaks at Tonset of 106.4°C with an enthalpy of about 47 J/g and Tonset of 118.8°C with an enthalpy of about 63 J/g. It shows about 0.3% weight loss at about 105°C. No residual solvent was detected by 1H NMR. Example 32: Bulk stability of Compound II Pattern 1 and Compound III Pattern 2 Compound II Pattern 1 and Compound III Pattern 2 were placed in an open container at 25°C/92% RH, in an open container at 40°C/75% RH, and in a closed container at 60°C for 1 week. Samples were characterized by XRPD and HPLC and inspected for color change. The results are presented in Table 50. Table 50. Stability: purity and appearance Parameter Physical form ge ge
Figure imgf000155_0002
Bulk (XRPD) No form change No form change (One peak disappeared) ° ge
Figure imgf000156_0001
Initial chemical and chiral purity Initial chemical purity of Compound II Pattern 1 and Compound III Pattern 2 is 99.7% and 98.8%, respectively. Chiral purity (%de) of Compound II Pattern 1 is 98.4%. Bulk stability Accelerated stability experiments were conducted at 25°C/92% RH in an open container, at 40°C/75% RH in an open container, and at 60°C in a tight container for one week. Compound II Pattern 1 showed good physical and chemical stability after exposure to the three conditions. Compound III Pattern 2 showed good physical stability at the above mentioned three conditions. Degradation product increased by 1.6% and 1.5% after exposure to 40°C/75% RH in an open container and 60°C in a tight container, respectively. Example 33: Solubility study of Compound II Pattern 1 and Compound III Pattern 2 Accurately 12 mg of Compound II Pattern 1, 12 mg of Compound III Pattern 2 were weighed into an 8 mL glass vial and to it 5 mL of solubility medium was added. The salt amount used is equivalent to 10 mg anhydrous free base. All the samples were clear solution in the media after 0.5 and 2h at 37°C. The obtained clear solutions were analyzed by pH meter for pH, and solubility was determined by observation. Table 51. Solubility at 37°C, target concentration 10 mg/5 mL, equilibration for 0.5 h and 2 h Compound II Pattern 1 Compound III Pattern 2 )
Figure imgf000156_0002
ES3 pH 6.8, 50 mM phosphate buffer >2 >2 (6.47) >2 >2 (6.44)
Figure imgf000157_0001
Solubility was tested in five media, pH 3.0 citrate buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, water, simulated vaginal fluid (pH 4.2) at 37°C for 0.5 h and 2 h. The two candidates, Compound II Pattern 1 and Compound III Pattern 2, are highly soluble in the media (>2 mg/mL). Example 34: Hygroscopicity of Compound II Pattern 1 and Compound III Pattern 2 Hygroscopicity was investigated by DVS at 25°C, using the following method: Method: 40-0-95-0-40%RH, dm/dt=0.002 Both Compound II Pattern 1 and Compound III Pattern 2 are slightly hygroscopic. Compound II Pattern 1 shows about 0.2% water uptake up to 95% RH. No form change after the DVS test was observed. Compound III Pattern 2 shows about 1.0% water uptake up to 95% RH. No form change after the DVS test was observed. Results are presented in Table 52. Table 52. Compound II Pattern 1 Compound III Pattern 2 2
Figure imgf000157_0002
90% 0.23 0.24 0.92 0.97 95% 0.24 0.24 1.01 1.01
Figure imgf000158_0001
Example 35: Polymorph screening of Compound II Pattern 1 and Compound III Pattern 2 Monofumarate (50 mg) was equilibrated in suitable amount of solvent or solvent mixture. Obtained suspensions were equilibrated for 1 week. Solids were isolated by centrifugation filtration. Wet cakes obtained after equilibration were analyzed by XRPD to determine crystal form change. Table 53. Mini-polymorph screening: Crystal modification after 1 week equilibration at 25°C (salt ratio is defined as free base : fumaric acid ratio Parameter Compound II Pattern 1 Compound III Pattern 2 Exp. y C,
Figure imgf000158_0002
Compound II Pattern 2 Compound III Pattern 4 Melting onset: 110.3°C, enthalpy: Melting onset: 95.7°C, enthalpy: y: y C, C, y: K II ue g; g;
Figure imgf000159_0002
Polymorphism evaluation of Compound II Pattern 1
Figure imgf000159_0001
equilibration experiments and 2 new potential polymorphs of the fumarate salts (Compound II Pattern 2 and Compound II Pattern 3) of Isomer I were obtained from acetonitrile and MEK, respectively. These both showed lower melting temperature than for Compound II Pattern 1. Both polymorphs of isomer 1 show unchanged high chiral purity. Polymorphism evaluation of Compound III Pattern 2 In this study, dissociation was observed during equilibration experiments, a hemifumarate (hemifumarate Pattern 2) of Isomer II was obtained. In addition, 4 new monofumarate salts (Compound III Pattern 3, Compound III Pattern 4, Compound III Pattern 5 and Compound III Pattern 6) of Isomer II were obtained. Example 36: X-ray Powder Diffraction (XRPD) The XRPD analysis was carried out on a Bruker D8 Advance diffractometer. Table 54 below provides the results of the XRPD performed on Compound II Pattern 1. The XRPD exhibited sharp peaks, indicating the sample was composed of crystalline material. Significant peaks were observed in the XRPD on Compound II Pattern 1 at about 3.1±0.2°, about 9.3±0.2°, about 12.1±0.2°, about 14.9±0.2°, about 15.1±0.2°, about 18.1±0.2°, about 19.8±0.2°, about 20.1±0.2°, about 25.1±0.2°, about 25.9±0.2°, and about 28.8±0.2°. Table 54. XRPD Peak List For Compound II Pattern 1 Pos. [°2θ] d-spacing [Å] Rel. Int. [%] Pos. [°2θ] d-spacing [Å] Rel. Int. [%] 3076 2869958 291 25863 344216 158
Figure imgf000160_0001
21.892 4.05666 4.9 36.622 2.45179 3.1 22.910 3.87861 13.7 37.419 2.40142 2.7
Figure imgf000161_0001
Table 55 below provides the results of the XRPD performed on Compound IV Pattern 1. Significant peaks were observed in the XRPD on Compound IV Pattern 1 at about 6.5±0.2°, about 12.1±0.2°, about 17.5±0.2°, about 18.1±0.2°, about 18.5±0.2°, about 19.6±0.2°, about 19.8±0.2°, about 20.2±0.2°, about 20.6±0.2°, and about 21.3±0.2. Table 55. XRPD Peak List For Compound IV Pattern 1 Pos. [°2θ] d-spacing [Å] Rel. Int. [%] Pos. [°2θ] d-spacing [Å] Rel. Int. [%] 4.651 18.98268 10.6 19.830 4.47357 30.2
Figure imgf000161_0002
Table 56 below provides the results of the XRPD performed on Compound III Pattern 1. Significant peaks were observed in the XRPD on Compound III Pattern 1 at about 9.5±0.2°, about 11.7±0.2°, about 14.6±0.2°, about 17.5±0.2°, about 18.0±0.2°, about 20.0±0.2°, about 20.4±0.2°, about 22.3±0.2°, about 23.7±0.2°, and about 25.5±0.2. Table 56. XRPD Peak List For Compound III Pattern 1 Pos. [°2θ] d-spacing [Å] Rel. Int. [%] Pos. [°2θ] d-spacing [Å] Rel. Int. [%] 5.848 15.10034 6.0 23.735 3.74577 12.7
Figure imgf000162_0001
Table 57 below provides the results of the XRPD performed on Compound III Pattern 2. The XRPD exhibited sharp peaks, indicating the sample was composed of crystalline material. Significant peaks were observed in the XRPD on Compound III Pattern 2 at about 8.9±0.2°, about 9.9±0.2°, about 11.7±0.2°, about 12.1±0.2°, about 15.1±0.2°, about 17.9±0.2°, about 18.2±0.2°, about 19.9±0.2°, about 25.1±0.2°, about 29.6±0.2°, and about 38.1±0.2°. Table 57. XRPD Peak List For Compound III Pattern 2 Pos. [°2θ] d-spacing [Å] Rel. Int. [%] Pos. [°2θ] d-spacing [Å] Rel. Int. [%]
Figure imgf000162_0002
13.646 6.48398 5.1 27.936 3.19118 13.4 14.629 6.05032 9.0 28.282 3.15297 19.0
Figure imgf000163_0001
Table 58 below provides the results of the XRPD performed on Compound V Pattern 1. Significant peaks were observed in the XRPD on Compound V Pattern 1 at about 5.0±0.2°, about 7.2±0.2°, about 10.1±0.2°, about 12.1±0.2°, about 17.5±0.2°, about 17.9±0.2°, about 19.3±0.2°, about 22.0±0.2°, about 24.3±0.2°, about 25.1±0.2°, and about 26.3±0.2. Table 58. XRPD Peak List For Compound V Pattern 1 Pos. [°2θ] d-spacing [Å] Rel. Int. [%] Pos. [°2θ] d-spacing [Å] Rel. Int. [%] 5041 1751522 634 18617 476222 270
Figure imgf000163_0002
Table 59 below provides the results of the XRPD performed on Compound V Pattern 2. Significant peaks were observed in the XRPD on Compound V Pattern 2 at about 5.1±0.2°, about 6.9±0.2°, about 7.6±0.2°, about 10.2±0.2°, about 11.6±0.2°, about 12.1±0.2°, about 15.1±0.2°, about 17.6±0.2°, about 18.1±0.2°, about 18.7±0.2°, about 19.5±0.2°, about 19.8±0.2°, and about 25.1±0.2°. Table 59. XRPD Peak List For Compound V Pattern 2 Pos. [°2θ] d-spacing [Å] Rel. Int. [%] Pos. [°2θ] d-spacing [Å] Rel. Int. [%] 5.101 17.31028 59.1 18.723 4.73547 33.2
Figure imgf000164_0001
Example 37. Tablet Stability Study Tablets were prepared using the excipients and excipient ratios shown in the first column of the following tables. The tablets were then stored under the conditions indicated and periodically sampled and tested for purity by HPLC. The tablets produced from Compound I monofumarate degraded less than tablets produced from Compound I across a range of different formulations. Tablets produced with Compound I Excipients 0 months 1 month 25C/60% RH 1 month 40C/75% RH
Figure imgf000164_0002
PC (49%) + MCC 98.64 97.16 46.11 (49%)
Figure imgf000165_0001
Excipients 0 months 1 month 25C/60% RH 1 month 40C/75% RH PEO-15 (25%) + MCC 99.03 97.76 83.13
Figure imgf000165_0002
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by referenced. Although the foregoing invention has been described in some detail by way of illustration and example for the purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teaching of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the invention as defined in the embodiments and/or claims.

Claims

CLAIMS: 1. A method of treating a cervical or vaginal HPV-induced infection or an associated condition in a female in need thereof, including cervical or vaginal intraepithelial neoplasia, comprising (i) administering to the base of the cervix an effective amount of dosage form of a therapeutic compound selected from:
Figure imgf000166_0001
(ii) and then inserting a retaining device placed in the vaginal area below the cervical base for a sufficient time to collect the post-treatment leakage of the therapeutic compound or its PMEG metabolite.
2. A method of treating a cervical or vaginal HPV-induced infection or an associated condition in a female in need thereof, including cervical or vaginal intraepithelial neoplasia, comprising (i) administering to the base of the cervix an effective amount of dosage form of a therapeutic compound selected from:
Figure imgf000167_0001
a pharmaceutically acceptable salt
Figure imgf000167_0002
(ii) inserting a retaining device placed in the vaginal area below the cervical base for a sufficient time to collect the post-treatment leakage of the therapeutic compound or its PMEG metabolite. 3. A method of treating a cervical or vaginal HPV-induced infection or an associated condition in a female in need thereof, including cervical or vaginal intraepithelial neoplasia, comprising (i) administering to the base of the cervix a dosage form with an effective amount of a therapeutic compound selected from:
Figure imgf000167_0003
a pharmaceutically acceptable salt
Figure imgf000167_0004
(ii) inserting a retaining device placed in the vaginal area below the cervical base for a sufficient time to collect the post-treatment leakage of the therapeutic compound or its PMEG metabolite. 4. The method of claim 2 wherein the pharmaceutically acceptable salt is selected from:
Figure imgf000168_0001
acceptable salt is selected from:
or . compound is in a pharmaceutically acceptable composition. 7. The method of claim 6, wherein the pharmaceutical composition is a solid dosage form. 8. The method of claim 6, wherein the pharmaceutical composition is a semi-solid dosage form. 9. The method of claim 7, wherein the solid pharmaceutical composition is in the form of a vaginal tablet. 10. The method of claims 1-9, wherein the pharmaceutical composition is administered with a vaginal applicator. 11. The method of claims 1-10, wherein the retaining device is selected from a menstrual cup, a menstrual disc, a tampon, diaphragm, cervical cap and a sponge. 12. The method of claims 1-11, wherein the retaining device is used as both the applicator and a retaining means. 13. The method of claim 11, wherein the retaining device is a menstrual cup. 14. The method of claim 11, wherein the retaining device is a menstrual disc. 15. The method of claim 11, wherein the retaining device is a tampon. 16. The method of claim 11, wherein the retaining device is a diaphragm. 17. The method of claim 11, wherein the retaining device is a cervical cap.
18. The method of claim 11, wherein the retaining device is a sponge. 19. The method of claims 1-5, wherein the compound is administered in a morphic form. 20. The method of claim 4, wherein the compound is an isolated morphic form of: wherein the
Figure imgf000170_0001
pattern comprising peaks independently selected from at least 3, 4, 5, or 6 of the following 2theta values 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. 21. The method of claim 5, wherein the compound is an isolated morphic form of: wherein the
Figure imgf000170_0002
pattern comprising peaks independently selected from at least 3, 4, 5, 6, 7, 8 or 9 of the following 2theta values 9.53+0.2°, 10.04+0.2°, 11.60+0.2°, 14.57+0.2°, 17.22+0.2°, 17.50+0.2°, 20.04+0.2°, 20.36+0.2°, 22.34+0.2°, 23.73+0.2°, 25.48+0.2°, 26.06+0.2°, 27.38+0.2°, and 32.20+0.2°.
22. The method of claim 5 wherein the compound is an isolated morphic form of:
Figure imgf000171_0001
wherein the isolated morphic form is characterized by an XRPD pattern comprising peaks independently selected from at least 3, 4, 5, 6, 7, 8 or 9 of the following 2theta values 8.94+0.2°, 9.89+0.2°, 9.91+0.2°, 11.66+0.2°, 12.11+0.2°, 15.13+0.2°, 17.85+0.2°, 18.15+0.2°, 19.90+0.2°, 20.38+0.2°, 22.94+0.2°, 25.09+0.2°, 26.54+0.2°, 26.90+0.2°, 27.38+0.2°, 28.28+0.2°, 28.95+0.2°, 29.64+0.2°, and 38.07+0.2°. 23. The method of claim 1, wherein the compound is an isolated morphic form of the compound of the formula: wherein the
Figure imgf000171_0002
pattern comprising peaks independently selected from at least 3, 4, 5, or 6 of the following 2theta values 3.08+0.2°, 9.30+0.2°, 12.08+0.2°, 14.92+0.2°, 15.10+0.2°, 20.14+0.2°, 25.14+0.2°, and 28.82+0.2°. 24. The method of claim 9, wherein the tablet disintegrates in less than about 250 μL of fluid. 25. The method of claim 9, wherein the table disintegrated in less than about 150 μL of fluid. 26. The method of claims 1-25, wherein about 0.05 to 1 mg of therapeutic compound or pharmaceutically acceptable salt thereof is administered.
27. The method of claims 1-25, wherein from about 0.1 to 1 mg of therapeutic compound is administered. 28. The method of claims 1-25, wherein from about 0.1 to 0.5 mg of therapeutic compound is administered. 29. The method of claims 1-25, wherein from about 0.2 to 0.3 mg of therapeutic compound are administered. 30. The method of claims 1-25, wherein about 0.3 mg of therapeutic compound is administered. 31. The method of claim 6, wherein the therapeutic compound comprises from about 0.01% to 10% of the pharmaceutical composition. 32. The method of claim 6, wherein the pharmaceutical composition comprises a polymer. 33. The method of claim 6, wherein the pharmaceutical composition comprises a disintegration enhancing excipient. 34. The method of claim 6, wherein the pharmaceutical composition comprises a penetration enhancing excipient. 35. The method of claim 6, wherein the pharmaceutical composition comprises an excipient that allows for controlled release. 36. The method of claim 6, wherein the pharmaceutical composition comprises at least one of a light mineral oil, propylparaben, Tefose 63, water, EDTA, methylparaben and Carbopol 974P. 37. The method of claim 6, wherein the pharmaceutical composition comprises at least one of EDTA, methyl paraben, Carbopol 974P, propylene glycol and sorbic acid. 38. The method of claim 6, wherein the pharmaceutical composition comprises mannitol, polycrystalline cellulose and magnesium stearate. 39. The method of claims 1-38, wherein the condition caused by a human papillomavirus is atypical squamous cells of undetermined significance (ASC-US). 40. The method of claims 1-38, wherein the condition caused by a human papillomavirus is atypical glandular cells (AGC). 41. The method of claims 1-38, wherein the condition caused by a human papillomavirus is low-grade squamous intraepithelial lesions (LSIL).
42. The method of claims 1-38, wherein the condition caused by a human papillomavirus is atypical squamous cells, and cannot exclude high grade squamous intraepithelial lesion (ASC-H). 43. The method of claims 1-38, wherein the condition caused by a human papillomavirus is high grade squamous intraepithelial lesions (HSIL). 44. The method of claims 1-38, wherein the condition caused by a human papillomavirus is adenocarcinoma in situ (AIS). 45. The method of claims 1-38, wherein the condition is cervical intraepithelial neoplasia. 46. The method of claims 1-38, wherein the condition is cervical intraepithelial neoplasia Grade 1. 47. The method of claims 1-38, wherein the condition is cervical intraepithelial neoplasia Grade 2. 48. The method of claims 1-38, wherein the condition is cervical intraepithelial neoplasia Grade 3. 49. The method of claims 1-38, wherein the condition is vaginal intraepithelial neoplasia. 50. The method of claims 1-38, further comprising applying a lubrication means to the epithelial tissue before inserting the dosage form in the affected area. 51. The method of claims 1-38, further comprising applying a lubrication means to the dosage form before inserting the dosage form in the affected area. 52. The method of claim 50 or 51, wherein the lubrication means is selected from water, a glycerol based lubricant, and a hydroxyethylcellulose-based lubricant. 53. The method of claims 1-52, wherein the dosage form is administered once per day. 54. The method of claim 53, wherein the dosage form is administered twice per week. 55. The method of claim 53, wherein the dosage form is administered three times or more per week. 56. The method of claims 53-55, wherein the dosage form is administered for about one week. 57. The method of any one of claims 53-55, wherein the dosage form is administered for about two weeks.
58. The method of any one of claims 53-55, wherein the dosage form is administered for about three weeks. 59. The method of any one of claims 53-55, wherein the dosage form is administered for about four weeks. 60. The method of any one of claims 53-55, wherein the dosage form is administered for about five weeks. 61. The method of any one of claims 53-55, wherein the dosage form is administered for about six weeks. 62. The method of any one of claims 53-55, wherein the dosage form is administered in a therapeutic cycle comprising: a. a treatment cycle comprising administering the compound, and b. a rest cycle, comprising a period of no treatment. 63. The method of claim 62, wherein the rest cycle is about one week. 64. The method of claim 62, wherein the rest cycle is about two weeks. 65. The method of claim 62, wherein the rest cycle is about three weeks. 66. A kit that includes a dosage form with a therapeutic compound of claims 1-5 and a retaining device. 67. The kit of claim 66, that also includes a vaginal applicator. 68. The kit of claim 66 or 67, wherein the dosage form is a vaginal tablet. 69. The kit of claims 66-68 wherein the retaining device is selected from a menstrual cup, a menstrual disc, a tampon, diaphragm, cervical cap and a sponge. 70. The kit of claims 66-69, wherein the retaining device is used as both the applicator and a retaining means. 71. The kit of claim 69, wherein the retaining device is a menstrual cup. 72. The kit of claim 69, wherein the retaining device is a menstrual disc. 73. The kit of claim 69, wherein the retaining device is a tampon. 74. The kit of claim 69, wherein the retaining device is a diaphragm. 75. The kit of claim 69, wherein the retaining device is a cervical cap. 76. The kit of claim 69, wherein the retaining device is a sponge. 77. The kit of claims 66-76, wherein the therapeutic compound is administered in a morphic form.
PCT/US2024/010538 2024-01-05 2024-01-05 Device system and use for hpv infection and cervical intraepithelial neoplasia Pending WO2025147262A1 (en)

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PCT/US2025/010220 WO2025147598A1 (en) 2024-01-05 2025-01-03 Device system and use for hpv infection and cervical intraepithelial neoplasia

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160083407A1 (en) * 2014-09-15 2016-03-24 The Regents Of The University Of California Nucleotide analogs
US20210246153A1 (en) * 2015-09-15 2021-08-12 The Regents Of The University Of California Nucleotide Analogs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160083407A1 (en) * 2014-09-15 2016-03-24 The Regents Of The University Of California Nucleotide analogs
US20210246153A1 (en) * 2015-09-15 2021-08-12 The Regents Of The University Of California Nucleotide Analogs

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