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WO2024264013A1 - Bioavailability enhancing ionic liquid formulations and uses thereof - Google Patents

Bioavailability enhancing ionic liquid formulations and uses thereof Download PDF

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Publication number
WO2024264013A1
WO2024264013A1 PCT/US2024/035153 US2024035153W WO2024264013A1 WO 2024264013 A1 WO2024264013 A1 WO 2024264013A1 US 2024035153 W US2024035153 W US 2024035153W WO 2024264013 A1 WO2024264013 A1 WO 2024264013A1
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Prior art keywords
acid
rpv
composition
ionic liquid
anionic
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PCT/US2024/035153
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French (fr)
Inventor
Abhijit DATE
Yogesh SUTAR
Joseph S. Adams
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University of Arizona
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University of Arizona
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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
    • A61P31/10—Antimycotics
    • 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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • 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
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives

Definitions

  • the present invention addresses this need.
  • An ionic liquid is a low-melting salt containing organic/inorganic cation and anion, and depending upon the composition, IL can also be liquid at room temperature (see, Example_2_References 25-26).
  • IL ionic liquid
  • APIs active pharmaceutical ingredients
  • API-ILs ILs
  • pharmaceutically acceptable counterions have emerged as a novel approach to modulate physicochemical and biopharmaceutical properties of ionizable drugs with poor solubility and/or permeability eventually leading to greater oral bioavailability (see, Example_2_References 27-33).
  • Rilpivirine is a potent antiretroviral drug used for the long-term management of HIV infection.
  • the high crystallinity and very low aqueous solubility of RPV are responsible for the highly variable pharmacokinetics of RPV seen in HIV-infected patients. While fatty meals can increase the absorption of RPV, the low lipid solubility of RPV precludes the development of oral lipid-based formulations such as self-nanoemulsifying systems (SNES).
  • SNES self-nanoemulsifying systems
  • Herpes simplex virus (HSV) keratitis is a recurrent and lifelong infection that results in visual morbidity, and it is the leading cause of infection-associated corneal blindness worldwide.
  • HSV keratitis can be categorized into epithelial, stromal, and endothelial keratitis.
  • HSV epithelial keratitis involves active HSV infection in the corneal epithelium and antiviral therapy is needed to diminish discomfort, minimize vision loss, and reduce the recurrence rate.
  • Topical eye drops of nucleoside analogs such as ganciclovir and trifluridine are FDA-approved for HSV epithelial keratitis therapy. However, these eye drops need to be administered 5- to 9-times a day to manage the ocular HSV infection which affects patient compliance and thus, treatment efficacy.
  • compositions comprising bioavailability
  • the present invention provides a composition comprising an ionic liquid formulation comprising a cationic component and an anionic component.
  • the cationic component is selected from a cationic or ionized therapeutic agent, a cationic or ionized amino acid, a cationic nutraceutical, a cationic agrochemical molecule, a cationic functional food, a cationic excipient, and a pharmaceutically acceptable cation.
  • the cationic component is a protonated form or cationic derivative of one of the following: D-glucamine, n-methyl-D-glucamine, N-ethyl-D-glucamine, N-octyl-d- glucamine, N-oxodecyl meglumine, D-glucosamine, D-mannosamine, D-galactosamine, L- carnitine, D-carnitine, Acetyl-L-carnitine, Propionyl-L-carnitine, O-hexanoyl-L-carnitine, Phosphatidyl-carnitine, Isovaleryl -L-carnitine, Propionyl-L-carnitine, Palmitoyl-L-carnitine, Sarcosine, Sarcosine methyl ester, Sarcosine ethyl ester, N-lauroyl arginine ethyl ester, N- lauroy
  • the cationic component is a protonated form or cationic derivative
  • the cationic component is selected from: In some embodiments, the cationic component is a phosphonium cation encompassed
  • the ionic liquid formulations comprise:
  • the ionic liquid formulations are associated with (e.g., encapsulated within) micelles prepared using micelle-forming agents such as but not limited to polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer (SoluPlus), polyethylene glycol-phospholipid conjugate, polyethylene glycol-polylactide, poly-lipoic acid or a PEG-poly-lipoic acid.
  • micelle-forming agents such as but not limited to polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer (SoluPlus), polyethylene glycol-phospholipid conjugate, polyethylene glycol-polylactide, poly-lipoic acid or a PEG-poly-lipoic acid.
  • the ionic liquid formulations are associated with a selfemulsifying composition
  • a selfemulsifying composition comprising a mixture of surfactant (such as polysorbate and poloxamer), cosurfactant (such as alcohol, glycol ether), lipids (such as triglycerides, mono- or diglycerides, fatty acids, and their esters), and ionic liquid according to any of the preceding claims that yields emulsion or nanoemulsion after dilution with water, buffer, 5% dextrose or other physiological fluids.
  • surfactant such as polysorbate and poloxamer
  • cosurfactant such as alcohol, glycol ether
  • lipids such as triglycerides, mono- or diglycerides, fatty acids, and their esters
  • the self-assembling ionic liquid is one of:
  • the self-assembling ionic liquid is one of:
  • any of the described ionic liquids can solubilize drugs, natural products or nutraceuticals with low solubility (less than 1 mg/ml) and/or low permeability ( ⁇ 1 * 10 6 cm/s) such as but not limited to cyclosporin A, docetaxel, paclitaxel, cabizataxel, dabrafenib, trametinib, sorafenib, venetoclax, coenzyme Q10, idebenone, triclabendazole, olaparib, urolithin A, myricetin, quercetin, resveratrol, genistein, pterostilbene, gefitinib, dolutegravir, cabotegravir, bictegravir, tenofovir alafenamide, adefovir dipivoxil, ivermectin, fluconazole, ibuprofen, niclosamide, idebenone
  • the composition further comprises at least one additional therapeutic agent.
  • the at least one additional therapeutic agent comprises any type or kind of therapeutic agent capable of inhibiting fungal activity.
  • the at least one additional therapeutic agent is selected from the following: a polyene, imidazole, triazole, thiazole, allylamine, echinocandin, among others.
  • Examples include Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, econazole, fenticonazole, isoconazole, kentoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, polygodial, tolnaftate, undec
  • the at least one additional therapeutic agent comprises any type or kind of agent capable of inhibiting reverse-transcriptase (RT) activity.
  • RT reverse-transcriptase
  • the at least one additional therapeutic agent comprises any type or kind of anti-viral agent.
  • the at least one additional therapeutic agent comprises any type or kind of anti-viral agent selected from helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, or ritonavir.
  • anti-viral agent selected from helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofo
  • the present invention provides a method of killing or inhibiting the growth of a fungus comprising contacting the fungus with a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions described herein.
  • the fungus is selected from Aspergillus spp., Fusarium spp., Malassezia spp., Candida spp., or Cryptococcus spp..
  • the present invention provides a use of a composition or pharmaceutical composition described herein, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a fungal infection.
  • the fungal infection is related to one or more of: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Fusarium moniliforme, Fusarium proliferation, Malassezia pachydermatis, Candida albicans, Candida glabrata infection, Candida tropicalis, Candida krusei, Candida auris, Cryptococcus neoformans, Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis, Sarcopodium oculorum, M. circinelloides, Rhizopus delemar, Rhizopus oryzae, and Lichtheimia corymbifera.
  • the present invention provides a method for treating or preventing herpes simplex virus (HSV) infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions recited in Claims 1-54.
  • HSV herpes simplex virus
  • the ionic liquid formulation comprises adefovir dipivoxil (ADV).
  • the method further comprises administering to the mammal one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir.
  • anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir
  • the ionic liquid formulation is configured for topical administration, transdermal, ocular, systemic administration, and/or oral administration.
  • the ionic liquid formulation is configured for topical administration, optical administration, and/or oral administration.
  • the present invention provides a method for treating or preventing HIV infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions described herein.
  • the ionic liquid formulation comprises tenofovir disoproxil, tenofovir alafenamide, and/or adefovir dipivoxil.
  • the method further comprises administering to the mammal one or more anti-HIV agents selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, doravirine, lenacapavir, and a combination thereof.
  • anti-HIV agents selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and
  • the ionic liquid formulation is configured for topical administration, optical administration, and/or oral administration.
  • the present invention provides a method for treating cancers such as melanoma, breast cancer, non-small cell lung cancer, hematological cancers, renal cancer, liver cancer and brain cancers in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions described herein.
  • cancers such as melanoma, breast cancer, non-small cell lung cancer, hematological cancers, renal cancer, liver cancer and brain cancers
  • the method further comprises administering to the mammal an antiparasitic agent.
  • the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal.
  • the present invention provides a method of treating or preventing neurocysticercosis in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions described herein.
  • the mammal is a human being.
  • the mammal is a human being suffering from or at risk of suffering from neurocysticercosis.
  • the method further comprises administering to the mammal an antiparasitic agent.
  • the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal.
  • the pharmaceutical composition containing ionic liquid is administered via oral, parenteral, transdermal, skin, bladder, nasal, cornea/general ophthalmic, intraocular, pulmonary, mucosal, transrectal/enemas, or vaginal route of administration.
  • FIG. 1 Hydrophilic metformin hydrochloride can he transformed into lip-ophilic ionic liquid (IL) metformin docus-ate with no crystallinity and high solubility in organic solvents and lipids with the FDA-approved fatty anion, sodium docusate.
  • IL lip-ophilic ionic liquid
  • FIG. 7 Outline of experiment to evaluate in vivo antiviral efficacy of SoluPlus nanomicelles containing TDF-IL (SN-TDF-IL).
  • FIG. 8 A general scheme for the synthesis of RPV-IL/salt/cocrystal using pharmaceutically acceptable biocompatible anions with different chemical structures. Additional details about the synthesis are included in methods section.
  • FIG. 13 RPV-ILs/salts/cocrystals retained the inherent antiretroviral activity of RPV in vitro.
  • FIG. 16 RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES significantly increased the oral bioavailability of RPV when compared to RPV suspension. Briefly, B6 male mice (n> 5 mice/group) were orally administered RPV suspension, RPV-Doc-MC60SNES or RPV-Doc - Cap90SNES (equivalent to 10 mg/kg of RPV).
  • RPV-Doc-MC60SNES as well as RPV-Doc- Cap90SNES showed rapid and significantly higher absorption of RPV.
  • the RPV-Doc- Cap90SNES showed lower clearance of RPV compared to RPV suspension and RPV-Doc- MC60SNES. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001.
  • FIG. 19 13 C NMR spectrum of RPV docusate (RPV-Doc).
  • FIG. 20 H NMR spectrum of RPV-DHA.
  • FIG. 22 H NMR spectrum of RPV geranate.
  • FIG. 23 13 C NMR spectrum of RPV-geranate.
  • FIG. 24 H NMR spectrum of RPV linoleate.
  • FIG. 25 13 C NMR spectrum of RPV-linoleate.
  • FIG. 26 H NMR spectrum of RPV-LS.
  • FIG. 27 13 C NMR spectrum of RPV-LS.
  • FIG. 28 H NMR spectrum of RPV oleate.
  • FIG. 29 13 C NMR spectrum of RPV oleate.
  • FIG. 30 The overlay of HPLC chromatograms of RPV, RPV-Doc, RPV-LS, RPV-DHA, RPV linoleate, RPV geranate, and RPV oleate (concentration: 30 pM; Retention time: 5.25) indicated the high purity.
  • FIG. 31 HRMS of RPV-Doc; HRMS m/z for C 4 2H 56 N6O7S [negative]: 788.38889.
  • FIG. 35 Dipivefrin, a pivalic acid ester prodrug of epinephrine is available as eye drops (Propine®).
  • Adefovir dipivoxil (ADV) is a pivalic acid ester prodrug of adefovir. Pivalic acid is marked in red.
  • FIG. 37 Hydrophilic metformin hydrochloride can be transformed into lipophilic ionic liquid (IL) metformin docusate with no crystallinity and high solubility in organic solvents and lipids with the FDA-approved fatty anion, sodium docusate.
  • IL lipophilic ionic liquid
  • FIG. 38 ADV was well tolerated by the human corneal epithelial cells after 24 h even at a concentration of 100 pM (> 85% cell viability) indicating corneal safety.
  • FIG. 39 ADV showed significantly higher in vitro efficacy against HSV-1 compared to other acyclic nucleoside phosphonates (ANPs), their prodrugs and ACV.
  • A HCE were infected with HSV-1 17 GFP at MOI of 1 and the cells were treated with a 5 pM concentration of various ANPs, prodrugs and ACV and the intracellular infectious viral titer was eventually determined using further assays.
  • B HCE were infected with P-galactosidase expressing recombinant HSV- 1 (KOS)tkl2 at MOI of 0.1 MOI and treated with various concentrations of ANPs, prodrugs, and ACV.
  • KOS P-galactosidase expressing recombinant HSV- 1
  • FIG. 40 Topical (ocular) administration of 1% w/v ADV solution showed robust antiviral effect in a mouse model of ocular HSV-1 infection.
  • 1% ADV solution (10 pL) was topically administered 3 times daily to C57BL/6 mice 1 day post ocular HSV-1 infection and ocular swabs were taken on days 2 and 4 (n -3 ) and vital titer was determined.
  • FIG. 41 (A) Schematic representation of the method to prepare lipophilic salt, MOX- PAM. MOX-PAM was obtained as a precipitate which was further washed with water and freeze dried for the preparation of NS. (B) Schematic of the process of using zirconium beads and laboratory scale wet milling method to transform lipophilic salt (MOX-PAM) into mucuspenetrating nanosuspension (MOX-PAM NS) NS. (C) The physicochemical properties of the MOX-PAM NS showing appropriate size, homogeneity, and surface charge. Data adapted from our recent paper with suitable modifications. 37
  • FIG. 42 Once a day topical treatment with moxifloxacin-pamoate nanosuspension (MOX-PAM NS) was more effective in treating ocular S. aureus infection.
  • MOX-PAM NS moxifloxacin-pamoate nanosuspension
  • rats were infected topically with .S', aureus 24 h prior to initiating treatment. Rats received either no treatment (Infection control), once a day Vigamox (0.5% w/v), three times a day Vigamox, or once a day MOX-PAM NS (equivalent to 0.5% w/v MOX).
  • corneal swabs were taken with a cotton-tipped applicator for the determination of bacterial load (n > 7 per group).
  • FIG. 43 A representative schematic of SoluPlus nanomiclles containing encapsulated drug or drug-based IL. The monomer of SoluPlus is included in the circle.
  • FIG. 44 SoluPlus nanomicelles containing lipophilic IL, OXF-Doc show greater efficacy compared to pure OXF and OXF-Doc in a murine cryptococcosis model.
  • CD-I mice were infected with Cryptococcus neoformans and then treated orally with various OXF or OXF-Doc formulations at a OXF dose of 25mg/kg.
  • the lung CFUs analyzed at 14 days post-infection.
  • *OXF-Doc in HPMC and OXF in SoluPlus are in suspended form and have low solubility.
  • FIG. 45 (A) Schematic of the method to prepare ADV lipophilic salt, ADV-PAM.
  • FIG. 46 (A) Structure of ADV ILs (ADV linoleate and ADV docosahexaenoate. (B) ’H-NMR spectrum of ADV linoleate shows chemical shifts in the proton corresponding to the -NH2 group of ADV (labeled as “a”) and disappearance of carboxylate proton of linoleic acid (labeled as “1”) indicating interaction between ADV and linoleic acid.
  • FIG. 47 TEM images of Meglumine-tocopherol (Meg-VES) self- assembled IL prepared in water.
  • FIG. 48 Various hydrophobic drugs could be solubilized in carnitine linoleate.
  • FIG. 49 SNES of carnitine salcaprozate containing solubilized sorafenib showed significant improvement in oral PK parameters (C ma x and AUC) compared to sorafenib tosylate suspension and sorafenib base indicating potential for oral drug delivery.
  • compositions comprising bioavailability (e.g., oral and local bioavailability) enhancing ionic liquid formulations and the use thereof for treating conditions or diseases in a subject.
  • bioavailability e.g., oral and local bioavailability
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by +10% and remain within the scope of the disclosed embodiments.
  • “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a compound or composition described herein to an appropriate control subject.
  • the term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the symptoms.
  • “treating” means an application or administration of the compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
  • a “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., humans and nonhumans) that may benefit from the administration of compositions contemplated herein.
  • mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • non-mammals include, but are not limited to, birds, fish, and the like.
  • the mammal is a human.
  • the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the compounds or compositions of the disclosure into a subject by a method or route which results in at least partial localization of the compounds or composition to a desired site.
  • the compounds or compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.
  • “Pharmacological composition” refers to a mixture of one or more of the compounds described herein or pharmaceutically acceptable salts thereof, with other chemical components, such as pharmaceutically acceptable carriers and/or excipients. The purpose of a pharmacological composition is to facilitate administration of a compound to an organism.
  • “Pharmaceutically acceptable salts” is a cationic salt formed at any acidic (e.g., carboxylic acid) group, or an anionic salt formed at any basic (e.g., amino) group.
  • Solvate is a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association often includes hydrogen bonding. In certain instances, the solvate is capable of isolation, for example, when one or more solvate molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include hydrates, ehanolates, and methanolates.
  • Prodrug refers to a pharmacologically inactive derivative of a parent “drug” molecule which requires biotransformation within the target physiological system to release, or to convert the prodrug into the active drug.
  • Prodrugs can address the problems associated with solubility, stability, cell permeability or bioavailability.
  • Prodrugs usually comprise an active drug molecule and a chemical masking group.
  • Prodrugs can be readily prepared from the parent compounds with well-known methods.
  • the term “ionic liquids” as used herein refers to organic sails or mixtures of organic sails which exist in a liquid state. Ionic liquids have been shown to be useful in a variety of fields, including in industrial processing, catalysis, pharmaceuticals, and electrochemistry.
  • the ionic liquids contain at least one anionic and at least one cationic component.
  • Ionic liquids can comprise an additional hydrogen bond donor (i.e. any molecule that can provide an — OH or an — NH group); examples include but are not limited to alcohols, fatty acids, and amines.
  • the anionic and the cationic component may be present in any molar ratio.
  • the present disclosure provides ionic liquid formulations comprising a cationic component and an anionic component.
  • the cationic component is selected from a cationic or ionized therapeutic agent, a cationic or ionized amino acid, a cationic nutraceutical, a cationic agrochemical molecule, a cationic functional food, a cationic excipient, and a pharmaceutically acceptable cation.
  • the cationic component is a cationic derivative of one of the
  • the cationic component is a cationic derivative of one of the following: D-glucamine, n-methyl-D-glucamine, N-ethyl-D-glucamine, N-octyl-d-glucamine, N-oxodecyl meglumine, D-glucosamine, D-mannosamine, D-galactosamine, L-camitine, D- carnitine, Acetyl-L-carnitine, Propionyl-L-camitine, O-hexanoyl-L-camitine, Phosphatidyl- carnitine, Tsovaleryl-L-carnitine, Propionyl-L-carnitine, Palmitoyl-L-carnitine, Sarcosine, Sarcosine methyl ester, Sarcosine ethyl ester, N-lauroyl arginine ethyl ester, N-lauroyl lysine Alb
  • the cationic component is a cationic molecule (e.g., linear, branched or cyclic) containing a primary, a secondary, a tertiary, or a quaternary ammonium group.
  • the cationic component is a cationic derivative (e.g., 1°, 2°, or 3°
  • the cationic component is a protonated form of a benzimidazole compound (e.g., anthelmintic benzimidazoles such as albendazole, mebendazole, flubendazole and triclabendazole) not including oxfendazole encompassed within the following: wherein Rl and R2 are each independently selected from hydrogen, alkyl, aryl, heteroalkyl and heteroaryl groups.
  • a benzimidazole compound e.g., anthelmintic benzimidazoles such as albendazole, mebendazole, flubendazole and triclabendazole
  • Rl and R2 are each independently selected from hydrogen, alkyl, aryl, heteroalkyl and heteroaryl groups.
  • the anionic component is selected from an anionic therapeutic agent, an anionic amino acid, an anionic nutraceutical, an anionic agrochemical molecule, an anionic functional food, an anionic excipient, and a pharmaceutically acceptable anion.
  • the anionic component is an anionic carboxylate, an anionic sulfonate, an anionic sulfate, an anionic phosphate, an anionic phosphonate, an anionic sulfamate, or a chemical moiety having negatively charged functional group.
  • the anionic component is a bile acid selected from cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, and tauroursodeoxycholic acid. In some embodiments, the anionic component is selected from:
  • the anionic component is an anionic carboxylate molecule selected from one of the following:
  • R - saturated or mono/di/poly unsaturated aliphatic R alky or aryl group
  • R -CH 2 - or -NH-;
  • Rj alkyl and/or aryl group -O-acyl or -O-carbamoyl or -O-carbonyl
  • R alkyl and/or aryl group
  • R alky or aryl group
  • R alkyl or aryl group
  • the anionic component is a negatively charged functional group selected from: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, undecylenic acid, oleic acid, linoleic acid, linolenic acid, myristoleic acid, ricinoleic acid, elaidic acid, N-decanoyl sarcosine, Lauryl sarcosine, docosahexaenoic acid, biotin, lactobionic acid, eicosapentaenoic acid, nervonic acid, Vitamin E succinate, 4-phenylbutyric acid, pamoic acid, a- lipoic acid, ibuprofen, naproxen, squalene acid, cholesterol hemisuccinate, capric acid, salcaprozic acid, docusic acid, cholic acid, glycocholic acid, taurocholic acid, tauroursodeoxycholic acid
  • the anionic component is an anionic carboxylate molecule
  • R C23H45 selected from one of the following:
  • the anionic component is selected from a saturated fatty acid derivative moiety (carboxylate), an unsaturated fatty acid derivative moiety, an aromatic acid derivative moiety, a sulfonate derivative moiety, a sulfate derivative moiety, a phosphate derivative moiety, and a sulfamate derivative moiety.
  • the anionic component is selected from a negatively charged functional group of saturated fatty acids selected from butyric acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid)lauric acid (dodecanoic acid), palmitic acid (hexadecenoic acid), and cholic acid.
  • a negatively charged functional group of saturated fatty acids selected from butyric acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid)lauric acid (dodecanoic acid), palmitic acid (hexadecenoic acid), and cholic acid.
  • the anionic component is selected from a negatively charged functional group of unsaturated fatty acids selected from: undecylenic acid, oleic acid, linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, nervonic acid, myristoleic acid, elaidic acid, and ricinoleic acid.
  • the anionic component is selected from a negatively charged functional group of aromatic acids selected from: salcaprozic acid, a-tocopherol succinate, 4- phenyl butyric acid, Ibuprofen, Naproxen, pamoic acid, Dolutegravir, Cabotegravir, and Bictegravir.
  • the anionic component is selected from a negatively charged functional group of sulfonate anions selected from: docusic acid, camphor sulfonic acid, taurocholic acid, tauroursodeoxycholic acid, and taurine.
  • the anionic component is selected from a negatively charged functional group of sulfate anions selected from: lauryl sulfate, and cholesterol sulfate.
  • the anionic component is selected from a negatively charged functional group of a phosphate anion selected from: a-tocopherol phosphate, 1 ,2-dioleoyl-sn- glycero- 3 -phosphate (DOPA), and thiamine phosphate.
  • a phosphate anion selected from: a-tocopherol phosphate, 1 ,2-dioleoyl-sn- glycero- 3 -phosphate (DOPA), and thiamine phosphate.
  • the anionic component is selected from a negatively charged functional group of sulfamate anions selected from: acesulfame, saccharin, and cyclamate.
  • the anionic component is selected from:
  • the anionic component is selected from a fatty anion, a docusate anion
  • the cationic components and anionic components are present in a ratio in the range of about 5:1 to about 1:5.
  • the ionic liquid formulations comprise one of more of: Meglumine-tocopherol succinate
  • the ionic formulations comprise one or more nucleotide analogues (antiviral agents) selected from:
  • the ionic liquid formulations are self- assembling ionic liquid formulations. In certain embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) nanoformulations. In certain embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) polymeric nanoformulations. In certain embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) nanomicelles (e.g., SoluPlus nanomicelles).
  • nanomicelles e.g., SoluPlus nanomicelles
  • the self-assembling liquid ionic formulations comprise meglumine & bile acid-based ionic liquids (which have ability to self-assemble).
  • the self-assembling liquid ionic formulation comprises one or more of- Meglumine-chenodeoxycliolate Meglumine-cholate
  • Megluimne-deoxyc.holate Meglumine-glyeocholate Megluinine-lithocliolate
  • the liquid ionic formulations comprise carnitine & bile acid based ionic liquids (which serves as a lipophilic solvent for self-nanoemulsifying system).
  • the liquid ionic formulation comprising carnitine & bile acid based ionic liquid are selected from:
  • the liquid ionic formulations comprise mitoquinone-based ionic liquids, which serves as a lipophilic solvent for self- nanoemulsifying system.
  • the mitoquinone-based ionic liquids are selected from:
  • the liquid ionic formulations comprise carnitine & dicarboxylic acid based ionic liquids, which serves as a lipophilic solvent for self-nanoemulsifying system.
  • the carnitine & dicarboxylic acid based ionic liquids are selected from:
  • the ionic formulations comprise carnitine & monocarboxylic acid/phosphonic acid based ionic liquids, which serve as a lipophilic solvent for self- nanoemulsifying system, wherein the carnitine & dicarboxylic acid based ionic liquids are selected front: Carnitine salcaprozate > Carnitine oleate
  • said self-assembling ionic liquids are meglumine & dicarboxylic acid-based ionic liquids, which have the ability to self-assemble, examples of which are: Meglumine azelate (2:1) Meglumine azelate (1:1)
  • the compounds within the ionic liquid formulations may exist as a stereoisomer wherein asymmetric or chiral centers are present.
  • the stereoisomer is “R” or “S” depending on the configuration of substituents around the chiral carbon atom.
  • R and S used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30.
  • Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers.
  • Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England (or more recent versions thereof), or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.
  • the ionic liquid formulations can be combined with another solvent to enhance solubility and/or delivery.
  • the solvent may be aqueous or non-aqueous.
  • the purpose of the solvent is to control the dose of the ionic liquid. Dilution of the ionic liquid by the solvent can serve the purpose of delivering a safe dose to the subject.
  • the purpose of the solvent is to improve solubility of the one or more drugs. Such improvements may come from the ability of the solvent to control the physicochemical environment of the ionic liquid to match the chemical properties of the one or more drugs.
  • the solvents used may include without limitation: sterile water, saline solution, glycerin, propylene glycol, ethanol, oils, ethyl oleate, isopropyl myristate, benzyl benzoate, or surfactants.
  • the solvent is chosen so as to not adversely impact the compatibility of the ionic liquid formulation.
  • a composition as described herein can further comprise a pharmaceutically acceptable excipient.
  • suitable excipients include, for example, water, saline, glycerol, ethanol, or the like, and combinations thereof.
  • the composition can contain minor amounts of additional excipients such as emulsifying agents, surfactants, pH buffering agents, and the like, which enhance the effectiveness of the ionic liquid formulation.
  • the ionic liquid formulation may be further encapsulated in a dosage form designed to facilitate delivery to an organism.
  • dosage forms include capsules, tablets, and syrups.
  • the ionic liquid formulation may require excipients sugars (such as lactose), starches (such as com starch), cellulose, cellulose derivatives (such as sodium carboxymethyl cellulose), gelatin, and other compatible substances.
  • excipients sugars such as lactose
  • starches such as com starch
  • cellulose such as sodium carboxymethyl cellulose
  • gelatin such as gelatin, and other compatible substances.
  • the ionic liquid formulation described herein further comprises one or more additional agents.
  • the one or more additional agents are selected from a nucleic acid, a small molecule, and a polypeptide.
  • the one or more additional agents comprise a nucleic acid.
  • the one or more additional agents comprise a small molecule.
  • the one or more additional agents comprise a polypeptide.
  • the polypeptide comprises an antibody.
  • the antibody comprises any one selected from Fragment Antigen-binding (Fab, F(ab')2), single chain variable fragment (scFv), and nanobodies.
  • the present disclosure also includes isotopically-labeled compounds, which is identical to those recited in Formula I but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes include those for hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to 2 H, 3 H, 13 C, 14 C, 1S N, 18 O, 17 0, 31 P, 32 P , 35 S, 18 F, and 36 C1, respectively.
  • the compound may incorporate positronemitting isotopes for medical imaging and positron-emitting tomography (PET) studies.
  • positron-emitting isotopes that can be incorporated in compounds of formula (I) are n C, 13 N, 15 O, and 18 F.
  • Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent.
  • the ionic liquid formulations described herein are associated (e.g., encapsulated) with biodegradable polymers (e.g., for purposes of enhancing bioavailability).
  • the biodegradable polymer is a lipid nanoemulsion.
  • the biodegradable polymer is nanomicelle.
  • the biodegradable polymer is SoluPlus nanomicelles.
  • Such ionic liquid formulations as described herein may be synthesized according to a variety of methods. Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Reactions can be worked up in the conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above described scheme.
  • an optically active form of a disclosed compound within the ionic liquid formulations When an optically active form of a disclosed compound within the ionic liquid formulations is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization, or enzymatic resolution).
  • a pure geometric isomer of a compound within the ionic liquid formulations it can be obtained by carrying out one of the above procedures using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation.
  • compositions may be suitable for administration to a subject (such as a patient, which may be a human or nonhuman).
  • the disclosed ionic liquid formulations may be incorporated into pharmaceutically acceptable compositions.
  • the pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the ionic liquid formulations.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the ionic liquid formulations are outweighed by the therapeutically beneficial effects.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • compositions and formulations may include pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, surfactant, cyclodextrins or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, com starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, hut not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; surfactants such as, but not limited to, cremophor EL, cremophor RH 60, Solutol HS 15 and polysorbate 80; cyclodextrins such as, but not limited to, alpha-CD, beta-CD, gamma-CD, HP-beta-CD, SBE-beta-
  • the route by which the disclosed ionic liquid formulations are administered and the form of the composition will dictate the type of carrier to be used.
  • the composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral injections) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).
  • the composition is for oral administration.
  • the composition is for subcutaneous administration.
  • the composition is for intravenous administration.
  • Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, cyclodextrins combinations thereof, and others. All carriers are optional in the compositions.
  • Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol.
  • the amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.
  • Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma.
  • the amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.
  • Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose.
  • the amount of binder(s) in a systemic composition is typically about 5 to about 50%.
  • Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins.
  • the amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%.
  • Suitable colorants include a colorant such as an FD&C dye.
  • the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.
  • Suitable flavors include menthol, peppermint, and fruit flavors.
  • the amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.
  • Suitable sweeteners include aspartame and saccharin.
  • the amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%.
  • Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E.
  • BHA butylated hydroxyanisole
  • BHT butylated hydroxytoluene
  • the amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%.
  • Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate.
  • the amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.
  • Suitable glidants include silicon dioxide.
  • the amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.
  • Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, dimethylacetamide and phosphate (or other suitable buffer).
  • the amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.
  • Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, Pa.) and sodium alginate.
  • the amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.
  • Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Del.
  • Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1 , Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239.
  • the amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.
  • Suitable cyclodextrins include alpha-CD, beta-CD, gamma-CD, hydroxypropyl betadex (HP-beta-CD), sulfobutyl-ether 0-cyclodextrin (SBE-beta-CD).
  • the amount of cyclodextrins in the systemic or topical composition is typically about 0% to about 40%.
  • systemic compositions include 0.01% to 50% of an active compound (e.g., an ionic liquid formulation as described herein) and 50% to 99.99% of one or more carriers.
  • Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.
  • compositions for oral administration can have various dosage forms.
  • solid forms include tablets, capsules, granules, and bulk powders.
  • These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives.
  • the oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.
  • Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof.
  • diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose.
  • Specific binders include starch, gelatin, and sucrose.
  • Specific disintegrants include alginic acid and croscarmellose.
  • Specific lubricants include magnesium stearate, stearic acid, and talc.
  • Specific colorants are the FD&C dyes, which can be added for appearance.
  • Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.
  • Capsules typically include an active ionic liquid formulation, and a carrier including one or more diluents disclosed above in a capsule comprising gelatin.
  • Granules typically comprise a disclosed ionic liquid formulation, and preferably glidants such as silicon dioxide to improve flow characteristics.
  • Implants can be of the biodegradable or the non-biodegradable type.
  • compositions may be coated by conventional methods, typically with pH or timedependent coatings, such that a disclosed ionic liquid formulation is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action.
  • the coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.
  • compositions for oral administration can have liquid forms.
  • suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non- effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like.
  • Liquid orally administered compositions typically include a disclosed ionic liquid formulation and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants.
  • Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.
  • compositions useful for attaining systemic delivery of the ionic liquid formulations to the subject include sublingual, buccal and nasal dosage forms.
  • Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol, and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose.
  • Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.
  • Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like.
  • Topical compositions include: a disclosed ionic liquid formulation, and a carrier.
  • the carrier of the topical composition preferably aids penetration of the ionic liquid formulation into the skin.
  • the carrier may further include one or more optional components.
  • the amount of the carrier employed in conjunction with a disclosed ionic liquid formulation is sufficient to provide a practical quantity of composition for administration per unit dose of the ionic liquid formulation.
  • Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).
  • a carrier may include a single ingredient or a combination of two or more ingredients.
  • the carrier includes a topical carrier.
  • Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like.
  • carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.
  • the carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.
  • Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane- 1,2-diol, butane- 1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, but
  • Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof.
  • the amount of propellant(s) in a topical composition is typically about 0% to about 95%.
  • Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof.
  • Specific solvents include ethyl alcohol and homotopic alcohols.
  • the amount of solvent(s) in a topical composition is typically about 0% to about 95%.
  • Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof.
  • Specific humectants include glycerin.
  • the amount of humectant(s) in a topical composition is typically 0% to 95%.
  • the amount of thickener(s) in a topical composition is typically about 0% to about 95%.
  • Suitable powders include beta-cyclodex trins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium poly acrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically -modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof.
  • the amount of powder(s) in a topical composition is typically 0% to 95%.
  • the amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1%.
  • Suitable pH adjusting additives include HC1 or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition.
  • compositions or formulations disclosed herein may further comprise at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is one or more other anti-fungal agents.
  • the composition comprises any one or more of the ionic liquid formulations, or a pharmaceutically acceptable salt thereof, and one or more other anti-fungal agent(s).
  • the other anti-fungal agent is an azole or an echinocandin. In some embodiments, the other anti-fungal agent is an azole. In some embodiments, the azole is itraconazole, posaconazole, voriconazole (VOR), or isavuconazole. In some embodiments, the azole is itraconazole. In some embodiments, the azole is posaconazole. In some embodiments, the azole is voriconazole. In some embodiments, the azole is isavuconazole. In some embodiments, the other anti-fungal agent is an echinocandin.
  • the echinocandin is caspofungin (CAS).
  • the other anti-fungal agent is nystatin, miconazole, Gentian violet, or amphotericin B.
  • the other antifungal agent is nystatin.
  • the other anti-fungal agent is miconazole.
  • the other anti-fungal agent is Gentian violet.
  • the other anti-fungal agent is amphotericin B. Additional anti-fungal agents include, but are not limited to, fosmanogepix, ibrexafungerp, olorofim, opelconazole, and rezafungin.
  • the other anti-fungal agent is fosmanogepix. In some embodiments, the other antifungal agent is ibrexafungerp. In some embodiments, the other anti-fungal agent is olorofim. In some embodiments, the other anti-fungal agent is opelconazole. In some embodiments, the other anti-fungal agent is rezafungin. In some embodiments, the other anti-fungal agent is Nikkomycin Z. Other anti-fungal agents include VT-1129, VT-1161, VT-1598, PC1244, SUBA- ITC, CAMB, MGCD290, T-2307, and VL-2397. Additional anti-fungal agents are disclosed in, for example, PCT Publication No. WO 2021/247781.
  • the antifungal agent is a known antifungal and not an ionic liquid formulation described herein, such as a polyene, imidazole, triazole, thiazole, allylamine, echinocandin, among others.
  • Examples include Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, econazole, fenticonazole, isoconazole, kentoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, polygodial, tolnaftate, undec
  • the at least one additional therapeutic agent is capable of inhibiting reverse-transcriptase (RT) activity.
  • RT reverse-transcriptase
  • An agent is capable of inhibiting RT can be useful for the treatment of HIV infection in humans by inhibiting HIV replication in infected cells or individuals.
  • the compounds approved for use in treating HIV infection and AIDS include nucleoside RT inhibitors (NRTI) such as 3 ’-azido-3 ‘-deoxythymidine ( AZT, also known as Zidovudine (ZDV), azidothymidine (AZT)), 2',3'-dideoxyinosine (ddl), 2',3 !
  • ddC dideoxycytidine
  • d4T dideoxycytidine
  • 3TC dideoxycytidine
  • abacavir emtricitabine
  • tenofovir disoproxil fumarate as well as nonnucleoside RT inhibitors (NNRTI) such as nevirapine, delavirdine, efavirenz, rilpivirine and doravirine
  • NRTI nonnucleoside RT inhibitors
  • the at least one additional therapeutic agent is an anti-HIV agent or an anti-viral agent.
  • anti-HIV agent refers to a compound, a mixture of one or more compounds, a formulation, a chemical agent or a biological agent such as antibody, protein, peptides, nucleotide, other biological compound, or a combination thereof, that can be directly or indirectly effective in the inhibition of HIV, the treatment or prophylaxis of HIV infection, and/or the treatment, prophylaxis or delay in the onset or progression of AIDS and/or diseases or conditions arising therefrom or associated therewith, an RNA virus infection, or a combination thereof.
  • the anti-HIV agents can comprise HIV antiviral agents, immunomodulators, anti-infectives, vaccines or a combination thereof useful for treating HIV infection or AIDS.
  • antiviral agents for Treating HIV infection or AIDS include, but are not limited to, under respective trademarks or registered trademarks with respective owners, atazanavir (Reyataz®), darunavir (Prezista®), dolutegravir (Tivicay®), doravirine, efavirenz (EFV, Sustiva®, Stocrin®), cabotegravir, bictegravir, emtricitabine (FTC, Emtriva®), rilpivirine (Edurant®), tenofovir hexadecyl oxy propyl (CMX- 157), tenofovir alafenamide fumarate, MK-8507, and lenacapavir.
  • An anti- HIV agent can have one or more activities such as entry inhibitor (El); a capsid inhibitor (CAI), fusion inhibitor (FI); integrase inhibitor (Ini); protease inhibitor (PI); nucleoside reverse transcriptase inhibitor (nRTI or NRTI) or non-nucleoside reverse transcriptase inhibitor (nnRTI or NNRTI).
  • El entry inhibitor
  • CAI capsid inhibitor
  • FI fusion inhibitor
  • PI integrase inhibitor
  • PI protease inhibitor
  • nRTI or NRTI nucleoside reverse transcriptase inhibitor
  • nnRTI or NNRTI non-nucleoside reverse transcriptase inhibitor
  • the anti-HIV agent is selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, lenacapavir, and a combination thereof.
  • the anti-viral agent is useful in treating or preventing herpes simplex virus (HSV) infection.
  • the ant- viral agent is selected from helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, or ritonavir.
  • the at least one additional therapeutic agent comprises a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid).
  • polynucleotide in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi- inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger mRNA
  • RNAi- inducing agents RNAi agents
  • siRNAs siRNAs
  • shRNAs shRNAs
  • miRNAs miRNAs
  • antisense RNAs antisense RNAs
  • ribozymes catalytic DNA
  • RNAs that induce triple helix formation aptamers, vectors, etc.
  • the at least one additional therapeutic agent is an RNA.
  • RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense RNAs, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.
  • siRNA small interfering RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • the at least one additional therapeutic agent is an mRNA.
  • An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide.
  • a polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity.
  • a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.
  • the at least one additional therapeutic agent is an siRNA.
  • An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest.
  • an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA.
  • An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest.
  • the siRNA may be an immunomodulatory siRNA.
  • the at least one additional therapeutic agent is an shRNA or a vector or plasmid encoding the same.
  • An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.
  • the disclosure further provides methods for treating a disease or disorder comprising administration of ionic liquid formulation as disclosed herein, to a subject in need thereof.
  • the subject is a human.
  • the present disclosure provides methods of treating or preventing a Cryptococcus fungal infection in a mammal comprising administering to the mammal in need thereof an ionic liquid formulation.
  • the present disclosure also provides methods of killing or inhibiting the growth of a Cryptococcus species comprising contacting the Cryptococcus species with ionic liquid formulations.
  • the present disclosure provides methods of treating or preventing a fungal infection in a mammal comprising administering to the mammal in need thereof ionic liquid formulations in combination with one or more other anti-fungal agent(s) (i.e., in the same ionic liquid formulation or in separate pharmaceutical compositions).
  • the present disclosure also provides methods of killing or inhibiting the growth of a fungus comprising contacting the fungus with ionic liquid formulations in combination with one or more other anti-fungal agent(s) (i.e., in the same ionic liquid formulation or in separate pharmaceutical compositions).
  • the fungus is, or the fungal infection is caused by, Aspergillus spp. (e.g., Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus), Fusarium spp. (e.g., Fusarium solani, Fusarium moniliforme, and Fusarium proliferatum), Malassezia spp. (e.g., Malassezia pachydermatis), Candida spp. (e.g., Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, and Candida auris), or Cryptococcus spp.
  • Aspergillus spp. e.g., Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus
  • Fusarium spp. e.g., Fusa
  • Mucorales such as Mucor spp. (e.g., M. circinelloides), Rhizopus spp. (e.g., Rhizopus delemar and Rhizopus oryzae), Lichtheimia spp. (e.g., Lichtheimia corymbifera), and Rhizomucor spp., or Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis, or Sarcopodium oculorum.
  • Mucor spp. e.g., M. circinelloides
  • Rhizopus spp. e.g., Rhizopus delemar and Rhizopus oryzae
  • Lichtheimia spp. e.g., Lichtheimia corymbifera
  • Rhizomucor spp. or Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis,
  • the fungus is, or the fungal infection is caused by, Aspergillus spp., Fusarium spp., Malassezia spp., Candida spp., or Cryptococcus spp.
  • the fungus is, or the fungal infection is caused by, Aspergillus spp.
  • the fungus is, or the fungal infection is caused by, Aspergillus fumigatus.
  • the fungus is, or the fungal infection is caused by, Aspergillus flavus.
  • the fungus is, or the fungal infection is caused by, Aspergillus niger.
  • the fungus is, or the fungal infection is caused by, Aspergillus terreus. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium spp. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium solani. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium moniliforme. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium proliferatum. In some embodiments, the fungus is, or the fungal infection is caused by, Malassezia spp.
  • the fungus is, or the fungal infection is caused by, Malassezia pachydermatis. In some embodiments, the fungus is, or the fungal infection is caused by, a Mucorales. In some embodiments, the fungus is, or the fungal infection is caused by, Mucor spp. In some embodiments, the fungus is, or the fungal infection is caused by, M. circinelloid.es . In some embodiments, the fungus is, or the fungal infection is caused by, Rhizopus spp. In some embodiments, the fungus is, or the fungal infection is caused by, Rhizopus delemar.
  • the fungus is, or the fungal infection is caused by, Rhizopus oryzae. In some embodiments, the fungus is, or the fungal infection is caused by, Lichtheimia spp. In some embodiments, the fungus is, or the fungal infection is caused by, Lichtheimia corymbifera. In some embodiments, the fungus is, or the fungal infection is caused by, Rhizomucor spp. In some embodiments, the fungus is, or the fungal infection is caused by, Candida spp. In some embodiments, the fungus is, or the fungal infection is caused by, Candida albicans. In some embodiments, the fungus is, or the fungal infection is caused by, Candida glabrata.
  • the fungus is, or the fungal infection is caused by, Candida tropicalis. In some embodiments, the fungus is, or the fungal infection is caused by, Candida krusei. In some embodiments, the fungus is, or the fungal infection is caused by, Candida auris. In some embodiments, the fungus is, or the fungal infection is caused by, Cryptococcus spp. In some embodiments, the fungus is, or the fungal infection is caused by, Cryptococcus neoformans. In some embodiments, the fungus is, or the fungal infection is caused by, Chrysosporium parvum.
  • the fungus is, or the fungal infection is caused by, Metarhizium anisopliae. In some embodiments, the fungus is, or the fungal infection is caused by, Phaeoisaria clematidis. In some embodiments, the fungus is, or the fungal infection is caused by Sarcopodium oculorum.
  • Additional pathogenic fungi include the genus Candida (examples include C. albicans, C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. parapsilosis, C. dubliniensis and C. auris), the genus Cryptococcus (examples include C. neoformans and C. gatti), the genus Trichosporon (examples include T. asahii, T. asteroides, T. cutaneum, T. dermatis, T. dohaense, T. inkin, T. loubieri, T. mucoides, and T.
  • Candida examples include C. albicans, C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. parapsilosis, C. dubliniensis and C. auris
  • the genus Cryptococcus examples include C.
  • the genus Coccidioides examples include C. immitis, and C. posadasii
  • the genus Pneumocystis include P. carinii and P. jiroveci
  • the genus Histoplasma examples include H. capsulatum
  • the genus Trichophyton examples include T. schoenleinii, T. mentagrophytes, T. verrucosum, and T. rub rum
  • the genus Rhizopus examples include R. oryzae and R.
  • the genus Apophysomyces examples include A. variabilis
  • the genus Rhizomucor examples include R. pusillus, R. regularior, and R. chlamydosporus
  • the genus Lichtheimia examples include L. ramose and L. corymbifera
  • the genus Scedosporium examples include S. apiospermum
  • the genus Lomentospora examples include L. prolificans.
  • the fungi is Mucorales (for which conventional therapy results are poor), and other lethal pathogens for which current therapy is poor or lacking (Fusarium, Scedosporium, Lomentospora, Acremonium, and Exserohilum).
  • the fungal species is resistant to a therapeutic agent. In some embodiments, the fungal species is resistant to an azole. In some embodiments, the fungal species is resistant to an echinocandin. In some embodiments, the fungal species is CAS- resistant. In some embodiments, the fungal species is VOR-resistant.
  • the present disclosure provides methods of treating or preventing human immunodeficiency virus (HIV) infection in a mammal comprising administering to the mammal in need thereof an ionic liquid formulation.
  • the ionic liquid formulation comprises rilpivirine.
  • the ionic liquid formulation comprises tenofovir.
  • the method further comprises administering to the subject one or more anti -HIV agents selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, lenacapavir, and a combination thereof.
  • the ionic liquid formulation is configured for oral administration.
  • the present disclosure provides methods of treating or preventing herpes simplex virus (HSV) infection in a mammal comprising administering to the mammal in need thereof an ionic liquid formulation.
  • the ionic liquid formulation comprises adefovir dipivoxil (ADV).
  • the method further comprises administering to the subject one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir.
  • the ionic liquid formulation is configured for topical administration.
  • the ionic liquid formulation is configured for optical administration.
  • the ionic liquid formulation is configured for oral administration.
  • the method further comprises administering to the subject one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir.
  • the ionic liquid formulation is configured for topical administration.
  • the ionic liquid formulation is configured for optical administration.
  • the ionic liquid formulation is configured for oral administration.
  • methods for treating or preventing a parasitic infection in a mammal comprises administering to the mammal in need thereof a therapeutically effective amount of an ionic liquid formulation.
  • the mammal is a human being.
  • the mammal is a human being suffering from or at risk of suffering from a parasitic infection.
  • the parasitic infection is selected from African trypanosomiasis, amoebiasis, ascariasis, babesiosis, Chagas disease, cryptosporidiosis, cutaneous larva migrans, dirofilariasis, echinococcosis, fasciolosis, filariasis, lymphatic filariasis, giardiasis, helminthiasis, hookworm infection, leishmaniasis, visceral leishmaniasis, malaria, neurocysticercosis, onchocerciasis, protozoan infection, schistosomiasis, taeniasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinosis, and zoonosis.
  • the method further comprises administering to the mammal an antiparasitic agent.
  • the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal.
  • the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole.
  • methods for treating or preventing neurocysticercosis in a mammal comprises administering to the mammal in need thereof a therapeutically effective amount of an ionic liquid formulation.
  • the mammal is a human being.
  • the mammal is a human being suffering from or at risk of suffering from neurocysticercosis.
  • the method further comprises administering to the mammal an antiparasitic agent.
  • the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal.
  • the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole.
  • suitable dosage ranges for intravenous (i.v.) administration are 0.01 mg to 500 mg per kg body weight, 0.1 mg to 100 mg per kg body weight, 1 mg to 50 mg per kg body weight, or 10 mg to 35 mg per kg body weight.
  • suitable dosage ranges for other modes of administration can be calculated based on the forgoing dosages as known by those skilled in the art.
  • recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal administration or administration by inhalation are in the range of 0.001 mg to 200 mg per kg of body weight, 0.01 mg to 100 mg per kg of body weight, 0.1 mg to 50 mg per kg of body weight, or 1 mg to 20 mg per kg of body weight.
  • Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.
  • the ionic liquid formulations described herein can be administered in any conventional manner by any route where they are active. Administration can be systemic, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants.
  • modes of administration for the ionic liquid formulations described herein can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.
  • injectable including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly
  • vaginal creams suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.
  • the selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician to obtain the desired clinical response.
  • the amount of ionic liquid formulations described herein to be administered is that amount which is therapeutically effective.
  • the dosage to be administered will depend on the characteristics of the subject being treated, e.g., the particular animal treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician).
  • the amount of a ionic liquid formulations described herein that will be effective in the treatment and/or prevention of a fungal infection will depend on the nature of the fungal infection, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • a suitable dosage range for oral administration is, generally, from about 0.001 milligram to about 200 milligrams per kilogram body weight.
  • the oral dose is from about 0.01 milligram to 100 milligrams per kilogram body weight, from about 0.01 milligram to about 70 milligrams per kilogram body weight, from about 0. 1 milligram to about 50 milligrams per kilogram body weight, from 0.5 milligram to about 20 milligrams per kilogram body weight, or from about 1 milligram to about 10 milligrams per kilogram body weight.
  • the oral dose is about 5 milligrams per kilogram body weight.
  • the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • kits comprising at least one disclosed ionic liquid formulation, and instructions for using the ionic liquid formulation.
  • the kits can also comprise other agents and/or products co-packaged, co-formulated, and/or co-delivered with other components.
  • a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed ionic liquid formulation and/or product and another agent for delivery to a patient.
  • kits can also comprise instructions for using the components of the kit.
  • the instructions are relevant materials or methodologies pertaining to the kit.
  • the materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents.
  • Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
  • kits can be employed in connection with the disclosed methods.
  • the kit may further contain containers or devices for use with the methods or compositions disclosed herein.
  • the kits optionally may provide additional components such as buffers and disposable single-use equipment (e.g., pipettes, cell culture plates or flasks).
  • kits provided herein are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.
  • TDF or TAF tenofovir prodrugs
  • cART combination antiretroviral therapy
  • TDF is an integral part of first-line therapy in low- and middle-income countries (L& MICs) due to its relatively lower cost and greater avail-ability 5,6 compared to TAF whereas TAF, due to its selectivity, greater potency, and lesser side effects, continues to replace TDF in developed countries.
  • L& MICs low- and middle-income countries
  • TAF due to its selectivity, greater potency, and lesser side effects
  • more than 15 FDA-approved oral products containing a combination of antiretroviral drugs contain either TDF or TAF 1 which further substantiates the importance of tenofovir prodrugs for the lifelong management of HIV infection.
  • a combination pill containing either TDF or TAF is also approved for oral HIV pre-exposure prophylaxis (PrEP) in different populations.
  • TDF and TAF have significant improvement in permeability and bioavailability compared to their parent drug (tenofovir; TFV), both prodrugs are still classified as Class Ill drugs by the Biopharmaceutics Classification System due to their high solubility and overall low permeability.
  • TFV tenofovir
  • TDF and TAF both are substrates for the P-gp mediated efflux which further limits their oral absorption.
  • 9-11 Furthermore, TDF, due to the high lability of phos-phonate diester side chains, undergoes premature hydrolysis in the intestine which further limits its oral absorp-tion.
  • ILs ionic liquids
  • Hydrophilic ionizable drugs including antiviral nucleotides such as tenofovir have high water solubility and low membrane permeability which eventually limit their bioavailability.
  • These hydrophilic ionizable drugs are also difficult to formulate into nanoformulations.
  • ILs are organic salts with a melting point of ⁇ 100°C and depending upon the cations and anions involved in the formation, ILs can even be liquid at room tempera-ture.
  • ILs ionic liquids
  • hydrophilic and lip-ophilic ionizable drugs with suboptimal solubility and permeability characteristics.
  • Example_l_Scheme 1 Hydrophobic and hydrophilic ionizable drugs with high crystallinity and low permeability can be converted to amphiphilic ILs using docusate
  • ExampIe_4A - Amphiphilic ILs can be efficiently incorporated into SoluPlus nanomicelles to further improve their in vivo efficacy. While the transformation of ionizable drugs with suboptimal solubility and permeability characteristics to amphiphilic IL(s) could improve their lipid solubility and permeability, it is necessary to package these ILs into nanoformulations to further improve the delivery, bioavailability, and efficacy of ILs.
  • SoluPlus is a biodegradable amphiphilic copolymer, which forms nanoscale micelles upon dilution with physiological fluids and can encapsulate a variety of hydrophobic drugs leading to improved solubility, permeability, and bioavailability.
  • mice were intranasally infected with Cryptococcus neoformans and the animals were treated with different OXF formulations for 14 days. After 14 days of therapy, the animals were euthanized, lungs were harvested and the C. neoformans burden in the lungs was measured (colony forming units or CFUs).
  • SoluPlus nanomicelles containing OXF-Doc (OXF-Doc in SoluPlus) showed the highest in vivo efficacy compared to the pure OXF and OXF-Doc (FIG. 36) indicating the importance of packaging of ILs into SoluPlus nanomicelles or to achieve enhanced drug delivery.
  • SoluPlus nanomicelles containing ILs of TDF/TAF will have improved oral bioavailability and in vivo antiviral efficacy.
  • Example_4B Daily oral administration of oral SoluPlus nanomicelles was well tolerated by mice.
  • Example_4C Develop, characterize, and evaluate SoluPlus nanomicelles containing TDF ILs or TAF ILs.
  • TDF/TAF water soluble ionizable TDF/TAF can electrostatically interact with generally regarded as safe (GRAS) fatty permeation enhancers to yield lipophilic ILs with reduced water solubility and improved permeability. Subsequent incorporation of these TDF/TAF- SoluPlus nanomicelles will further improve their oral delivery leading to improved oral bioavailability and phar-macokinetics.
  • GRAS safe fatty permeation enhancers
  • Example_l_Scheme 2 shows that TDF as well as TAF can readily interact with GRAS fatty permeation enhancers such as oleic acid, capric acid, undecylenic acid, and salcaprozic acid to yield ILs. While Example_l_Scheme 2 only depicts the formation of TDF oleate and TAF oleate as an IL, the other fatty acids such as capric acid, undecylenic acid, and salcaprozic acid also readily transformed TDF/TAF to TDF/TAF IL within 10 minutes.
  • GRAS fatty permeation enhancers such as oleic acid, capric acid, undecylenic acid, and salcaprozic acid
  • Example_l_Scheme 2 Synthesizing TDF/TAF ionic liquids using fatty acid permeation enhancers as a counterion.
  • TDF/TAF oleate is readily formed within 10 minutes after mixing TDF/TAF and oleic in methanol followed by complete evaporation of ethanol. The same scheme can be used to synthesize other TDF/TAF ILs.
  • TDF oleate TDF caprate, TDF undecylenate, TDF salcaprozate
  • TAF oleate TAF caprate
  • TAF undecylenate TAF salcaprozate
  • TDF/TAF-SoluPlus nanomicelles containing TDF/TAF-ILs and a total of 4 formulations of SoluPlus nanomicelles (2 formulations each with TDF ILs or TAF ILs) with the lowest size, polydispersity index, and surface charge and highest physicochemical and chemical stability will be selected for the PK and relative oral bioavailability studies.
  • mice 27 Based on the previous reports on the pharmacokinetics of TDF and TAF in mice 27 , we plan to study the pharmacokinetics at a 60 mg/kg dose of TDF and 5 mg/kg of TAF.
  • TDF 10 mg/mL
  • TAF 4 mg/ml
  • the optimized SoluPlus nanomicelles containing TDF or TAF ILs will be prepared to obtain 10 mg/ml of TDF or 4 mg/ml of TAF.
  • six- to 8-weeks old Balb/c mice will be randomly divided into 6 groups (n > 10 mice per group; 5 male and 5 female).
  • mice will receive either receive 100 pL oral gavage of TDF solution, TAF solution, SoluPlus nanomicelles containing TDF IL (2 formulations), or SoluPlus nanomicelles containing TAF IL (2 formulations).
  • TDF solution aqueous fetal calf serum
  • SoluPlus nanomicelles containing TAF IL aqueous fetal calf serum
  • the blood, lymph nodes, vaginal tissue, and brain will be collected from the eu-thanized animals for the study of tissue distribution. Blood will be centrifuged at 2000 g and 4°C for 15 min and the recovered plasma and other tissues will be stored at -20°C until further analysis.
  • the concentration of TAF, tenofovir and tenofovir diphosphate in plasma, mesenteric lymph node, vaginal tissue, and brain homogenate will be determined using LC-MS/MS.
  • the PK of TAF, tenofovir and tenofovir diphosphate in plasma, lymph node, vaginal tissue, and brain after delivery of oral TDF/TAF nanoformulations will be carried out at the University of Arizona Cancer Center Analytical Chemistry Shared Resource.
  • Example_4D Evaluate in vivo antiviral efficacy of oral SoluPlus nanomicelles containing TDF IL.
  • the hu-BLT mice reconstitute all lineages of immune cells in peripheral blood, secondary lymphoid, and mucosal tissues, of which T cells have undergone ‘education’ in human thymic tissues.
  • Dr. Li’s group has over 10 years of experience in developing new hu-BLT mice models, such as double hu-BLT mice 30 and hu-BLT-hIL34 mice. 31 His group has also used hu- BLT mice to study HIV-1 transmission, pathogenesis, and latency, and to evaluate the efficacy of ART.
  • HFT human fetal liver and thymic tissues
  • HFT Humanized-mice are the only small animal models that are susceptible to HIV infection directly. Among the different hu-mice, human T cells in hu-HSC mice are less abundant compared to hu-BLT mice, especially in mucosal tissues. Further, human T cells are “uneducated” by the human thymus. In contrast, the hu-BLT mice reconstitute all lineages of immune cells in peripheral blood, secondary lymphatic, and mucosal tissues. 29,42-45 Notably, these human T cells have undergone positive and negative selections, differentiation, and development in human thymic tissues. 27,28 Thus, without HFT, the research goals of this project may not be adequately accomplished.
  • TDF dose is selected based on a previous report on hu-BLT mice. 50 We will collect blood on days 0, 2, 3, 7, 10, and 14 for quantifying plasma viral RNA load and partial plasma samples will also be used to quantify the TDF concentration. We will weigh the mice daily during the treatment period. At the end of the experiment after monitoring for 14 days post treatment, all the animals will be euthanized. Tissues of lymph nodes, spleen, and ileum gut will be harvested, of which two third will be fixed in 10% neutral formalin for vRNA quantification using HIV RNAscope as we previously reported and one third will be freshly homogenized for vRNA and TDF quantification using qPCR, RT-qPCR, and the assay. The efficacy will be indicated by the decay of pVL (vRNA copies) and infected cell frequency in lymph nodes and spleen tissues.
  • Example_4 The following references pertain to the numerical reference citations recited in Example_4:
  • HIV human immune deficiency virus
  • cART combination antiretroviral therapy
  • PK pharmacokinetics
  • Rilpivirine is a potent non-nucleoside reverse transcriptase inhibitor (NNRTI) prescribed for the first-line treatment of HIV-1 infection in naive patients with a viral load (VL) of less than 100,000 copies/mm. 9-11 Furthermore, due to its good safety profile and dosing convenience (once daily 25 mg RPV tablet), RPV is commonly used in virologically suppressed pre-treated patients when treatment simplification is desired. 12 15 Additionally, an RPV-based 2- drug regimen (Juluca) is commonly used for the long-term maintenance of virologic suppression in patients.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • RPV has low tissue penetration ratios ( ⁇ 0.1) in the brain, spleen, gut-associated lymphoid tissue (GALT), and testes compared to plasma.
  • the anatomical sites such as the brain, spleen, GALT, and testes are known as the HIV sanctuary sites where the replication-competent virus persists. 18, 19 20
  • Oral lipid-based formulations of antiretroviral drugs such as saquinavir, ritonavir, and tipranavir were successfully translated to the clinic.
  • SNES self-nanoemulsifying systems
  • Ionic liquid is a low-melting salt containing organic/inorganic cation and anion, and depending upon the composition, IL can also be liquid at room temperature. 25, 26
  • APIs ionizable active pharmaceutical ingredients
  • APLILs ILs
  • Example 2A Materials and Methods Related to Example 2
  • Rilpivirine was purchased from Ambeed (Arlington Heights, IL, USA). Sodium docusate and Linoleic acid (99%) were obtained from TCI chemicals (Portland, OR, USA). Sodium lauryl sulfate (SLS) was received as a gift sample from BASF. Oleic acid and geranic acid were purchased from Thermo Fischer (MA, USA). Docosahexaenoic acid (DHA) was received from Nu-Check Prep (MN, USA). Methanol (AR grade), acetonitrile (AR grade), acetone (AR grade), and dichloromethane (AR grade) were purchased from VWR International (PA, USA).
  • the nylon syringe filters with glass fiber prefilter (0.22 pm) were purchased from Simsii Inc. (CA, USA).
  • Capryol 90, Labrafac MC60, Peceol and Labrasol, Plurol Oleique CC 497, Transcutol and Lauroglycol 90 (Gattefosse USA, NI, USA), Capmul MCM-C8 (Abitec Corp., OH, USA) were received as gift samples.
  • Kolliphor ELP, Kolliphor PS80, Kolliphor PS20, Kolliphor HS15, Kollisolv PEG400, Kolliphor RH40 was received as a gift sample from BASF. All other chemicals used were of analytical grade unless otherwise indicated. Animals.
  • C57BL/6 (B6) mice male, 10- week-old mice were obtained from breeding stocks maintained at the University of Arizona. All mice were housed under conditions of controlled temperature (22 °C) with on-off light cycle, with food and water provided ad libitum. Mice were fasted for 12h before oral administration of the drugs. All animal studies were approved by the University of Arizona Animal Care and Use Committee.
  • HPLC High-pressure liquid chromatography
  • the working solutions were prepared by diluting the stock solution with methanol (HPLC grade) to achieve a concentration equivalent to 30 uM RPV.
  • the chromatographic separation was achieved using Gemini (150mm x 4.6 mm, 3pm, 110 A ) C18 column (Phenomenex, Torrance, CA).
  • the mobile phase was composed of acetonitrile and 25 mM potassium dihydrogen phosphate solution (70:30 v/v).
  • the RPV was monitored at 290 nm at a flow rate of 0.6 mL/min and a column oven temperature of 35 °C.
  • a standard curve was prepared by injecting 1-25 pg/mL of RPV. All the experiments were performed in triplicate. The inter-day and intra-day variability for the standard curve was always ⁇ 5%.
  • RPV solubility in aqueous and lipid vehicles The saturation solubility of RPV in different aqueous and lipid vehicles such as water, pH 1.2 buffer, pH 4.5 buffer, pH 6.8 buffer, Capryol 90, Labrasol ALF, Labrafac MC60, Capmul MCM C8 and Peceol was determined in triplicate. Briefly, an excess amount of RPV ( ⁇ 5 mg) was transferred to an Eppendorf tube containing 1 ml of aqueous or lipid vehicle. The contents were mixed for 30 seconds to ensure a uniform dispersion. The dispersions were incubated in a temperature- controlled orbital shaker set at 37 °C and 50 rpm speed for 24 hours.
  • RPV-IL/lipophilic salts Spectroscopic characterization of RPV-IL/lipophilic salts.
  • the developed RPV- IL/lipophilic salts were characterized using FTIR spectroscopy and NMR spectroscopy using the procedures described in our recently published papers. 32, 33
  • octanol-water partition coefficient (LogP). The octanol-water partition coefficient of RPV, and its ILs/salts was determined using the previously described method. 32 Before beginning the experiment, water and 1 -octanol were equilibrated for 24 hours using an orbital shaker. Octanol (0.5 ml) and 10 mg of RPV or RPV ILs/salts were mixed in an Eppendorf tube and equilibrated for 24 hours at room temperature.
  • Solubility of RPV-ILs/salts/cocrystals in aqueous vehicles The saturation solubility of RPV-ILs/salts/cocrystals in ultra-pure water, pH 1.2, pH 4.8, and pH 6.8 buffer was determined using previously reported procedures.
  • each well was infected with 2 ng HIV- IBAL and simultaneously treated with the indicated RPV (1 nM), RPV-IL/salt (equivalent to 1 nM RPV) or vehicle control and left for 48 hours. After 48 h, each well was lysed with IX Passive Lysis Buffer for 10-15 mins at 37°C, 5% CO2, and the lysate was collected. The luciferase assay was performed using a Berthhold Detection Systems Sirius Luminometer. To each assay tube, 50 pl of luciferase substrate (Promega) was added followed by 20 pl cell lysate. The mix was immediately vortexed and assessed by the luminometer. All experiments were carried out in triplicate.
  • RPV or RPV ILs/salts stock was prepared in ethanol: acetone (1: 1; RPV concentration: 5 mg/mL), and 1 mL of this solution was added to the previously prepared ethanolic solution of lipid vehicle. The mixture was vortexed, evaporated on a rotary evaporator, and dried in a vacuum oven overnight to remove traces of organic solvents. The vacuum-dried lipid solution was inspected visually for the solubilization/precipitation of RPV or RPV ILs/salts. The addition of RPV or RPV ILs/salts in the lipid vehicle was continued in 5 mg increments until precipitation was observed.
  • a range of co-surfactants were evaluated for their ability to facilitate the formation of nanoemulsion. Briefly, 100 mg of RPV-Doc:Labrafac MC60 (1 :2 w/w) mixture, 50 mg of surfactant (selected from the previous screening), and 50 mg of each cosurfactant underwent homogenization through heating at 70-80°C. A portion of this isotropic mixture (10 mg) was weighed and diluted with 10 mL of distilled water to form a fine emulsion. Following a 2 h settling period, its transmittance was measured at 638.2 nm using a UV-visible spectrophotometer with distilled water as a reference.
  • ternary phase diagram Construction of the ternary phase diagram.
  • RPV-Doc:Labrafac MC60 oil phase
  • Kolliphor ELP surfactant
  • Transcutol HP cosurfactant
  • compositions generated for the phase diagram were diluted with 10 mL of distilled water, and the globule size of the resulting dispersions was determined using dynamic light scattering (Litesizer 500 particle analyzer, Anton-Paar USA, Inc., Torrance, CA). The compositions that yielded nanoemulsion with an average globule size ⁇ 250 nm and uniform size distribution were identified.
  • a ternary phase diagram highlighting the optimal RPV-Doc-MC60SNES compositions was generated using Origin Pro software.
  • the ternary phase diagram identified seven optimal formulations suitable for further evaluation.
  • shortlisted compositions (10 mg each) were diluted with 10 ml of water, pH 1.2 buffer, pH 4.5 buffer, and pH 6.8 buffer.
  • the globule size of the resulting dispersions was determined using dynamic light scattering (Litesizer 500 particle analyzer, Anton-Paar USA, Inc., Torrance, CA), and the composition that had the highest amount of oil phase and yielded a nanoemulsion in all dilution mediums irrespective of their pH value was selected for the PK and biodistribution studies.
  • RPV-Doc-Cap90SNES For the preparation of RPV-Doc-Cap90SNES, RPV-Doc-Capryol 90 mixture (125 mg), Kolliphor ELP (93.7 mg), and Capmul MCM (31.5 mg) were weighed in a microcentrifuge tube and homogenized with heating (70-75 °C).
  • the RPV-Doc-Cap90SNES 50 mg was diluted to 50 mL with distilled water, pH 1.2 buffer, pH 4.5 buffer, pH 6.8 buffer, or pH 7.4 buffer and vortexed at 1500 rpm for 1 min to yield nanoemulsion.
  • the globule size and polydispersity index (PDI) of the nanoemulsion were evaluated using a Litesizer 500 particle analyzer (Anton-Paar USA, Inc., Torrance, CA). All batches were carried out in triplicate.
  • RPV-Doc-MC60SNES, or RPV-Doc-Cap90SNES were diluted with ultrapure Millipore water (5x and 2x respectively) and the diluted nanoemulsion (7.5 pl) was applied to copper grids (Electron Microscopy Sciences, PA, USA) and left to dry at room temperature overnight. Images were obtained at the Microscopy Core facility at the University of Arizona using a FEI Tecnai G2 Spirit BT TEM (FEI, Hilsboro, OR, USA) at an accelerating voltage of lOOkV. TIFF images were captured using an AMT XR41 side-mounted camera at various magnifications to obtain suitable TEM images. All samples were imaged within 24 hours of rehydration.
  • RPV concentration 2 mg/ml
  • HPMC hydroxypropyl methylcellulose
  • RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES were diluted with ultrapure water to obtain a nanoemulsion containing RPV-Doc equivalent to 2 mg/mL. of RPV. Briefly, B6 male mice were randomly divided into 3 groups (n > 4 mice per group).
  • mice received either 100 pL oral gavage of RPV suspension, RPV-Doc - MC60SNES, or RPV-Doc-Cap90SNES (RPV dose: 10 mg/kg).
  • the blood samples were collected through the tail vein using heparinized capillary tubes at 0, 0.5, 1, 3, 6, 9, and 12 h.
  • the collected blood was centrifuged at 10000 g and 4°C for 15 min and the recovered plasma samples were stored at -20°C until further analysis.
  • mice Male mice were randomly divided into 6 groups (n > 3 mice per group). The mice received either 100 pL oral gavage of RPV suspension, RPV-Doc- MC60SNES, or RPV-Doc-Cap90SNES (RPV dose: 10 mg/kg). Tire mice were euthanized using carbon dioxide at 1 h or 6 h after drug administration. The blood was first collected from the heart, and various tissues such as the hrain, mesenteric lymph node (MLN), and testis were collected. All samples were stored at -80 °C until further analysis.
  • MNN mesenteric lymph node
  • MLNs were homogenized in 100 pl PBS with a glass homogenizer by hand.
  • the plasma samples (5 pl each) or tissue homogenates (10 pl each) were mixed with 50 pl methanol and 10 pl of internal standard (IS, d6-rilpivirine 100 ng/ml).
  • the mixture was vortexed for 30 seconds, diluted with 950 pL Milli Q water, and loaded onto an OASIS® HLB cartridge (Cl 8, 1 ml/30mg, Waters Corporation, Milford, MA) pre-conditioned with 1 mL methanol followed by 1 mL water. The cartridge was then washed with 1 mL water and the analytes were finally eluted from the cartridges with 1 ml methanol for LC-MS/MS analysis.
  • OASIS® HLB cartridge Cl 8, 1 ml/30mg, Waters Corporation, Milford, MA
  • the LC-MS system consisted of an Agilent 1290 UPLC system (Agilent Technologies, Santa Clara, CA) and a Sciex Qtrap6500+ Mass Spectrometer (AB SCIEX, Framingham, MA). Analytes were separated on an EclipsePlus Cl 8 column (2.1x50 mm, 1.8 pm, Agilent) at a temperature of 35 °C, with mobile phase A containing 0.1% formic acid (v/v) in water and mobile phase B containing 0.1% formic acid (v/v) in acetonitrile.
  • Elution was at a flow rate of 0.2 mL/min as follows: 10% B (0-0.2 min), 10% B ⁇ 95% B (0.2-5 min), 95% B (5-7 min), 95% B ⁇ 10% B (7-7.1 min), 10% B (7.1-10 min).
  • the MS was operated in the positive ion mode, using electrospray ionization.
  • the ion spray voltage and temperature were set at 5000 V and 500°C, respectively.
  • Curtain gas, ion source gas 1, and ion source gas 2 were set at 25, 50, 50 psi, respectively.
  • RPV and the IS were detected using Multiple Reaction Monitoring (MRM), with a dwell time of 200 msec per transition, at m/z 367.1/224.1 (Collision Energy 38eV) and 373.1/230.1 (Collision Energy 40eV), respectively. Retention times for RPV and the IS were 3.53 min.
  • MRM Multiple Reaction Monitoring
  • the standards (10 to 10000 ng/mL in 5 pl methanol), ppi methanol), along with 10 pl IS (at 100 ng/mL in methanol), were added to 5 pl of blank mouse plasma to construct the calibration curve.
  • the correlation coefficient of RPV in plasma was >0.99 across a range of 10-10,000 ng/ml.
  • the limit of quantification (LOQ) was established at 10 ng/ml with a signal to noise ratio >10:1. The recovery was more than 85% under the current sample preparation method.
  • Example 2B - RPV showed extremely low solubility in aqueous vehicles and low solubility in lipid vehicles.
  • Example 2C Only sodium docusate could convert RPV into amphiphilic IL, RPV docusate (RPV-Doc).
  • the significant peak at 3315 cm' 1 in the RPV spectrum was attributed to the -NH stretch of the secondary amine (FIG.
  • the NMR spectra of RPV-doc and RPV-LS revealed significant displacements in the -CH proton signal of the pyrimidine ring (adjacent to the tertiary amine [proton: a]) and displacement of the secondary amine proton signal (proton: b) of RPV (FIG. 10) whereas, RPV-DHA, RPV oleate, RPV geranate, and RPV-linoleate exhibited very slight displacements in the -CH proton signal of the pyrimidine ring (FIG. 10).
  • Example 2E Thermal characterization techniques and X-ray diffractometry studies confirmed that RPV-Doc is an ionic liquid.
  • Example_2_Table SI Globule Size and Polydispersity Index of RPV-Doc:Labrafac MC60 containing SNES at different pH conditions*
  • composition R3 which yielded nanoemulsions with acceptable size and low polydispersity index irrespective of the pH of the dilution medium was selected for further evaluation.
  • Example 21 - RPV-Doc could be successfully incorporated into the previously reported SNES composition containing Capryol 90.
  • Example 2J - TEM analysis confirmed the formation of lipid nanoemulsion after dilution of RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES.
  • the TEM images (FIG. 15A-B) corroborated the globule size data obtained from the dynamic light scattering and showed the spherical morphology of the nanoemulsions.
  • HIV sanctuary sites such as mesenteric lymph nodes (MLNs), brain, and testis.
  • RPV-Doc-MC60SNES had to be slightly modified from composition R3 (FIG. 14D).
  • the RPV-Doc-MC60SNES used for the PK and biodistribution studies contained RPV- Doc:Labrafac MC60:Kolliphor ELP:Transcutol HP (46:39: 15% w/w ratio).
  • Example_2_Table 6 Pharmacokinetic parameters obtained after oral administration of RPV suspension, RPV-Doc-MC60SNES, or RPV Doc-Cap90SNES to B6 mice (data expressed as mean ⁇ S.D.; n >5).
  • RPV is a potent NNRTI that is now commonly used in combination with dolutegravir as a part of the 2-drug regimen (Juluca®). Additionally, RPV can also be used as a part of the first- line antiretroviral therapy for HIV-infected patients with plasma HIV RNA less than 100,000 copies/ml. While the low therapeutic dose (25 mg) and once-daily administration highlight the advantages of RPV-based regimens, current oral RPV formulations result in highly variable PK and suboptimal therapeutic RPV levels in 20-30% of patients eventually leading to treatment failure. 8, 16, 17 It is noteworthy that the absolute oral bioavailability of RPV in humans is unknown.
  • RPV has limited penetration into various HIV sanctuary sites such as MLNs, and the brain. 18, 19 A few studies have examined the potential of cyclodextrin nanosponges to improve the oral bioavailability of RPV 37 ’ 38 but these systems were not evaluated for the delivery to the HIV sanctuary sites. Our studies show that RPV has very low solubility in water and buffers representing pH in the gastrointestinal environment. Previous studies show that fatty meals can improve oral bioavailability of RPV 39 indicating the potential of oral lipid-based formulations. However, our studies showed that RPV has low solubility in lipids which precludes the development of oral lipid-based formulations. RPV is a classic example of crystalline hydrophobic ionizable drugs with low solubility in water and lipids.
  • Example 2 In the experiments described in Example 2 (Examples 2A-2K), we wanted to evaluate the ability of several anionic bulky counterions with different chemical structures, pKa values, and anionic functional groups to form ionic liquids (ILs) with RPV, highly crystalline hydrophobic weak base (pK a : 5.6).
  • ILs ionic liquids
  • RPV highly crystalline hydrophobic weak base
  • pK a highly crystalline hydrophobic weak base
  • oleic acid pK a : 5
  • linoleic acid pK a : 4.77
  • docosahexaenoic acid DHA; pK a : 4.89
  • geranic acid pK a : 5.266
  • geranic acid-based ILs such as choline geranate
  • RPV hydrochloride salt in situ and used sodium salt of fatty anions.
  • the geranic acid, DHA, and linoleic acid were converted to sodium salt in situ and then interacted with RPV hydrochloride.
  • RPV-Doc was obtained as a viscous liquid
  • RPV-LS and RPV-DHA were obtained as soft solids
  • RPV oleate, RPV geranate, and RPV linoleate were obtained as solids.
  • RPV-Doc proved to be an ionic liquid.
  • RPV-LS showed partial crystallinity in the XRD and due to the ApK a > 2 between RPV and lauryl sulfate, RPV-LS could be regarded as partially amorphous lipophilic salt.
  • the PXRD spectra of RPV oleate, RPV geranate, RPV-DHA, and RPV linoleate showed crystalline or partially crystalline nature and the DSC thermograms showed melting endotherms that were distinctly different than RPV.
  • RPV oleate, RPV linoleate and RPV geranate and RPV-DHA should be considered as lipid cocrystals.
  • fatty acids under right conditions, have been shown to form lipid cocrystals with weakly basic drugs such as itraconazole. 43 - 44
  • RPV-IL/lipophilic salt/lipid cocrystals showed considerably different aqueous solubility and apparent LogP values compared to pure RPV. Furthermore, compared to pure RPV, RPV- IL/lipophilic salt/lipid cocrystals also showed considerably higher kinetic solubility in the lipids used for the SNES development with RPV-Doc showing solubility as high as -500 mg/g.
  • lipids with higher molecular volume and long-chain fatty acid esters such as glyceryl monooleate (Peceol) were not efficient in solubilizing RPV-IL/lipophilic salt/lipid cocrystals compared to lipids containing esters of short-chain fatty acids (Capryol 90, Labrafac MC60, Labrasol, and Campul MCMCs).
  • the high lipid solubility of RPV-Doc opened the possibility of developing oral lipid-based formulations such as SNES.
  • the transformation of RPV into RPV-IL/lipophilic salt/lipid cocrystals did not alter the inherent antiretroviral activity of RPV in vitro.
  • SNES is an anhydrous isotropic mixture containing oil, surfactant(s), co-surfactants, and drug in a suitable proportion which can be incorporated into hard gelatin or soft gelatin capsules for oral delivery.
  • SNES composition is optimized to form an oil-in-water nanoemulsion upon dilution with the aqueous phase upon gentle agitation.
  • Kolliphor ELP and Transcutol HP are well-known pharmaceutical excipients which are used in FDA-approved pharmaceutical products.
  • the optimized RPV-Doc-MC60SNES yielded nanoemulsion with size ⁇ 250 nm irrespective of the pH of the dilution medium.
  • RPV-Doc-Cap90SNES resulted in considerably quicker absorption (tma X : ⁇ 0.7 h) compared to RPV-Doc-MC60SNES and RPV suspension (Example_2_Table 6). This difference could be due to the lower globule size ( ⁇ 100 nm) of nanoemulsion obtained upon the dilution of RPV-Doc-Cap90SNES compared to RPV-Doc- MC60SNES which yielded nanoemulsion with size ⁇ 250 nm.
  • DOI 10.1007/s00228-017-2405-l
  • Lipid-Based Formulations Preferentially Increase Lipid Solubility, In Vitro Solubilization Behavior and In Vivo Exposure. Pharmaceutics 2019, 72 (1). DOI:
  • Example 3 Ionic Liquid Formulations including Adefovir Dipivoxil
  • HSV can cause recurrent and lifelong ocular infection leading to HSV keratitis and ocular HSV infection is the most common cause of infection-associated vision loss worldwide.
  • ANPs FDA-approved acyclic nucleoside phosphonates
  • ANPs are a class of broad-spectrum antiviral drugs that are active against a variety of DNA viruses including HSV and retroviruses.
  • ANPs contain a phosphonate (FIG. 34) stably attached to the acyclic nucleoside. 18-23
  • the ANPs bypass the critical step of viral kinase-mediated activation and the host cellular kinases transform ANPs into triphosphate mimicking ANPs eventually leading to the inhibition of viral DNA polymerase.
  • ANPs such as cidofovir, adefovir, and tenofovir or their prodrugs, adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are used for the treatment of various viral infections but not for HSV infections.
  • ADV adefovir dipivoxil
  • TDF tenofovir disoproxil fumarate
  • tenofovir has low potency against HSV-1 and would not be useful for ocular herpes therapy.
  • adefovir has at least 10-fold higher potency against HSV compared to tenofovir 26 but like tenofovir, its extreme hydrophilicity and low tissue permeability 27-30 can further limit its potential for ocular delivery.
  • the pivalic acid ester prodrug is used for ocular delivery.
  • Dipivefrin a pivalic acid ester prodrug (FIG. 35) was developed to improve the ocular tissue permeability of epinephrine, a polar molecule with limited permeability 31 and this prodrug is commercially available as Propine® eye drops indicating the potential of pivalic acid ester prodrugs in ocular delivery.
  • ADV is an FDA-approved pivalic acid prodrug which is currently approved for the treatment of chronic hepatitis B infection. Our preliminary studies showed that ADV has remarkably higher in vitro antiviral activity (FIG. 36) compared to adefovir (AFV) and acyclovir (ACV). Hence, ADV can be repurposed for ocular herpes therapy.
  • Poloxamer 407-coated mucus-penetrating ocular nanosuspensions have been translated to the clinic.
  • the presence of the mucin layer in the tear film poses a significant barrier to the ocular delivery of various drugs.
  • the mucin layer due to its complex mesh-like structure, can trap foreign molecules including drugs and particles due to adhesive interactions; thus, preventing effective delivery to the corneal epithelium.
  • Poloxamer 407 is an FDA-approved amphiphilic polymeric surfactant approved for oral, parenteral, and topical (vaginal, dermal, and ocular) routes.
  • Transformation of hydrophilic ionizable drugs into lipophilic salts or ionic liquids (ILs) is a pharmaceutically viable strategy to improve their transformation into nanoformulations and delivery.
  • Many pharmaceutical drugs including those present in the clinically approved topical eye drops are available as water-soluble salts 42-45 and in the context of ocular delivery, these water soluble salts often show increased systemic absorption and rapid drug elimination. 44,45 Water soluble drug salts are also difficult to formulate into nanoparticles or other sustained release drug delivery systems.
  • ILs are organic salts with a melting point of ⁇ 100°C and depending upon the cations and anions involved in the formation, ILs can even be liquid at room temperature. Transformation of highly water soluble salts such as metformin hydrochloride into metformin docusate, a lipophilic IL (FIG. 37) using pharmaceutically acceptable fatty anions is a novel and pharmaceutically viable strategy to improve their permeability, bioavailability, and efficacy. 49,50 Furthermore, a transdermal anesthetic patch containing IL of lidocaine has successfully completed the Phase III clinical trial 51 and is waiting for approval indicating the pharmaceutical viability of the IL approach.
  • Example 3B Topical administration of 1 % w/v ADV solution showed robust antiviral efficacy in a mouse model of ocular herpes infection.
  • Example 3C - Moxifloxacin hydrochloride the water soluble antibiotic salt, can be converted to a lipophilic salt, MOX pamoate (MOX-PAM) which can be further transformed into a mucus-penetrating nanosuspension (NS).
  • MOX Moxifloxacin hydrochloride
  • MOX-PAM MOX pamoate
  • NS mucus-penetrating nanosuspension
  • MOX eye drops must be administered at least 3 times daily to be effective.
  • MOX being a water soluble salt, is rapidly drained away which results in a higher systemic exposure and rapid decline in ocular levels necessitating multiple administrations per day.
  • GRAS generally regarded as safe
  • PAM disodium pamoate
  • FIG. 41 A we found that disodium pamoate (PAM) resulted in a near 100% transformation of MOX into lipophilic MOX-PAM salt (FIG. 41 A) and this salt was further processed using freeze drying.
  • PAM disodium pamoate
  • MOX-PAM NS hydrophobic drugs/lipophilic salts
  • Example 3D - Topical administration of MOX-PAM NS showed improved ocular pharmacokinetics (PK) compared to Vigamox®.
  • Example 3E Once daily topical administration of MOX-PAM NS was more or equally effective compared to thrice daily topical Vigamox eyedrops in the rat model of Staphylococcus aureus infection.
  • Example_3_Scheme 1 Synthesis of lipophilic docusate-based ILs of anthelmintic benzimidazoles and antidiabetic biguanide.
  • Example 3F - Lipophilic ILs can be efficiently incorporated into SoluPlus nanomicelles to further improve their in vivo efficacy.
  • SoluPlus is a biodegradable amphiphilic copolymer, which forms nano-scale micelles upon dilution with physiological fluids and can encapsulate a variety of hydrophobic drugs leading to improved solubility, permeability, and bioavailability.
  • HSV-1 The replication and spread of HSV-1 in these tissues will be tested by (i) detecting the infectious virus in homogenized tissue by plaque assay, (ii) measuring HSV-1 DNA present by quantitative real-time (RT-) PCR, (iii) detecting HSV-1 protein expression in tissues by immunohistochemistry using a polyclonal HSV-1 antibody, and (iv) detecting and estimating LAT expression by in situ hybridization.
  • Example 3H Develop, characterize, and evaluate topical nanomicelles containing ADV- fatty acid IL for the treatment of ocular HSV infection
  • Example_3_Scheme 4 Scheme for synthesizing ADV-fatty acid ionic liquids ADV oleate is readily formed within 10 minutes after mixing ADV and oleic in ethanol followed by complete evaporation of ethanol.
  • Example_3_Table 3 Summary of ocular PK experiments for ADV solution and SoluPlus nanomicelles containing ADV IL.
  • Sigel A Sigel H, Sigel RKO. Coordination Chemistry of Nucleotides and Antivirally Active Acyclic Nucleoside Phosphonates, including Mechanistic Considerations. Molecules. 2022 Apr 19;27(9):2625.
  • Example_5_Table 1 Meglumine & dicarboxylic acid-based ILs self-assembly in water at various concentrations.
  • Example_5_Table 2 Various drugs solubility in Meglumine & bile acid-based ILs.
  • Example_5_Table 3 Niclosamide dissolved in Meglumine Cholate IL self-assemble in water at various dilutions.
  • This example describes self-assembling meglumine containing ionic liquids and their applications to drug delivery.
  • the self-assembling ionic liquid, meglumine vitamin E succinate (Meg-VES-IL) was dissolved in water at a concentration > 1.5 mg/ml to yield self-assembled nanostructures (see, Example_6_ Figure 1).
  • the drug-loaded Meg-VES self-assembling nanostructures were prepared by codissolving drug and Meg-VES in organic solvents such as methanol/ethanol and adding this mixture to the water followed by removal of organic solvent to yield nanostructures (Example_6_Table 1).
  • Example_6_Table 1 Size, and polydispersity index (PDI) of blank and drug-loaded meglumine-tocopherol succinate (Meg-VES) self-assembling IL prepared in water.
  • PDI polydispersity index
  • This example describes the preparation of self-assembling meglumine ionic liquids using stabilizers such as PEGylated phospholipid (DSPE-PEG2000) and polysorbate 80.
  • stabilizers such as PEGylated phospholipid (DSPE-PEG2000) and polysorbate 80.
  • Example_6_Table 2 Size, and poly dispersity index (PDI) and surface charge of selfassembling meglumine containing ILs prepared with additional stabilizers.
  • This example describes the development of carnitine-fatty acid amphiphilic ionic liquids and their transformation into self-nanoemulsifying systems to improve oral bioavailability of hydrophobic drugs.
  • Carnitine is a GRAS amino acid used as a nutraceutical in adults and infant formula.
  • amphiphilic ILs using carnitine and anionic fatty permeation enhancers such as is a hitherto unexplored approach to improving solubility and delivery of hydrophobic drugs.
  • Example_7_Scheme 1 Scheme for the synthesis of carnitine fatty acid ionic liquids
  • This example describes the kinetic solubility of various hydrophobic drugs in carnitine-fatty acid ionic liquids.
  • Example_7_Table 1 Solubility of various drugs in carnitine salcaprozate
  • This example describes the improvement of oral bioavailability of hydrophobic drugs when packaged into carnitine fatty acid ionic liquid self-nanoemulsifying systems.

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Abstract

This invention is in the field of medicinal chemistry and drug delivery. In particular, the invention relates to compositions comprising bioavailability (e.g., oral and local bioavailability) enhancing ionic liquid formulations and the use thereof for treating conditions or diseases in a subject.

Description

BIOAVAILABILITY ENHANCING IONIC LIQUID FORMULATIONS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Application No. 63/509,738, filed June 22, 2023, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
This invention is in the field of medicinal chemistry and drug delivery. Disclosed herein are compositions comprising bioavailability (e.g., oral and local bioavailability) enhancing ionic liquid formulations and the use thereof for treating conditions or diseases in a subject.
INTRODUCTION
The term “ionic liquids (ILs)” as used herein refers to organic salts or mixtures of organic salts which have melting point less than 150°C and depending upon the cations and anions used for the formation, IL can also exhibit liquid state at room temperature. There is an urgent need to enhance solubility, and bioavailability (e.g., oral and local bioavailability) of known pharmaceutical agents for purposes of improving the therapeutic effectiveness of the known pharmaceutical agent.
Through providing improved ionic liquid formulations with enhanced bioavailability, the present invention addresses this need.
SUMMARY OF THE INVENTION
An ionic liquid (IL) is a low-melting salt containing organic/inorganic cation and anion, and depending upon the composition, IL can also be liquid at room temperature (see, Example_2_References 25-26). Over the last 15 years, pharmaceutical applications of ILs have been on the rise. Conversion of ionizable active pharmaceutical ingredients (APIs) into ILs (API-ILs) using pharmaceutically acceptable counterions has emerged as a novel approach to modulate physicochemical and biopharmaceutical properties of ionizable drugs with poor solubility and/or permeability eventually leading to greater oral bioavailability (see, Example_2_References 27-33).
Lifelong treatment with a combination of oral antiretroviral drugs is the mainstay of the current strategies for the management of people living with HIV-1 infection. However, the treatment cost associated with newer oral antiretroviral drugs considerably limits their use in low- and middle-income countries (L& MICs). Additionally, many oral antiretroviral drugs have low and variable bioavailability which leads to suboptimal drug utilization. Hence, strategies to improve oral bioavailability and minimize intra- and/or inter-individual variability in the pharmacoki-netics (PK) of antiretroviral drugs are gravely needed to achieve a reduction in therapeutic dose further leading to significant cost savings, especially in L& MICs. The development of clinically viable formulations to improve the oral bioavailability of antiretroviral drugs which will eventually lead to a reduction in therapeutic dose and significant cost savings, especially in L& MICs.
Rilpivirine (RPV) is a potent antiretroviral drug used for the long-term management of HIV infection. The high crystallinity and very low aqueous solubility of RPV are responsible for the highly variable pharmacokinetics of RPV seen in HIV-infected patients. While fatty meals can increase the absorption of RPV, the low lipid solubility of RPV precludes the development of oral lipid-based formulations such as self-nanoemulsifying systems (SNES). To improve the oral delivery of RPV, experiments were conducted that evaluated the potential of six biocompatible bulky anions to transform RPV into amphiphilic RPV ionic liquids with high lipid solubility and only sodium docusate successfully yielded amphiphilic RPV ionic liquid (IL), RPV docusate (RPV-Doc). Spectroscopic, chromatographic, and thermal characterization techniques confirmed the formation of RPV-Doc as an IL. RPV-Doc showed remarkably higher (-100 to 200-fold higher) solubility in lipids compared to pure RPV. RPV-Doc was incorporated into two SNES formulations which, depending upon the composition of the SNES formulation, yielded nanoemulsion of size < 100 nm or < 250 nm irrespective of the pH of the dilution medium. Oral pharmacokinetics and biodistribution studies in mice showed that both SNES formulations containing RPV-Doc yielded rapid and significantly higher oral bioavailability (~ 6-fold higher Cmax and AUC) of RPV compared to RPV suspension. Furthermore, compared to RPV suspension, both SNES formulations containing RPV-Doc resulted in significantly higher and sustained RPV levels in the HIV sanctuary sites such as mesenteric lymph nodes and the brain. Taken together, our innovative approach can be used to improve the oral bioavailability and tissue penetration of RPV which can eventually result in a reduction in the pharmacokinetic variability and therapeutic dose of RPV leading to optimal drug utilization.
Tenofovir prodrugs, tenofovir disoproxil fumarate (TDF), or tenofovir alafenamide fumarate (TAF) are the backbone of oral combination antiretroviral therapy (cART) for the longterm HIV management. Tenofovir prodrugs also play a critical role in the oral pre-exposure prophylaxis of HIV infection. However, factors such as high aqueous solubility, poor permeability, premature degradation in the gastrointestinal tract (in the case of TDF), and susceptibility to P- gly coprotein (P-gp) mediated efflux are responsible for the low oral bioavailability of TDF (25%) and TAF (40%). TDF remains to be the mainstay of cART in L& MICs. It is estimated that the bioavailability enhancing strategy that can reduce the therapeutic dose of TDF from 300 mg to 225 mg would lead to $50-75 million in cost savings per annum. TAF, due to its higher potency, greater metabolic stability, and tolerability, continues to replace TDF in cART in developed countries but its low oral bioavailability warrants further formulation development. Hence, a strategy to improve the oral bioavailability of tenofovir prodrugs is highly needed. Experiments described herein demonstrated that TDF as well as TAF can readily interact with GRAS fatty permeation enhancers such as oleic acid, capric acid, undecylenic acid, and salcaprozic acid to yield ILs.
Herpes simplex virus (HSV) keratitis is a recurrent and lifelong infection that results in visual morbidity, and it is the leading cause of infection-associated corneal blindness worldwide. Depending upon the anatomical location, HSV keratitis can be categorized into epithelial, stromal, and endothelial keratitis. HSV epithelial keratitis involves active HSV infection in the corneal epithelium and antiviral therapy is needed to diminish discomfort, minimize vision loss, and reduce the recurrence rate. Topical eye drops of nucleoside analogs such as ganciclovir and trifluridine are FDA-approved for HSV epithelial keratitis therapy. However, these eye drops need to be administered 5- to 9-times a day to manage the ocular HSV infection which affects patient compliance and thus, treatment efficacy.
Due to the emergence of viral strains resistant to nucleoside analogs, there is a dire need to develop new therapies to treat HSV infections. Acyclic nucleoside phosphonates (ANPs) are broad- spectrum antiviral drugs that are active against a variety of DNA viruses including HSV and retroviruses. The FDA-approved ANPs or their prodrugs such as cidofovir, adefovir (AFV) and tenofovir (TFV), adefovir dipivoxil (ADV), and tenofovir disoproxil fumarate (TDF) are not used to treat HSV infections. Experiments described herein demonstrated that ADV, an off- patent prodrug of AFV currently used only for the treatment of chronic hepatitis B virus (HBV) infections, has potent (EC50 < 0.625 pM) and significantly higher antiviral activity against HSV-1 when compared to ACV and other ANPs and their prodrugs and excellent safety to human corneal epithelial cells even at 100 pM. Furthermore, topical administration of 1% ADV solution (thrice daily) showed robust antiviral activity in a murine model of ocular HSV-1 infection. Experiments demonstrated that ADV-pamoate (PAM) can be successfully synthesized and transformed into ADV-PAM nanosuspension (ADV-PAM-NS; size: - 260 nm) containing Poloxamer 407, a mucoinert stabilizer. Experiments will continue the development and optimization of ADV-PAM-NS to obtain desired ADV loading. Experiments further demonstrated that mono- and polyunsaturated fatty acids can interact with ADV to spontaneously form lipophilic IL and that these ADV-ILs can be efficiently packaged into a SoluPlus (a mucoinert biodegradable polymer) nanomicelles (size < 70-150 nm).
Accordingly, the present invention relates to compositions comprising bioavailability
(e.g., oral bioavailability) enhancing ionic liquid formulations and the use thereof for treating conditions or diseases in a subject.
In certain embodiments, the present invention provides a composition comprising an ionic liquid formulation comprising a cationic component and an anionic component.
In some embodiments, wherein the cationic component is selected from a cationic or ionized therapeutic agent, a cationic or ionized amino acid, a cationic nutraceutical, a cationic agrochemical molecule, a cationic functional food, a cationic excipient, and a pharmaceutically acceptable cation.
In some embodiments, the cationic component is a protonated form or cationic derivative of one of the following:
Figure imgf000005_0001
Figure imgf000005_0002
In some embodiments, the cationic component is a protonated form or cationic derivative of one of the following: D-glucamine, n-methyl-D-glucamine, N-ethyl-D-glucamine, N-octyl-d- glucamine, N-oxodecyl meglumine, D-glucosamine, D-mannosamine, D-galactosamine, L- carnitine, D-carnitine, Acetyl-L-carnitine, Propionyl-L-carnitine, O-hexanoyl-L-carnitine, Phosphatidyl-carnitine, Isovaleryl -L-carnitine, Propionyl-L-carnitine, Palmitoyl-L-carnitine, Sarcosine, Sarcosine methyl ester, Sarcosine ethyl ester, N-lauroyl arginine ethyl ester, N- lauroyl lysine Albendazole, mebendazole, flubendazole, triclabendazole, Alexidine, Chlorhexidine, Picloxydine Metformin, Phenformin, Buformin, Proguanil, Moroxydine, BX- 795, Rilpivirine, etravirine, nucleotide analogues (antiviral agents) (e.g., Tenofovir, Tenofovir disoproxil, Tenofovir alafenamide, Adefovir, Adefovir dipivoxil Acyclovir, Ganciclovir, Penciclovir, Lamivudine, abacavir, Emtricitabine, Zalcitabine, and Cytidine monophosphate), Imiquimod, Resiquimod, Gardiquimod, Pentamidine, Furamidine, nafamostat Pafuramidine, Diminazene, indoximod, rasagiline, ropinirole, venetoclax, navitoclax, obatoclax, moxifloxacin, levodopa, imeglimin, cyloguanil, clofazimine, bedaquiline, dabrafenib, vemurafenib, trametinib, sorafenib, and hexaminolevulinate hydrochloride.
In some embodiments, the cationic component is a cationic molecule (e.g., linear, branched or cyclic) containing a primary, a secondary, a tertiary, or a quaternary ammonium group.
In some embodiments, the cationic component is a cationic carnitine derivative of the following formula:
Figure imgf000006_0001
wherein Rl, R2, and R3 are each independently selected from hydrogen, C1-C6 alkyl group, a C1-C3 alkyl group, a C1-C6 alkyl group, and a methyl group; wherein R is selected from hydrogen, CH3C=O, CH3 (CH2)MC=0, and a -(0=0- C1-C15 alkyl group.
In some embodiments, the cationic component is a protonated form or cationic derivative
(e.g., 1°, 2°, or 3° ammonium salt or imine salt) selected from:
Figure imgf000007_0001
Figure imgf000007_0002
Figure imgf000008_0001
Figure imgf000008_0002
In some embodiments, the cationic component is selected from:
Figure imgf000008_0003
In some embodiments, the cationic component is a phosphonium cation encompassed
Figure imgf000009_0002
R: alkyl group or alkyl alcohol or alkyl acid or heteroalkyl
. , . , _ ,, R| to Ri<: -H or halides or alkvl or -NO within the following: ' - * 2
In some embodiments, the cationic component is selected from mitoquinone, mitoquinone mesylate, and SKQ1.
In some embodiments, the cationic component is selected from.
Figure imgf000009_0003
In some embodiments, the cationic component is a protonated form of a benzimidazole compound (e.g., anthelmintic benzimidazoles such as albendazole, mebendazole, flubendazole and triclabendazole) not including oxfendazole; wherein the benzimidazole compound is
Figure imgf000009_0001
encompassed within the following: ; wherein Rl, R2 are each independently selected from hydrogen, alkyl, aryl, heteroalkyl and heteroaryl groups.
In some embodiments, the anionic component is selected from an anionic therapeutic agent, an anionic amino acid, an anionic nutraceutical, an anionic agrochemical molecule, an anionic functional food, an anionic excipient, and a pharmaceutically acceptable anion.
In some embodiments, the anionic component is an anionic carboxylate, an anionic sulfonate, an anionic sulfate, an anionic phosphate, an anionic phosphonate, an anionic sulfamate, or a chemical moiety having negatively charged functional group.
Figure imgf000010_0001
In some embodiments, the anionic component is K>= OH <” -tior-NHcii2cti2s63,-NiicH2cod
In some embodiments, the anionic component is a bile acid selected from cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, and tauroursodeoxycholic acid. In some embodiments, the anionic component is selected from:
Figure imgf000010_0002
In some embodiments, the anionic component is a negatively charged functional group (e.g. carboxylate) selected from one of the following:
HOOC-R
HOOC-R
R - saturated or mono/di/poly unsaturated aliphatic R = alky or aryl group
Figure imgf000010_0003
R = -CH2- or -NH-; Rj = alkyl and/or aryl group -O-acyl or -O-carbamoyl or -O-carbonyl
Figure imgf000010_0004
R = alkyl and/or aryl group R = alky or aryl group R - alkyl or aryl group an j
Figure imgf000011_0001
R = alky l or aryl/heterocycle
R* = -H or alkyl or aryl/heterocycle
In some embodiments, the anionic component is a molecule with negatively charged functional group selected from: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, undecylenic acid, oleic acid, linoleic acid, linolenic acid, myristoleic acid, ricinoleic acid, elaidic acid, N-decanoyl sarcosine, Lauryl sarcosine, docosahexaenoic acid, biotin, lactobionic acid, eicosapentaenoic acid, nervonic acid, Vitamin E succinate, 4- phenylbutyric acid, pamoic acid, a-lipoic acid, ibuprofen, naproxen, squalene acid, cholesterol hemisuccinate, capric acid, salcaprozic acid, docusic acid, cholic acid, glycocholic acid, taurocholic acid, tauroursodeoxy cholic acid and other anionic bile acids, taurine, camphor sulfonic acid lauryl sulfate, cholesterol sulfate, dioleoyl phosphatidic acid (DOPA), vitamin E phosphate, thiamine phosphate, saccharine sodium, acesulfame potassium, cyclamate sodium, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, gallic acid, vanillic acid, and phthalic acid.
In some embodiments, the anionic component is an anionic carboxylate molecule selected from one of the following:
HOOC-R
Caproic acid: R = C5H^
Caprylic acid: R = C7H15
Lauric acid: R = CnH23
Oleic acid: R = C17H33 HOOC-R
Docosahexaenoic acid: R - C2tH31 a-tocopherol succinate: R - CjH^COO-vit E Nervonic acid: R - C23H45 4-phenylbutyric acid: R = CsHefCgHg)
Salcaprozic acid
Figure imgf000011_0002
In some embodiments, the anionic component is selected from a saturated fatty acid derivative moiety (carboxylate), an unsaturated fatty acid derivative moiety, an aromatic acid derivative moiety, a sulfonate derivative moiety, a sulfate derivative moiety, a phosphate derivative moiety, and a sulfamate derivative moiety. In some embodiments, the anionic component is selected from a negatively charged functional group of saturated fatty acids selected from butyne acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid)lauric acid (dodecanoic acid), palmitic acid (hexadecenoic acid), and cholic acid.
In some embodiments, the anionic component is selected from a negatively charged functional group of unsaturated fatty acids selected from: undecylenic acid, oleic acid, linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, nervonic acid, myristoleic acid, elaidic acid, and ricinoleic acid.
In some embodiments, the anionic component is selected from a negatively charged functional group of aromatic acids selected from: salcaprozic acid, a-tocopherol succinate, 4- phenyl butyric acid, Ibuprofen, Naproxen, pamoic acid, Dolutegravir, Cabotegravir, and Bictegravir.
In some embodiments, the anionic component is selected from a negatively charged functional group of sulfonate anions selected from: docusic acid
Figure imgf000012_0001
In some embodiments, the anionic component is selected from a negatively charged functional group of sulfate anions selected from: lauryl sulfate
Figure imgf000013_0002
Lauryl sulfate: R = C«2H25 > „
( ), and cholesterol sulfate
Figure imgf000013_0003
In some embodiments, the anionic component is selected from a negatively charged functional group of a phosphate anion selected from: a-tocopherol phosphate, 1 ,2-dioleoyl-sn- glycero-3-phosphate (DOPA; diamine
Figure imgf000013_0004
phosphate.
In some embodiments, the anionic component is selected from a negatively charged
Figure imgf000013_0005
functional group of sulfamate anions selected from: acesulfame ( Acesulfame
Figure imgf000013_0001
saccharin, and cyclamate.
In some embodiments, the anionic component is selected from:
Figure imgf000014_0001
In some embodiments, the cationic components and anionic components are present in a ratio in the range of about 5:1 to about 1:5.
In some embodiments, the ionic liquid formulations comprise one of more of:
Figure imgf000015_0001
Alexidine docusate Alexidine oleate Metformin docusate
Figure imgf000015_0002
Figure imgf000015_0003
Figure imgf000015_0004
Meglumine-tocopherol succinate
Figure imgf000015_0005
Meglumine-tocopherol phosphate Meglumine-salcaprozate
Figure imgf000015_0006
Meglumine-oteate Meglumine-oleate (1 :2)
Figure imgf000016_0001
hexaenoate
Figure imgf000016_0002
Meglumine-cholesterol sulfate anj imiquimod oleate
In some embodiments, the ionic liquid formulations comprise one or more nucleotide analogues (antiviral agents) selected from:
Figure imgf000016_0003
ADV Docosahexanoate an j Adefovir dipivoxil Oleate In some embodiments, the ionic liquid formulations comprise:
Figure imgf000017_0001
Mitoquinone caprate Mitoquinone caproate Mitoquinone linoleate
Figure imgf000017_0002
In some embodiments, the ionic liquid formulations comprise a carnitine or carnitine derivative, and an anionic component described herein; wherein the carnitine or carnitine derivative is encompassed within the following formula:
Figure imgf000017_0003
wherein Rl, R2, and R3 are each independently selected from hydrogen, C1-C6 alkyl group, a C1-C3 alkyl group, a C1-C6 alkyl group, and a methyl group; wherein R is selected from hydrogen, CH3C=O, CH3 (CH2)i4C=O, and a -(O=C)- C1-C15 alkyl group.
In some embodiments, the ionic liquid formulations comprise of carnitine and fatty anions:
Figure imgf000018_0001
Carnitine salcaprozate Carnitine oleate Carnitine caprate
Figure imgf000018_0002
Carnitine nervonate Carnitine linoleate Carnitine laurate
Figure imgf000018_0003
Carnitine tocopherol succinate In some embodiments, ionic liquid formulations comprise of carnitine as a cation and bile acids an anion:
Figure imgf000019_0001
In some embodiments, the ionic liquid formulations comprise of carnitine as a cation and
5 dicarboxylic acids as an anion:
Figure imgf000019_0002
Carnitine azelate (2:1) Carnitine azelate (1:1)
Figure imgf000019_0003
Figure imgf000020_0001
In some embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) nanoformulations prepared from polymers, peptides, lipids and/or inorganic materials.
In some embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) polymeric nanoformulations such as but not limited to polylactide-co- glycolide (PLGA) nanoparticles, polymethylmethacrylate nanoparticles.
In some embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) micelles prepared using micelle-forming agents such as but not limited to polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer (SoluPlus), polyethylene glycol-phospholipid conjugate, polyethylene glycol-polylactide, poly-lipoic acid or a PEG-poly-lipoic acid.
In some embodiments, the ionic liquid formulations are associated with a selfemulsifying composition comprising a mixture of surfactant (such as polysorbate and poloxamer), cosurfactant (such as alcohol, glycol ether), lipids (such as triglycerides, mono- or diglycerides, fatty acids, and their esters), and ionic liquid according to any of the preceding claims that yields emulsion or nanoemulsion after dilution with water, buffer, 5% dextrose or other physiological fluids.
In some embodiments, the ionic liquid formulations undergo self-assembly to form nanostructures of size less than 900 nm (or less than 500 nm) over a period of 48 hours or less.
In some embodiments, the self-assembling ionic liquids contain cationic meglumine derivative of Formula II or Ila and one or more anionic molecules (e.g. carboxylates, phosphates, sulfate, sulfonates, sulfamate, etc): (Formula II) and
Figure imgf000020_0002
Figure imgf000021_0004
(Formula Ila); wherein R”, Rl, R2, and R3 are independently a C1-C6 alkyl group, a C1-C3 alkyl group, or a methyl group. In some embodiments, R1=R2=H and R3 = C1-C6 alkyl group, a C1-C3 alkyl group, or a methyl group.
In some embodiments, the self-assembling ionic liquid is one of:
Figure imgf000021_0001
Meglumine-tocopherol succinate Meglumine-tocopherol phosphate
Figure imgf000021_0002
Meglumine-salcaprozate Meglumine-oteate
Figure imgf000021_0003
Meglumine-docosahexaenoate Meglumine-cholesterol sulfate In some embodiments, the self-assembling ionic liquid is one of:
Figure imgf000022_0001
Meglumine-chenodeoxycholate 5 Meglumine-cholate Meglumine-dcoxycholate
Figure imgf000022_0002
Meglumine-glycocholate Meglumine-lithocholate Meglnm in c-ta u rocholate
Figure imgf000022_0003
Figure imgf000022_0004
Meglumine-ursodeoxycholate
In some embodiments, the self-assembling ionic liquid is one of:
Figure imgf000022_0005
Meglumine azelate (2:1) Meglumine azelate (1:1)
Figure imgf000023_0001
In some embodiments, the concentration of the self-assembling IL is between about 0.01 to about 10 mg/ml in water, buffer or other aqueous vehicles.
In some embodiments, any of the described ionic liquids can solubilize drugs, natural products or nutraceuticals with low solubility (less than 1 mg/ml) and/or low permeability (< 1 * 106 cm/s) such as but not limited to cyclosporin A, docetaxel, paclitaxel, cabizataxel, dabrafenib, trametinib, sorafenib, venetoclax, coenzyme Q10, idebenone, triclabendazole, olaparib, urolithin A, myricetin, quercetin, resveratrol, genistein, pterostilbene, gefitinib, dolutegravir, cabotegravir, bictegravir, tenofovir alafenamide, adefovir dipivoxil, ivermectin, fluconazole, ibuprofen, niclosamide, idebenone, niclosamide oleate, niclosamide lipoate, at least at a concentration of 25 mg/ml of ionic liquid.
In certain embodiments, the present invention provides a pharmaceutical composition (e.g. cream, emulsion, self-nanoemulsifying system, transdermal patch, liquid-filled capsule, solid dispersion, hydrogel, oleogel, aerosol, powder, microneedles, foam, film, aqueous solution, etc.) comprising an effective amount of a ionic liquid composition of any one of claims 1-45, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
In some embodiments, the composition further comprises at least one additional therapeutic agent.
In some embodiments, the at least one additional therapeutic agent comprises any type or kind of therapeutic agent capable of inhibiting fungal activity. In some embodiments, the at least one additional therapeutic agent is selected from the following: a polyene, imidazole, triazole, thiazole, allylamine, echinocandin, among others. Examples include Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, econazole, fenticonazole, isoconazole, kentoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, polygodial, tolnaftate, undecylenic acid, and crystal violet.
In some embodiments, the at least one additional therapeutic agent comprises any type or kind of agent capable of inhibiting reverse-transcriptase (RT) activity.
In some embodiments, the at least one additional therapeutic agent comprises any type or kind of anti-HIV agent or an anti-viral agent.
In some embodiments, the at least one additional therapeutic agent comprises any type or kind of anti-HIV agent selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, lenacapavir, and a combination thereof.
In some embodiments, the at least one additional therapeutic agent comprises any type or kind of anti-viral agent.
In some embodiments, the at least one additional therapeutic agent comprises any type or kind of anti-viral agent selected from helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, or ritonavir.
In certain embodiments, the present invention provides a method of killing or inhibiting the growth of a fungus comprising contacting the fungus with a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions described herein.
In some embodiments, the fungus is selected from Aspergillus spp., Fusarium spp., Malassezia spp., Candida spp., or Cryptococcus spp..
In some embodiments, the fungus is selected from Aspergillus fumigatus , Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Fusarium moniliforme, Fusarium proliferatum , Malassezia pachydermatis, Candida albicans, Candida glabrata infection, Candida tropicalis, Candida krusei, Candida auris, Cryptococcus neoformans, Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis, Sarcopodium oculorum, M. circinelloides, Rhizopus delemar, Rhizopus oryzae, and Lichtheimia corymbifera.
In certain embodiments, the present invention provides a use of a composition or pharmaceutical composition described herein, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a fungal infection.
In some embodiments, the fungal infection is related to one or more of: Aspergillus spp., Fusarium spp., Malassezia spp., Candida spp., and Cryptococcus spp..
In some embodiments, the fungal infection is related to one or more of: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Fusarium moniliforme, Fusarium proliferation, Malassezia pachydermatis, Candida albicans, Candida glabrata infection, Candida tropicalis, Candida krusei, Candida auris, Cryptococcus neoformans, Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis, Sarcopodium oculorum, M. circinelloides, Rhizopus delemar, Rhizopus oryzae, and Lichtheimia corymbifera.
In certain embodiments, the present invention provides a method for treating or preventing herpes simplex virus (HSV) infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions recited in Claims 1-54.
In some embodiments, the ionic liquid formulation comprises adefovir dipivoxil (ADV).
In some embodiments, the method further comprises administering to the mammal one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir.
In some embodiments, the ionic liquid formulation is configured for topical administration, transdermal, ocular, systemic administration, and/or oral administration.
In certain embodiments, the present invention provides a method for treating or preventing epithelial keratitis related to HSV infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions described herein, the ionic liquid formulation comprises adefovir dipivoxil (ADV).
In some embodiments, the method further comprises administering to the mammal one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir.
In some embodiments, the ionic liquid formulation is configured for topical administration, optical administration, and/or oral administration.
In certain embodiments, the present invention provides a method for treating or preventing HIV infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions described herein.
In some embodiments, the ionic liquid formulation comprises tenofovir disoproxil, tenofovir alafenamide, and/or adefovir dipivoxil.
In some embodiments, the method further comprises administering to the mammal one or more anti-HIV agents selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, doravirine, lenacapavir, and a combination thereof.
In some embodiments, the ionic liquid formulation is configured for topical administration, optical administration, and/or oral administration.
In certain embodiments, the present invention provides a method for treating cancers such as melanoma, breast cancer, non-small cell lung cancer, hematological cancers, renal cancer, liver cancer and brain cancers in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions described herein.
In certain embodiments, the present invention provides a method for treating or preventing a parasitic infection in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions described herein.
In some embodiments, the mammal is a human being. In some embodiments, the mammal is a human being suffering from or at risk of suffering from a parasitic infection. In some embodiments, the parasitic infection is selected from African trypanosomiasis, amoebiasis, ascariasis, babesiosis, Chagas disease, cryptosporidiosis, cutaneous larva migrans, dirofilariasis, echinococcosis, fasciolosis, filariasis, lymphatic filariasis, giardiasis, helminthiasis, hookworm infection, leishmaniasis, visceral leishmaniasis, malaria, neurocysticercosis, onchocerciasis, protozoan infection, schistosomiasis, taeniasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinosis, and zoonosis. In some embodiments, the method further comprises administering to the mammal an antiparasitic agent. In some embodiments, the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal. In some embodiments, the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole.
In certain embodiments, the present invention provides a method of treating or preventing neurocysticercosis in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions described herein. In some embodiments, the mammal is a human being. In some embodiments, the mammal is a human being suffering from or at risk of suffering from neurocysticercosis.
In some embodiments, the method further comprises administering to the mammal an antiparasitic agent. In some embodiments, the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal. In some embodiments, the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole.
In certain embodiments, the present invention provides a method for treating parasitic infections such as malaria, human filariasis, cysticercosis, and human parasitosis in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions described herein.
In some embodiments, the pharmaceutical composition containing ionic liquid is administered via oral, parenteral, transdermal, skin, bladder, nasal, cornea/general ophthalmic, intraocular, pulmonary, mucosal, transrectal/enemas, or vaginal route of administration.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 : Hydrophilic metformin hydrochloride can he transformed into lip-ophilic ionic liquid (IL) metformin docus-ate with no crystallinity and high solubility in organic solvents and lipids with the FDA-approved fatty anion, sodium docusate.
FIG. 2: A representative schematic of SoluPlus nanomiclles containing encapsulated drug or drug-based IL. The monomer of SoluPlus is included in the circle.
FIG. 3: SoluPlus nanomicelles containing amphiphilic IL, OXF-Doc show greater efficacy compared to pure OXF and OXF-Doc in a murine cryptococcosis model. CD-I mice were infected with Cryptococcus neoformans and then treated orally with various OXF or OXF- Doc formulations at a OXF dose of 25mg/kg. The lung CFUs analyzed at 14 days post-infection. * OXF-Doc in HPMC and OXF in SoluPlus are in suspended form and have low solubility.
FIG. 4: Safety of oral SoluPlus: CD-I outbred mice were treated by oral daily gavage with SoluPlus (975 mg/kg), or water, and weights monitored to determine toxicity. Mean weight lines are plotted. No significant difference between in weight between SoluPlus and water were observed.
FIG. 5: Structures of TDF and TAF ILs that have been successfully synthesized using method used in Example_l_Scheme 2.
FIG. 6: (A) ' H-NMR spectrum of TAF oleate shows chemical shifts in the proton corresponding to the -NH2 group of TAF (labeled as “a”) and disappearance of carboxylate proton of oleic acid (labeled as “1”) indicating interaction between TAF and oleic acid. (B) Transformation of pure solid TAF to IL, TAF oleate and subsequent incorporation of TAF oleate into SoluPlus nanomi-celles (size ~ 130 nm, PDI: < 0.25 and surface charge: ~ -15 mV).
FIG. 7: Outline of experiment to evaluate in vivo antiviral efficacy of SoluPlus nanomicelles containing TDF-IL (SN-TDF-IL).
FIG. 8: A general scheme for the synthesis of RPV-IL/salt/cocrystal using pharmaceutically acceptable biocompatible anions with different chemical structures. Additional details about the synthesis are included in methods section.
FIG. 9: FT-IR characterization of RPV, sodium docusate, sodium lauryl sulfate (SLS), DHA, oleic acid, linoleic acid, geranic acid, and their corresponding IL/salt/cocrystal. The FT-IR spectra of RPV IL/salt/cocrystal show significant shifts in -N-H- stretching of RPV (3315 cm 1), and in -C=O/-O-H/-S=O stretching of counterions suggesting strong interaction between RPV and the biocompatible anions.
FIG. 10: The ’ H NMR spectra confirmed the interaction between RPV and biocompatible anions. The interaction of RPV with each anion resulted in a noticeable shift in the -CH proton peak (proton a) of the pyrimidine ring with RPV-Doc and RPV-LS showing a greater displacement. The 1 H NMR spectra RPV-ILs/salts/cocrystals with carboxylic acid anion showed disappearance or significant displacement of carboxylic acid (-COOH) proton, indicating strong ionic interactions between RPV and its counterions. However, only RPV-Doc and RPV-LS resulted in a significant displacement in the -NH proton peaks (proton b) of RPV indicating the highest level of interaction.
FIG. 11: (a) Thermogravimetric analysis of RPV, and their ILs/salts. (b) Ts% of RPV and its ILs/salts/cocrystals observed in the TGA analysis (c) Differential Scanning calorimetry (DSC) thermogram of RPV and its ILs/salts/cocrystals. The characteristic peak from RPV was either absent or altered in the case of RPV ILs/salts/cocrystals indicating the interaction between RPV and counterions, (d) Determination of the glass transition temperature (Tg) of RPV-Doc.
FIG. 12: The powder X-ray diffractograms of RPV, and its salts/ILs/cocrystals. The RPV-Doc showed the absence of characteristic RPV peaks indicating the formation of amorphous IL whereas other ILs/salts/cocrystals showed the absence, shifting, or suppression of characteristic RPV peaks indicating the interaction between RPV and counterions.
FIG. 13: RPV-ILs/salts/cocrystals retained the inherent antiretroviral activity of RPV in vitro. TZM-bl cells were infected with HIV- IB AL strain and treated with RPV or RPV- ILs/salts/cocrystals (equivalent to 1 nM RPV) solubilized in DMSO and the viral titer was measured 48 hours after the infection. (Data expressed as mean + S.D.; n = 3).
FIG. 14: Development and optimization of SNES containing RPV-Doc and Labrafac MC60 (RPV-Doc-MC60SNES). (a) Evaluation of pharmaceutically acceptable surfactants for their ability to emulsify RPV-Doc: Labrafac MC60 mixture. The higher % transmittance values indicate greater emulsification capability, (b) Screening of cosurfactants using RPV- Doc:Labrafac MC60/Kolliphor ELP/cosurfactant (2:1:1 w/w) mixtures. The higher % transmittance values indicate greater emulsification capability, (c) The ternary phase diagram of RPV-Doc:Labrafac MC60 (oil phase), Kolliphor ELP (surfactant), and Transcutol HP (cosurfactant) to identify optimal SNES compositions for further development. The red region represents SNES compositions that yielded uniform nanoemulsion of size < 250 nm in water, (d) Details of the SNES compositions selected to evaluate the impact of the pH of the dilution medium on the nanoemulsion size and stability (e) Globule size and polydispersity index (PDI) of seven optimized SNES formulae at different pH conditions. #: indicates sample precipitation.
FIG. 15: A representative TEM image of a) RPV-Doc-Cap90SNES and b) RPV-Doc- MC60SNES (scale bar = 500 nm). FIG. 16: RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES significantly increased the oral bioavailability of RPV when compared to RPV suspension. Briefly, B6 male mice (n> 5 mice/group) were orally administered RPV suspension, RPV-Doc-MC60SNES or RPV-Doc - Cap90SNES (equivalent to 10 mg/kg of RPV). RPV-Doc-MC60SNES as well as RPV-Doc- Cap90SNES showed rapid and significantly higher absorption of RPV. The RPV-Doc- Cap90SNES showed lower clearance of RPV compared to RPV suspension and RPV-Doc- MC60SNES. * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001.
FIG. 17: RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES increased the delivery of RPV to various HIV sanctuary sites such as the MLNs, brain and testis when compared to RPV suspension. Briefly, B6 male mice (n=3 mice/time point/group) were orally administered RPV suspension, RPV-Doc-MC60SNES, or RPV-Doc-Cap90SNES (equivalent to 10 mg/kg of RPV). RPV-Doc-MC60SNES as well as RPV-Doc-Cap90SNES showed significantly higher RPV levels in the plasma, MLNs, and brain at 1 hour. At 6 h, RPV levels were significantly higher only in plasma for both SNES formulations and in the brain for RPV-Doc-Cap90SNES. Both SNES formulations showed higher RPV levels in the testis compared to RPV suspension but the differences were not significant. * P < 0.05, ** P< 0.01, *** P< 0.001.
FIG. 18: H NMR spectrum of RPV docusate (RPV-Doc).
FIG. 19: 13C NMR spectrum of RPV docusate (RPV-Doc).
FIG. 20: H NMR spectrum of RPV-DHA.
FIG. 21: 13C NMR spectrum of RPV-DHA.
FIG. 22: H NMR spectrum of RPV geranate.
FIG. 23: 13C NMR spectrum of RPV-geranate.
FIG. 24: H NMR spectrum of RPV linoleate.
FIG. 25: 13C NMR spectrum of RPV-linoleate.
FIG. 26: H NMR spectrum of RPV-LS.
FIG. 27: 13C NMR spectrum of RPV-LS.
FIG. 28: H NMR spectrum of RPV oleate.
FIG. 29: 13C NMR spectrum of RPV oleate.
FIG. 30: The overlay of HPLC chromatograms of RPV, RPV-Doc, RPV-LS, RPV-DHA, RPV linoleate, RPV geranate, and RPV oleate (concentration: 30 pM; Retention time: 5.25) indicated the high purity.
FIG. 31 : HRMS of RPV-Doc; HRMS m/z for C42H56N6O7S [negative]: 788.38889.
FIG. 32: HRMS of RPV-LS; HRMS m/z for C34H44N6O4S [negative]: 631.30720 FIG. 33: The DSC thermogram demonstrating the Tg and melting endotherm of RPV-LS.
FIG. 34: Representative structure of acyclic nucleoside and acyclic nucleoside.
FIG. 35: Dipivefrin, a pivalic acid ester prodrug of epinephrine is available as eye drops (Propine®). Adefovir dipivoxil (ADV) is a pivalic acid ester prodrug of adefovir. Pivalic acid is marked in red.
FIG. 36: ADV showed significantly higher in vitro antiviral activity compared to adefovir (AFV) and ACV in HSV-2 infected HeLa cells (n=3) and was well tolerated by HeLa cells even at 200 pM.
FIG. 37: Hydrophilic metformin hydrochloride can be transformed into lipophilic ionic liquid (IL) metformin docusate with no crystallinity and high solubility in organic solvents and lipids with the FDA-approved fatty anion, sodium docusate.
FIG. 38: ADV was well tolerated by the human corneal epithelial cells after 24 h even at a concentration of 100 pM (> 85% cell viability) indicating corneal safety.
FIG. 39: ADV showed significantly higher in vitro efficacy against HSV-1 compared to other acyclic nucleoside phosphonates (ANPs), their prodrugs and ACV. (A) HCE were infected with HSV-1 17 GFP at MOI of 1 and the cells were treated with a 5 pM concentration of various ANPs, prodrugs and ACV and the intracellular infectious viral titer was eventually determined using further assays. (B) HCE were infected with P-galactosidase expressing recombinant HSV- 1 (KOS)tkl2 at MOI of 0.1 MOI and treated with various concentrations of ANPs, prodrugs, and ACV.
FIG. 40: Topical (ocular) administration of 1% w/v ADV solution showed robust antiviral effect in a mouse model of ocular HSV-1 infection. 1% ADV solution (10 pL) was topically administered 3 times daily to C57BL/6 mice 1 day post ocular HSV-1 infection and ocular swabs were taken on days 2 and 4 (n -3 ) and vital titer was determined.
FIG. 41: (A) Schematic representation of the method to prepare lipophilic salt, MOX- PAM. MOX-PAM was obtained as a precipitate which was further washed with water and freeze dried for the preparation of NS. (B) Schematic of the process of using zirconium beads and laboratory scale wet milling method to transform lipophilic salt (MOX-PAM) into mucuspenetrating nanosuspension (MOX-PAM NS) NS. (C) The physicochemical properties of the MOX-PAM NS showing appropriate size, homogeneity, and surface charge. Data adapted from our recent paper with suitable modifications.37
FIG. 42: Once a day topical treatment with moxifloxacin-pamoate nanosuspension (MOX-PAM NS) was more effective in treating ocular S. aureus infection. To assess therapeutic efficacy, rats were infected topically with .S', aureus 24 h prior to initiating treatment. Rats received either no treatment (Infection control), once a day Vigamox (0.5% w/v), three times a day Vigamox, or once a day MOX-PAM NS (equivalent to 0.5% w/v MOX). (A) On the third day of treatment, corneal swabs were taken with a cotton-tipped applicator for the determination of bacterial load (n > 7 per group). (C) Twenty-four hours after the end of treatment (day 4), the eyes were enucleated and were prepared for the determination of bacterial burden (n > 7 per group). Once a day MOX-PAM NS had similar treatment efficacy as three times a day Vigamox. Data expressed as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001. FIG. 42 is reproduced from our recently published paper.37
FIG. 43: A representative schematic of SoluPlus nanomiclles containing encapsulated drug or drug-based IL. The monomer of SoluPlus is included in the circle.
FIG. 44: SoluPlus nanomicelles containing lipophilic IL, OXF-Doc show greater efficacy compared to pure OXF and OXF-Doc in a murine cryptococcosis model. CD-I mice were infected with Cryptococcus neoformans and then treated orally with various OXF or OXF-Doc formulations at a OXF dose of 25mg/kg. The lung CFUs analyzed at 14 days post-infection. *OXF-Doc in HPMC and OXF in SoluPlus are in suspended form and have low solubility.
FIG. 45: (A) Schematic of the method to prepare ADV lipophilic salt, ADV-PAM.
(B) 'H-NMR spectrum of ADV pamoate show chemical shifts in the proton corresponding to the -NH2 group of ADV (labeled as “a”) and aromatic protons of pamoic acid (labeled as “1”) indicating interaction between ADV and pamoic acid. (C) Schematic of the lab-scale wet milling method using zirconium beads to transform ADV-PAM into mucus-penetrating nanosuspension (ADV-PAM NS). (D) The physicochemical properties of a prototype ADV- PAM NS.
FIG. 46: (A) Structure of ADV ILs (ADV linoleate and ADV docosahexaenoate. (B) ’H-NMR spectrum of ADV linoleate shows chemical shifts in the proton corresponding to the -NH2 group of ADV (labeled as “a”) and disappearance of carboxylate proton of linoleic acid (labeled as “1”) indicating interaction between ADV and linoleic acid. (C) Transformation of pure solid ADV to IL, ADV linoleate and subsequent incorporation of ADV linoleate into SoluPlus nanomicelles (size - 77 nm, PDI: < 0.25 and surface charge: - -15 mV).
FIG. 47: TEM images of Meglumine-tocopherol (Meg-VES) self- assembled IL prepared in water.
FIG. 48: Various hydrophobic drugs could be solubilized in carnitine linoleate. FIG. 49: SNES of carnitine salcaprozate containing solubilized sorafenib showed significant improvement in oral PK parameters (Cmax and AUC) compared to sorafenib tosylate suspension and sorafenib base indicating potential for oral drug delivery.
FIG. 50: Venetoclax (Ven) plasma concentration after oral administration of Ven suspension, Ven-DOPA SNES and SNES containing carinitine linoleate and venetoclax. (Carlino IL-Ven SNES) to CD-I mice (data expressed as mean ± S.D.; n = 4). A significant improvement in oral bioavailability is observed.
DETAILED DESCRIPTION OF THE INVENTION
This invention is in the field of medicinal chemistry and drug delivery. Disclosed herein are compositions comprising bioavailability (e.g., oral and local bioavailability) enhancing ionic liquid formulations and the use thereof for treating conditions or diseases in a subject.
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
1. Definitions
Certain terms employed in the specification, examples, and appended claims are further described here in the present invention. These definitions should be read in light of the entire invention and as would be understood by a person skilled in art.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.
As used herein, the terms “a” or “an” means “at least one” or “one or more” unless the context clearly indicates otherwise.
As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by +10% and remain within the scope of the disclosed embodiments.
As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a compound or composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the symptoms. As such, “treating” means an application or administration of the compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., humans and nonhumans) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.
As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the compounds or compositions of the disclosure into a subject by a method or route which results in at least partial localization of the compounds or composition to a desired site. The compounds or compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.
Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2nd edition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7th Edition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.
“Pharmacological composition” refers to a mixture of one or more of the compounds described herein or pharmaceutically acceptable salts thereof, with other chemical components, such as pharmaceutically acceptable carriers and/or excipients. The purpose of a pharmacological composition is to facilitate administration of a compound to an organism.
“Pharmaceutically acceptable salts” is a cationic salt formed at any acidic (e.g., carboxylic acid) group, or an anionic salt formed at any basic (e.g., amino) group.
“Solvate” is a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association often includes hydrogen bonding. In certain instances, the solvate is capable of isolation, for example, when one or more solvate molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include hydrates, ehanolates, and methanolates.
“Prodrug” refers to a pharmacologically inactive derivative of a parent “drug” molecule which requires biotransformation within the target physiological system to release, or to convert the prodrug into the active drug. Prodrugs can address the problems associated with solubility, stability, cell permeability or bioavailability. Prodrugs usually comprise an active drug molecule and a chemical masking group. Prodrugs can be readily prepared from the parent compounds with well-known methods. The term “ionic liquids” as used herein refers to organic sails or mixtures of organic sails which exist in a liquid state. Ionic liquids have been shown to be useful in a variety of fields, including in industrial processing, catalysis, pharmaceuticals, and electrochemistry. The ionic liquids contain at least one anionic and at least one cationic component. Ionic liquids can comprise an additional hydrogen bond donor (i.e. any molecule that can provide an — OH or an — NH group); examples include but are not limited to alcohols, fatty acids, and amines. The anionic and the cationic component may be present in any molar ratio.
2. Ionic Liquid Formulations
In certain aspects, the present disclosure provides ionic liquid formulations comprising a cationic component and an anionic component.
Such embodiments are not limited to a particular cationic component. In some embodiments, the cationic component is selected from a cationic or ionized therapeutic agent, a cationic or ionized amino acid, a cationic nutraceutical, a cationic agrochemical molecule, a cationic functional food, a cationic excipient, and a pharmaceutically acceptable cation.
In some embodiments, the cationic component is a cationic derivative of one of the
Figure imgf000036_0001
In some embodiments, the cationic component is a cationic derivative of one of the following: D-glucamine, n-methyl-D-glucamine, N-ethyl-D-glucamine, N-octyl-d-glucamine, N-oxodecyl meglumine, D-glucosamine, D-mannosamine, D-galactosamine, L-camitine, D- carnitine, Acetyl-L-carnitine, Propionyl-L-camitine, O-hexanoyl-L-camitine, Phosphatidyl- carnitine, Tsovaleryl-L-carnitine, Propionyl-L-carnitine, Palmitoyl-L-carnitine, Sarcosine, Sarcosine methyl ester, Sarcosine ethyl ester, N-lauroyl arginine ethyl ester, N-lauroyl lysine Albendazole, mebendazole, flubendazole, triclabendazole, Alexidine, Chlorhexidine, Picloxydine Metformin, Phenformin, Buformin, Proguanil, Moroxydine, BX-795, Rilpivirine, etravirine nucleotide analogues (antiviral agents) e.g., Tenofovir, Tenofovir disoproxil, Tenofovir alafenamide, Adefovir, Adefovir dipivoxil Acyclovir, Ganciclovir, Penciclovir, Lamivudine, abacavir, Emtricitabine, Zalcitabine, and Cytidine monophosphate, Imiquimod, Resiquimod, Gardiquimod, Pentamidine, Furamidine, nafamostat Pafuramidine, Diminazene, indoximod, rasagiline, ropinirole, venetoclax, moxifloxacin, imeglimin, cyloguanil, clofazimine, bedaquiline, dabrafenib, vemurafenib, trametinib, sorafenib, and hexaminolevulinate hydrochloride.
In some embodiments, the cationic component is a cationic molecule (e.g., linear, branched or cyclic) containing a primary, a secondary, a tertiary, or a quaternary ammonium group.
In some embodiments, the cationic component is a cationic carnitine derivative of the following formula:
Figure imgf000037_0001
; wherein Rl, R2, and R3 are each independently selected from hydrogen, C1-C6 alkyl group, a C1-C3 alkyl group, a C1-C6 alkyl group, and a methyl group; wherein R is selected from hydrogen, CH3C=0, CH3 (CH2)i4C=O, and a -(O=C)-C1-C15 alkyl group.
In some embodiments, the cationic component is a cationic derivative (e.g., 1°, 2°, or 3°
N-methyl-D-g I ucam i ne
Figure imgf000037_0002
R — — “ LH3»f O jy<j • —* «vri3 ammonium salt or imine salt) selected from: L-Carnitine Alexidine
Figure imgf000038_0001
Nucleotide analogue
Figure imgf000038_0002
Hudoside analogue Lamivudine
Acyclovir:
Figure imgf000039_0001
-2-yl)methanol
Ganciclovir: R = -NH2; R^ = -CH2OCH(CH2OH)CH2OH R2 = -H
Pentamidine imiqutmod
Figure imgf000039_0002
In some embodiments, the cationic component is selected from:
Figure imgf000039_0003
In some embodiments, the cationic component
Figure imgf000040_0002
some embodiments, the cationic component is selected from idebenone, mitoquinone, and mitoquinone mesylate. In some embodiments, the cationic component is
Figure imgf000040_0003
In some embodiments, the cationic component is a protonated form of a benzimidazole compound (e.g., anthelmintic benzimidazoles such as albendazole, mebendazole, flubendazole and triclabendazole) not including oxfendazole encompassed within the following:
Figure imgf000040_0001
wherein Rl and R2 are each independently selected from hydrogen, alkyl, aryl, heteroalkyl and heteroaryl groups.
Such embodiments are not limited to a particular anionic component. In some embodiments, the anionic component is selected from an anionic therapeutic agent, an anionic amino acid, an anionic nutraceutical, an anionic agrochemical molecule, an anionic functional food, an anionic excipient, and a pharmaceutically acceptable anion.
In some embodiments, the anionic component is an anionic carboxylate, an anionic sulfonate, an anionic sulfate, an anionic phosphate, an anionic phosphonate, an anionic sulfamate, or a chemical moiety having negatively charged functional group.
In some embodiments, the anionic component
Figure imgf000040_0004
i
In some embodiments, the anionic component is a bile acid selected from cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, and tauroursodeoxycholic acid. In some embodiments, the anionic component is selected from:
Figure imgf000041_0001
In some embodiments, the anionic component is an anionic carboxylate molecule selected from one of the following:
HOOC-R
HOOC-R
R - saturated or mono/di/poly unsaturated aliphatic R = alky or aryl group
Figure imgf000041_0002
R = -CH- or -O- or -NH-; R' = -OH or -O-alkyl or
R = -CH2- or -NH-; Rj = alkyl and/or aryl group -O-acyl or -O-carbamoyl or -O-carbonyl
Figure imgf000041_0003
R = alkyl and/or aryl group R = alky or aryl group R = alkyl or aryl group
Sulfamate
Figure imgf000041_0004
In some embodiments, the anionic component is a negatively charged functional group selected from: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, undecylenic acid, oleic acid, linoleic acid, linolenic acid, myristoleic acid, ricinoleic acid, elaidic acid, N-decanoyl sarcosine, Lauryl sarcosine, docosahexaenoic acid, biotin, lactobionic acid, eicosapentaenoic acid, nervonic acid, Vitamin E succinate, 4-phenylbutyric acid, pamoic acid, a- lipoic acid, ibuprofen, naproxen, squalene acid, cholesterol hemisuccinate, capric acid, salcaprozic acid, docusic acid, cholic acid, glycocholic acid, taurocholic acid, tauroursodeoxycholic acid and other anionic bile acids, taurine, camphor sulfonic acid lauryl sulfate, cholesterol sulfate, DOPA, vitamin E phosphate, thiamine phosphate, saccharine sodium, acesulfame potassium, cyclamate sodium, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, gallic acid, vanillic acid, and phthalic acid.
In some embodiments, the anionic component is an anionic carboxylate molecule
HOOC-R
Caproic acid: R - CgHn Caprylic acid: R = C7H15 Lauric acid: R ~ C^Hgs Oleic acid: R ~
Docosahexaenoic acid: R = C21H3I
, . , p,, e „ Nervonic acid: R = C23H45 selected from one of the following: ,
Figure imgf000042_0002
Figure imgf000042_0001
In some embodiments, the anionic component is selected from a saturated fatty acid derivative moiety (carboxylate), an unsaturated fatty acid derivative moiety, an aromatic acid derivative moiety, a sulfonate derivative moiety, a sulfate derivative moiety, a phosphate derivative moiety, and a sulfamate derivative moiety.
In some embodiments, the anionic component is selected from a negatively charged functional group of saturated fatty acids selected from butyric acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid)lauric acid (dodecanoic acid), palmitic acid (hexadecenoic acid), and cholic acid.
In some embodiments, the anionic component is selected from a negatively charged functional group of unsaturated fatty acids selected from: undecylenic acid, oleic acid, linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, nervonic acid, myristoleic acid, elaidic acid, and ricinoleic acid.
In some embodiments, the anionic component is selected from a negatively charged functional group of aromatic acids selected from: salcaprozic acid, a-tocopherol succinate, 4- phenyl butyric acid, Ibuprofen, Naproxen, pamoic acid, Dolutegravir, Cabotegravir, and Bictegravir.
In some embodiments, the anionic component is selected from a negatively charged functional group of sulfonate anions selected from: docusic acid, camphor sulfonic acid, taurocholic acid, tauroursodeoxycholic acid, and taurine.
In some embodiments, the anionic component is selected from a negatively charged functional group of sulfate anions selected from: lauryl sulfate, and cholesterol sulfate.
In some embodiments, the anionic component is selected from a negatively charged functional group of a phosphate anion selected from: a-tocopherol phosphate, 1 ,2-dioleoyl-sn- glycero- 3 -phosphate (DOPA), and thiamine phosphate.
In some embodiments, the anionic component is selected from a negatively charged functional group of sulfamate anions selected from: acesulfame, saccharin, and cyclamate.
In some embodiments, the anionic component is selected from:
Figure imgf000044_0001
In some embodiments, the anionic component is selected from a fatty anion, a docusate anion
Figure imgf000044_0002
Figure imgf000045_0001
Figure imgf000045_0003
Figure imgf000045_0002
In some embodiments, the cationic components and anionic components are present in a ratio in the range of about 5:1 to about 1:5.
In some embodiments, the ionic liquid formulations comprise one of more of:
Figure imgf000046_0001
Figure imgf000046_0002
Figure imgf000046_0003
Meglumine-tocopherol succinate
Figure imgf000046_0004
Meglumine-tocopherol phosphate Meglumine-salcaprozate
Figure imgf000047_0001
Meglumine-oleate Meglumine-oleate (1:2)
Figure imgf000047_0002
hexaenoate
Figure imgf000047_0003
Meglumine-cholesterol sulfate , and Imiquimod oleate
In some embodiments, the ionic formulations comprise one or more nucleotide analogues (antiviral agents) selected from:
Figure imgf000048_0001
ADV Docosahexanoate ;in(| Adefovir dipivoxil Oleate
In certain embodiments, the ionic liquid formulations are self- assembling ionic liquid formulations. In certain embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) nanoformulations. In certain embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) polymeric nanoformulations. In certain embodiments, the ionic liquid formulations are associated with (e.g., encapsulated within) nanomicelles (e.g., SoluPlus nanomicelles).
In some embodiments, the self-assembling liquid ionic formulations comprise meglumine & bile acid-based ionic liquids (which have ability to self-assemble).
In some embodiments, the self-assembling liquid ionic formulation comprises one or
Figure imgf000048_0002
more of- Meglumine-chenodeoxycliolate Meglumine-cholate
Figure imgf000049_0001
Megluimne-deoxyc.holate Meglumine-glyeocholate Megluinine-lithocliolate
Figure imgf000049_0002
In some embodiments, the liquid ionic formulations comprise carnitine & bile acid based ionic liquids (which serves as a lipophilic solvent for self-nanoemulsifying system). In some embodiments, the liquid ionic formulation comprising carnitine & bile acid based ionic liquid are selected from:
Figure imgf000049_0003
Figure imgf000050_0001
In some embodiments, the liquid ionic formulations comprise mitoquinone-based ionic liquids, which serves as a lipophilic solvent for self- nanoemulsifying system. In some embodiments, the mitoquinone-based ionic liquids are selected from:
Figure imgf000050_0002
Mitoquinone undecylenate anj Mitoquinone niclosamide
In some embodiments, the liquid ionic formulations comprise carnitine & dicarboxylic acid based ionic liquids, which serves as a lipophilic solvent for self-nanoemulsifying system. In some embodiments, the carnitine & dicarboxylic acid based ionic liquids are selected from:
Figure imgf000051_0001
Carnitine azelate (2:1) Carnitine azelate (1:1)
Figure imgf000051_0002
In some embodiments, the ionic formulations comprise carnitine & monocarboxylic acid/phosphonic acid based ionic liquids, which serve as a lipophilic solvent for self- nanoemulsifying system, wherein the carnitine & dicarboxylic acid based ionic liquids are
Figure imgf000051_0003
selected front: Carnitine salcaprozate > Carnitine oleate
Figure imgf000051_0004
Carnitine nervonate Carnitine linoleate Carnitine laurate
Figure imgf000052_0001
Carnitine docoshexaenoate Carnitine caprate
Figure imgf000052_0002
In some embodiments, said self-assembling ionic liquids are meglumine & dicarboxylic acid-based ionic liquids, which have the ability to self-assemble, examples of which are:
Figure imgf000052_0003
Meglumine azelate (2:1) Meglumine azelate (1:1)
Figure imgf000052_0004
Meglumine suberate (2:1) Meglumine suberate (1:1)
The compounds within the ionic liquid formulations may exist as a stereoisomer wherein asymmetric or chiral centers are present. The stereoisomer is “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The disclosure contemplates various stereoisomers and mixtures thereof and these are specifically included within the scope of this disclosure. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England (or more recent versions thereof), or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.
It should be understood that the compounds may possess tautomeric forms, as well as geometric isomers, and that these also constitute embodiments of the disclosure.
In some embodiments, the ionic liquid formulations can be combined with another solvent to enhance solubility and/or delivery. The solvent may be aqueous or non-aqueous. In some embodiments, the purpose of the solvent is to control the dose of the ionic liquid. Dilution of the ionic liquid by the solvent can serve the purpose of delivering a safe dose to the subject. In some embodiments, the purpose of the solvent is to improve solubility of the one or more drugs. Such improvements may come from the ability of the solvent to control the physicochemical environment of the ionic liquid to match the chemical properties of the one or more drugs.
The solvents used may include without limitation: sterile water, saline solution, glycerin, propylene glycol, ethanol, oils, ethyl oleate, isopropyl myristate, benzyl benzoate, or surfactants.
In some embodiments, the solvent is chosen so as to not adversely impact the compatibility of the ionic liquid formulation.
In some embodiments, a composition as described herein, e.g., a composition comprising ionic liquid formulations and one or more drugs, can further comprise a pharmaceutically acceptable excipient. Suitable excipients include, for example, water, saline, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the composition can contain minor amounts of additional excipients such as emulsifying agents, surfactants, pH buffering agents, and the like, which enhance the effectiveness of the ionic liquid formulation.
In some embodiments, the ionic liquid formulation may be further encapsulated in a dosage form designed to facilitate delivery to an organism. Non-limiting examples of such dosage forms include capsules, tablets, and syrups.
In some embodiments, the ionic liquid formulation may require excipients sugars (such as lactose), starches (such as com starch), cellulose, cellulose derivatives (such as sodium carboxymethyl cellulose), gelatin, and other compatible substances.
In some embodiments, the ionic liquid formulation described herein further comprises one or more additional agents. In some embodiments, the one or more additional agents are selected from a nucleic acid, a small molecule, and a polypeptide. In some embodiments, the one or more additional agents comprise a nucleic acid. In some embodiments, the one or more additional agents comprise a small molecule. In some embodiments, the one or more additional agents comprise a polypeptide. In some embodiments the polypeptide comprises an antibody. In some embodiments, the antibody comprises any one selected from Fragment Antigen-binding (Fab, F(ab')2), single chain variable fragment (scFv), and nanobodies.
The present disclosure also includes isotopically-labeled compounds, which is identical to those recited in Formula I but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes include those for hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to 2H, 3H, 13C, 14C, 1SN, 18O, 170, 31P, 32P , 35S, 18F, and 36C1, respectively. Substitution with heavier isotopes such as deuterium, for example, 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. The compound may incorporate positronemitting isotopes for medical imaging and positron-emitting tomography (PET) studies. Suitable positron-emitting isotopes that can be incorporated in compounds of formula (I) are nC, 13N, 15O, and 18F. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent.
In certain embodiments, the ionic liquid formulations described herein are associated (e.g., encapsulated) with biodegradable polymers (e.g., for purposes of enhancing bioavailability). In some embodiments, the biodegradable polymer is a lipid nanoemulsion. In some embodiments, the biodegradable polymer is nanomicelle. In some embodiments, the biodegradable polymer is SoluPlus nanomicelles.
Such ionic liquid formulations as described herein may be synthesized according to a variety of methods. Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Reactions can be worked up in the conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above described scheme.
Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the disclosure. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4th ed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the ionic liquid formulations of the disclosure can be accomplished by methods analogous to those described in the synthetic schemes described hereinabove and in specific examples.
When an optically active form of a disclosed compound within the ionic liquid formulations is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization, or enzymatic resolution). Similarly, when a pure geometric isomer of a compound within the ionic liquid formulations is required, it can be obtained by carrying out one of the above procedures using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation.
3. Compositions
The disclosed ionic liquid formulations may be incorporated into compositions that may be suitable for administration to a subject (such as a patient, which may be a human or nonhuman).
3a. Pharmaceutical Compositions
The disclosed ionic liquid formulations may be incorporated into pharmaceutically acceptable compositions. The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the ionic liquid formulations. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the ionic liquid formulations are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
The pharmaceutical compositions and formulations may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, surfactant, cyclodextrins or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, com starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, hut not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; surfactants such as, but not limited to, cremophor EL, cremophor RH 60, Solutol HS 15 and polysorbate 80; cyclodextrins such as, but not limited to, alpha-CD, beta-CD, gamma-CD, HP-beta-CD, SBE-beta-CD; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
The route by which the disclosed ionic liquid formulations are administered and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral injections) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis). In some embodiments, the composition is for oral administration. In some embodiments, the composition is for subcutaneous administration. In some embodiments, the composition is for intravenous administration.
Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, cyclodextrins combinations thereof, and others. All carriers are optional in the compositions.
Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.
Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%. Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.
Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%.
Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.
Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.
Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%.
Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%.
Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.
Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.
Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, dimethylacetamide and phosphate (or other suitable buffer). The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.
Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, Pa.) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.
Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Del. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1 , Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.
Suitable cyclodextrins include alpha-CD, beta-CD, gamma-CD, hydroxypropyl betadex (HP-beta-CD), sulfobutyl-ether 0-cyclodextrin (SBE-beta-CD). The amount of cyclodextrins in the systemic or topical composition is typically about 0% to about 40%.
Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of an active compound (e.g., an ionic liquid formulation as described herein) and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.
Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.
Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.
Capsules (including implants, time release and sustained release formulations) typically include an active ionic liquid formulation, and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise a disclosed ionic liquid formulation, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the non-biodegradable type.
The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention. Solid compositions may be coated by conventional methods, typically with pH or timedependent coatings, such that a disclosed ionic liquid formulation is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.
Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non- effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include a disclosed ionic liquid formulation and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.
Other compositions useful for attaining systemic delivery of the ionic liquid formulations to the subject include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol, and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.
The disclosed ionic liquid formulations can be topically administered. Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed ionic liquid formulation, and a carrier. The carrier of the topical composition preferably aids penetration of the ionic liquid formulation into the skin. The carrier may further include one or more optional components.
The amount of the carrier employed in conjunction with a disclosed ionic liquid formulation is sufficient to provide a practical quantity of composition for administration per unit dose of the ionic liquid formulation. Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).
A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.
The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.
Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane- 1,2-diol, butane- 1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95%.
Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in a topical composition is typically about 0% to about 95%.
Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95%. Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) in a topical composition is typically 0% to 95%.
The amount of thickener(s) in a topical composition is typically about 0% to about 95%.
Suitable powders include beta-cyclodex trins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium poly acrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically -modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95%.
The amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1%.
Suitable pH adjusting additives include HC1 or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition.
3a. Additional Therapeutic Agents
Any of the above compositions or formulations disclosed herein may further comprise at least one additional therapeutic agent.
In some embodiments the at least one additional therapeutic agent is one or more other anti-fungal agents. In some embodiments, the composition comprises any one or more of the ionic liquid formulations, or a pharmaceutically acceptable salt thereof, and one or more other anti-fungal agent(s).
In some embodiments, the other anti-fungal agent is an azole or an echinocandin. In some embodiments, the other anti-fungal agent is an azole. In some embodiments, the azole is itraconazole, posaconazole, voriconazole (VOR), or isavuconazole. In some embodiments, the azole is itraconazole. In some embodiments, the azole is posaconazole. In some embodiments, the azole is voriconazole. In some embodiments, the azole is isavuconazole. In some embodiments, the other anti-fungal agent is an echinocandin. In some embodiments, the echinocandin is caspofungin (CAS). In some embodiments, the other anti-fungal agent is nystatin, miconazole, Gentian violet, or amphotericin B. In some embodiments, the other antifungal agent is nystatin. In some embodiments, the other anti-fungal agent is miconazole. In some embodiments, the other anti-fungal agent is Gentian violet. In some embodiments, the other anti-fungal agent is amphotericin B. Additional anti-fungal agents include, but are not limited to, fosmanogepix, ibrexafungerp, olorofim, opelconazole, and rezafungin. In some embodiments, the other anti-fungal agent is fosmanogepix. In some embodiments, the other antifungal agent is ibrexafungerp. In some embodiments, the other anti-fungal agent is olorofim. In some embodiments, the other anti-fungal agent is opelconazole. In some embodiments, the other anti-fungal agent is rezafungin. In some embodiments, the other anti-fungal agent is Nikkomycin Z. Other anti-fungal agents include VT-1129, VT-1161, VT-1598, PC1244, SUBA- ITC, CAMB, MGCD290, T-2307, and VL-2397. Additional anti-fungal agents are disclosed in, for example, PCT Publication No. WO 2021/247781.
In embodiments, the antifungal agent is a known antifungal and not an ionic liquid formulation described herein, such as a polyene, imidazole, triazole, thiazole, allylamine, echinocandin, among others. Examples include Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, econazole, fenticonazole, isoconazole, kentoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, polygodial, tolnaftate, undecylenic acid, and crystal violet, among others.
In some embodiments, the at least one additional therapeutic agent is capable of inhibiting reverse-transcriptase (RT) activity. An agent is capable of inhibiting RT can be useful for the treatment of HIV infection in humans by inhibiting HIV replication in infected cells or individuals. Examples of the compounds approved for use in treating HIV infection and AIDS include nucleoside RT inhibitors (NRTI) such as 3 ’-azido-3 ‘-deoxythymidine ( AZT, also known as Zidovudine (ZDV), azidothymidine (AZT)), 2',3'-dideoxyinosine (ddl), 2',3!- dideoxycytidine (ddC), d4T, 3TC, abacavir, emtricitabine, and tenofovir disoproxil fumarate, as well as nonnucleoside RT inhibitors (NNRTI) such as nevirapine, delavirdine, efavirenz, rilpivirine and doravirine (DHHS guidelines: https://aidsinfo.nib.gov/understanding- hiv-aids, lyidogan &. Anderson, Viruses, 6, 4095-4139, 2014, doi: 10.3390/v6104095: Hayakawa et al., Antiviral Client & Chemotherapy, 15: 169-187, 2004; Ohrul et al., J. Med. Client. 43, 4516- 4525, 2000; Pauwels, Antiviral Research, 71, 77-89, 2006).
In some embodiments, the at least one additional therapeutic agent is an anti-HIV agent or an anti-viral agent. The term "anti-HIV agent", “anti-viral agent” or a grammatical variant refers to a compound, a mixture of one or more compounds, a formulation, a chemical agent or a biological agent such as antibody, protein, peptides, nucleotide, other biological compound, or a combination thereof, that can be directly or indirectly effective in the inhibition of HIV, the treatment or prophylaxis of HIV infection, and/or the treatment, prophylaxis or delay in the onset or progression of AIDS and/or diseases or conditions arising therefrom or associated therewith, an RNA virus infection, or a combination thereof. The anti-HIV agents can comprise HIV antiviral agents, immunomodulators, anti-infectives, vaccines or a combination thereof useful for treating HIV infection or AIDS. Examples of antiviral agents for Treating HIV infection or AIDS include, but are not limited to, under respective trademarks or registered trademarks with respective owners, atazanavir (Reyataz®), darunavir (Prezista®), dolutegravir (Tivicay®), doravirine, efavirenz (EFV, Sustiva®, Stocrin®), cabotegravir, bictegravir, emtricitabine (FTC, Emtriva®), rilpivirine (Edurant®), tenofovir hexadecyl oxy propyl (CMX- 157), tenofovir alafenamide fumarate, MK-8507, and lenacapavir. Some of the anti-HIV agents shown above can be used in a salt form; for example, atazanavir sulfate, tenofovir alafenamide fumarate or other salts. An anti- HIV agent can have one or more activities such as entry inhibitor (El); a capsid inhibitor (CAI), fusion inhibitor (FI); integrase inhibitor (Ini); protease inhibitor (PI); nucleoside reverse transcriptase inhibitor (nRTI or NRTI) or non-nucleoside reverse transcriptase inhibitor (nnRTI or NNRTI).
In some embodiments, the anti-HIV agent is selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, lenacapavir, and a combination thereof.
In some embodiments, the anti-viral agent is useful in treating or preventing herpes simplex virus (HSV) infection. In some embodiments, the ant- viral agent is selected from helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, or ritonavir.
In some embodiments, the at least one additional therapeutic agent comprises a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide,” in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi- inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
In some embodiments, the at least one additional therapeutic agent is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense RNAs, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.
In certain embodiments, the at least one additional therapeutic agent is an mRNA. An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.
In other embodiments, the at least one additional therapeutic agent is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.
In some embodiments, the at least one additional therapeutic agent is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.
4. Methods of Use
The disclosure further provides methods for treating a disease or disorder comprising administration of ionic liquid formulation as disclosed herein, to a subject in need thereof. In some embodiments, the subject is a human.
The present disclosure provides methods of treating or preventing a Cryptococcus fungal infection in a mammal comprising administering to the mammal in need thereof an ionic liquid formulation. The present disclosure also provides methods of killing or inhibiting the growth of a Cryptococcus species comprising contacting the Cryptococcus species with ionic liquid formulations.
The present disclosure provides methods of treating or preventing a fungal infection in a mammal comprising administering to the mammal in need thereof ionic liquid formulations in combination with one or more other anti-fungal agent(s) (i.e., in the same ionic liquid formulation or in separate pharmaceutical compositions).
The present disclosure also provides methods of killing or inhibiting the growth of a fungus comprising contacting the fungus with ionic liquid formulations in combination with one or more other anti-fungal agent(s) (i.e., in the same ionic liquid formulation or in separate pharmaceutical compositions).
In some embodiments, the fungus is, or the fungal infection is caused by, Aspergillus spp. (e.g., Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus), Fusarium spp. (e.g., Fusarium solani, Fusarium moniliforme, and Fusarium proliferatum), Malassezia spp. (e.g., Malassezia pachydermatis), Candida spp. (e.g., Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, and Candida auris), or Cryptococcus spp. (e.g., Cryptococcus neoformans), Mucorales such as Mucor spp. (e.g., M. circinelloides), Rhizopus spp. (e.g., Rhizopus delemar and Rhizopus oryzae), Lichtheimia spp. (e.g., Lichtheimia corymbifera), and Rhizomucor spp., or Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis, or Sarcopodium oculorum. In some embodiments, the fungus is, or the fungal infection is caused by, Aspergillus spp., Fusarium spp., Malassezia spp., Candida spp., or Cryptococcus spp. In some embodiments, the fungus is, or the fungal infection is caused by, Aspergillus spp. In some embodiments, the fungus is, or the fungal infection is caused by, Aspergillus fumigatus. In some embodiments, the fungus is, or the fungal infection is caused by, Aspergillus flavus. In some embodiments, the fungus is, or the fungal infection is caused by, Aspergillus niger. In some embodiments, the fungus is, or the fungal infection is caused by, Aspergillus terreus. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium spp. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium solani. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium moniliforme. In some embodiments, the fungus is, or the fungal infection is caused by, Fusarium proliferatum. In some embodiments, the fungus is, or the fungal infection is caused by, Malassezia spp. In some embodiments, the fungus is, or the fungal infection is caused by, Malassezia pachydermatis. In some embodiments, the fungus is, or the fungal infection is caused by, a Mucorales. In some embodiments, the fungus is, or the fungal infection is caused by, Mucor spp. In some embodiments, the fungus is, or the fungal infection is caused by, M. circinelloid.es . In some embodiments, the fungus is, or the fungal infection is caused by, Rhizopus spp. In some embodiments, the fungus is, or the fungal infection is caused by, Rhizopus delemar. In some embodiments, the fungus is, or the fungal infection is caused by, Rhizopus oryzae. In some embodiments, the fungus is, or the fungal infection is caused by, Lichtheimia spp. In some embodiments, the fungus is, or the fungal infection is caused by, Lichtheimia corymbifera. In some embodiments, the fungus is, or the fungal infection is caused by, Rhizomucor spp. In some embodiments, the fungus is, or the fungal infection is caused by, Candida spp. In some embodiments, the fungus is, or the fungal infection is caused by, Candida albicans. In some embodiments, the fungus is, or the fungal infection is caused by, Candida glabrata. In some embodiments, the fungus is, or the fungal infection is caused by, Candida tropicalis. In some embodiments, the fungus is, or the fungal infection is caused by, Candida krusei. In some embodiments, the fungus is, or the fungal infection is caused by, Candida auris. In some embodiments, the fungus is, or the fungal infection is caused by, Cryptococcus spp. In some embodiments, the fungus is, or the fungal infection is caused by, Cryptococcus neoformans. In some embodiments, the fungus is, or the fungal infection is caused by, Chrysosporium parvum. In some embodiments, the fungus is, or the fungal infection is caused by, Metarhizium anisopliae. In some embodiments, the fungus is, or the fungal infection is caused by, Phaeoisaria clematidis. In some embodiments, the fungus is, or the fungal infection is caused by Sarcopodium oculorum.
Additional pathogenic fungi include the genus Candida (examples include C. albicans, C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. parapsilosis, C. dubliniensis and C. auris), the genus Cryptococcus (examples include C. neoformans and C. gatti), the genus Trichosporon (examples include T. asahii, T. asteroides, T. cutaneum, T. dermatis, T. dohaense, T. inkin, T. loubieri, T. mucoides, and T. oroides), the genus Malassezia (examples include M. globose and M. restricta), the genus Aspergillus (examples include A. fumigatus, A. flaws, A. terreus and A. niger), the genus Fusarium (examples include F. solani, F. falciforme, F. oxysporum, F. verticillioides, and F. proliferation), the genus Mucor (examples include M. circinelloides, M. ramosissimus, M. indicus, M. rasemosus, and M. piriformis), the genus Blastomyces (examples include B. dermatitidis and B. brasiliensis), the genus Coccidioides (examples include C. immitis, and C. posadasii), the genus Pneumocystis (examples include P. carinii and P. jiroveci), the genus Histoplasma (examples include H. capsulatum), the genus Trichophyton (examples include T. schoenleinii, T. mentagrophytes, T. verrucosum, and T. rub rum), the genus Rhizopus (examples include R. oryzae and R. stolonifera), the genus Apophysomyces (examples include A. variabilis), the genus Rhizomucor (examples include R. pusillus, R. regularior, and R. chlamydosporus), the genus Lichtheimia (examples include L. ramose and L. corymbifera), the genus Scedosporium (examples include S. apiospermum), and the genus Lomentospora (examples include L. prolificans).
In some embodiments, the fungi is Mucorales (for which conventional therapy results are poor), and other lethal pathogens for which current therapy is poor or lacking (Fusarium, Scedosporium, Lomentospora, Acremonium, and Exserohilum).
In some embodiments, the fungal species is resistant to a therapeutic agent. In some embodiments, the fungal species is resistant to an azole. In some embodiments, the fungal species is resistant to an echinocandin. In some embodiments, the fungal species is CAS- resistant. In some embodiments, the fungal species is VOR-resistant.
The present disclosure provides methods of treating or preventing human immunodeficiency virus (HIV) infection in a mammal comprising administering to the mammal in need thereof an ionic liquid formulation. In some embodiments, the ionic liquid formulation comprises rilpivirine. In some embodiments, the ionic liquid formulation comprises tenofovir. In some embodiments, the method further comprises administering to the subject one or more anti -HIV agents selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, lenacapavir, and a combination thereof. In some embodiments, the ionic liquid formulation is configured for oral administration.
The present disclosure provides methods of treating or preventing herpes simplex virus (HSV) infection in a mammal comprising administering to the mammal in need thereof an ionic liquid formulation. In some embodiments, the ionic liquid formulation comprises adefovir dipivoxil (ADV). In some embodiments, the method further comprises administering to the subject one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir. In some embodiments, the ionic liquid formulation is configured for topical administration. In some embodiments, the ionic liquid formulation is configured for optical administration. In some embodiments, the ionic liquid formulation is configured for oral administration.
The present disclosure provides methods of treating or preventing epithelial keratitis related to HSV infection in a mammal comprising administering to the mammal in need thereof an ionic liquid formulation. In some embodiments, the ionic liquid formulation comprises adefovir dipivoxil (ADV). In some embodiments, the method further comprises administering to the subject one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir. In some embodiments, the ionic liquid formulation is configured for topical administration. In some embodiments, the ionic liquid formulation is configured for optical administration. In some embodiments, the ionic liquid formulation is configured for oral administration.
In certain embodiments, methods for treating or preventing a parasitic infection in a mammal are provided. In such embodiments, the method comprises administering to the mammal in need thereof a therapeutically effective amount of an ionic liquid formulation. In some embodiments, the mammal is a human being. In some embodiments, the mammal is a human being suffering from or at risk of suffering from a parasitic infection. In some embodiments, the parasitic infection is selected from African trypanosomiasis, amoebiasis, ascariasis, babesiosis, Chagas disease, cryptosporidiosis, cutaneous larva migrans, dirofilariasis, echinococcosis, fasciolosis, filariasis, lymphatic filariasis, giardiasis, helminthiasis, hookworm infection, leishmaniasis, visceral leishmaniasis, malaria, neurocysticercosis, onchocerciasis, protozoan infection, schistosomiasis, taeniasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinosis, and zoonosis.
In some embodiments, the method further comprises administering to the mammal an antiparasitic agent. In some embodiments, the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal. In some embodiments, the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole. In certain embodiments, methods for treating or preventing neurocysticercosis in a mammal are provided. In such embodiments, the method comprises administering to the mammal in need thereof a therapeutically effective amount of an ionic liquid formulation. In some embodiments, the mammal is a human being. In some embodiments, the mammal is a human being suffering from or at risk of suffering from neurocysticercosis.
In some embodiments, the method further comprises administering to the mammal an antiparasitic agent. In some embodiments, the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal. In some embodiments, the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole.
In some embodiments, suitable dosage ranges for intravenous (i.v.) administration are 0.01 mg to 500 mg per kg body weight, 0.1 mg to 100 mg per kg body weight, 1 mg to 50 mg per kg body weight, or 10 mg to 35 mg per kg body weight. Suitable dosage ranges for other modes of administration can be calculated based on the forgoing dosages as known by those skilled in the art. For example, recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal administration or administration by inhalation are in the range of 0.001 mg to 200 mg per kg of body weight, 0.01 mg to 100 mg per kg of body weight, 0.1 mg to 50 mg per kg of body weight, or 1 mg to 20 mg per kg of body weight. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.
The ionic liquid formulations described herein can be administered in any conventional manner by any route where they are active. Administration can be systemic, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants. Thus, modes of administration for the ionic liquid formulations described herein (either alone or in combination with other pharmaceuticals) can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams. The selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician to obtain the desired clinical response. The amount of ionic liquid formulations described herein to be administered is that amount which is therapeutically effective. The dosage to be administered will depend on the characteristics of the subject being treated, e.g., the particular animal treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician). The amount of a ionic liquid formulations described herein that will be effective in the treatment and/or prevention of a fungal infection will depend on the nature of the fungal infection, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the fungal infection, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, a suitable dosage range for oral administration is, generally, from about 0.001 milligram to about 200 milligrams per kilogram body weight. In some embodiments, the oral dose is from about 0.01 milligram to 100 milligrams per kilogram body weight, from about 0.01 milligram to about 70 milligrams per kilogram body weight, from about 0. 1 milligram to about 50 milligrams per kilogram body weight, from 0.5 milligram to about 20 milligrams per kilogram body weight, or from about 1 milligram to about 10 milligrams per kilogram body weight. In some embodiments, the oral dose is about 5 milligrams per kilogram body weight.
It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
5. Kits
In another aspect, the disclosure provides kits comprising at least one disclosed ionic liquid formulation, and instructions for using the ionic liquid formulation. The kits can also comprise other agents and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed ionic liquid formulation and/or product and another agent for delivery to a patient.
The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may further contain containers or devices for use with the methods or compositions disclosed herein. The kits optionally may provide additional components such as buffers and disposable single-use equipment (e.g., pipettes, cell culture plates or flasks).
The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.
EXAMPLES
The following examples are illustrative, but not limiting, of the ionic liquid formulations, and methods of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention. The use of pronouns such as “I”, “we”, and “our”, for example, refer to one or more of the inventors.
Example_4 - Ionic Liquid Formulations Including Tenofovir
At present, tenofovir prodrugs (TDF or TAF) are an indispensable part of the combination antiretroviral therapy (cART) and more than 20 million HIV infected individuals received either TDF or TAF in 2017.1-6 TDF is an integral part of first-line therapy in low- and middle-income countries (L& MICs) due to its relatively lower cost and greater avail-ability5,6 compared to TAF whereas TAF, due to its selectivity, greater potency, and lesser side effects, continues to replace TDF in developed countries. Importantly, more than 15 FDA-approved oral products containing a combination of antiretroviral drugs contain either TDF or TAF1 which further substantiates the importance of tenofovir prodrugs for the lifelong management of HIV infection. Finally, a combination pill containing either TDF or TAF is also approved for oral HIV pre-exposure prophylaxis (PrEP) in different populations.
There is a need to improve the oral bioavailability of TDF as well as TAF for effective long-term HIV management. Although TDF and TAF have significant improvement in permeability and bioavailability compared to their parent drug (tenofovir; TFV), both prodrugs are still classified as Class Ill drugs by the Biopharmaceutics Classification System due to their high solubility and overall low permeability.5-8 TDF and TAF both are substrates for the P-gp mediated efflux which further limits their oral absorption.9-11 Furthermore, TDF, due to the high lability of phos-phonate diester side chains, undergoes premature hydrolysis in the intestine which further limits its oral absorp-tion.2,5,6 Due to these aforementioned factors, the oral bioavailability of TDF (-j 25%) and TAF (-j 40%) is still considerably low.2-6 Given that these prodrugs need to be administered for a long period, efforts to increase their oral bioavailability to achieve optimal drug utilization and reduction in therapeutic dose will eventually lead to a significant reduction in drug- associated cost and side effects, especially in L& MICs.5,6 For example, it is estimated that a 33% improvement in oral bioavailability of TDF (therapeutic dose reduction from 300 mg to 225 mg) will result in $50-75 million in cost saving every year.6 Hence, there is a dire need to develop strategies to improve the oral bioavailability of TDF and TAF. However, the hydrophilic nature of TDF and TAF preclude/min-imize their incorporation in particles or nanoformulations making the oral bioavailability improvement challenging.
There are safety and tolerability issues with subcutaneous long-acting TAF formulations that further highlight the need for oral delivery of tenofovir prodrugs. Antiretroviral long-acting injectables are actively being developed as an alternative to minimize adherence issues associated with oral antiretroviral therapy. In congruence with these efforts, several subcutaneous long-acting TAF formulation technologies were developed and evaluated in pre-clinical settings.12 Several preclinical studies indicated that subcutaneous delivery of TAF could result in local safety (site lesions) and/or tolerability issues during clinical development.13-15 At the Bill and Melinda Gates Foundation Meeting in 2020, it was concluded that long-acting TAF formulations may not be worth pursuing for HIV prevention.12 Hence, it is even more critical to focus on improving the oral bioavailability of tenofovir prodrugs. Transformation of hydrophilic ionizable drugs into ionic liquids (ILs) is a pharmaceutically viable strategy to improve their transformation into nanoformulations and delivery. Hydrophilic ionizable drugs including antiviral nucleotides such as tenofovir have high water solubility and low membrane permeability which eventually limit their bioavailability. These hydrophilic ionizable drugs are also difficult to formulate into nanoformulations. ILs are organic salts with a melting point of < 100°C and depending upon the cations and anions involved in the formation, ILs can even be liquid at room tempera-ture.16,17 Transformation of highly water soluble salts such as metformin hydrochloride into metformin docusate, a lipophilic IL (FIG. 1) using pharmaceutically acceptable fatty anions is a novel and pharmaceutically viable strategy to improve their permeability, bioavailability, and efficacy.18 Furthermore, the transformation of hydrophilic ionizable drugs into amphiphilic ILs facilitates incorporation into various nanoformulations. Finally, a transdermal anesthetic patch containing IL of lidocaine has successfully completed the Phase III clinical trial19 and is waiting for approval indicating the pharmaceutical viability of the IL approach.
Experiments were conducted with a proposal to develop amphiphilic IL(s) of TDF and TAF with pharmaceutically acceptable fatty permeation enhancers to reduce water solubility, improve permeability and enable subsequent incorporation of IL(s) of TDF or TAF into polymeric nanomicelles to achieve enhanced oral bioavailability and in vivo antiviral activity.
A majority of the antiviral nucleotides and their prodrugs including TDF and TAF have high water solubility and poor membrane permeability which result in low oral bioavailability.5'8 In this grant, we for the first time, will explore the potential of fatty permeation enhancers such as undecylenic acid,20 and salcaprozic acid, a fatty permeation enhancer used in an FDA-approved oral product (Rybelsus®), 21,22 to form ionic liquids. Interestingly, our preliminary data confirmed that TDF and TAF can rapidly interact with oleic acid, capric acid, undecylenic acid, and salcaprozic acid to yield amphiphilic ILs. To our knowledge, we are the first group to develop ILs of ionizable nucleotide prodrugs such as TDF and TAF using anionic fatty permeation enhancers and this strategy can be universally applied to other ionizable hydrophilic drugs to improve their permeability and bioavailability. Hence, this innovative approach will open new paradigms in the oral delivery of hydrophilic ionizable drugs.
To our knowledge, we are the first group to demonstrate the ability of SoluPlus, to package amphiphilic ILs to yield polymeric nanomicelles25 that can be delivered as an oral solution or can be freeze dried to obtain a solid powder. Our unpublished preliminary data also show that orally delivered SoluPlus nanomicelles containing oxfendazole docusate (amphiphilic IL) resulted in significantly higher in vivo efficacy compared to pure drug which further underscores the benefits of our approach. Given that TDF and TAF are both P-gp substrates with low oral bioavailability, the development of So-luPlus nanomicelles containing TDF/TAF ILs is an innovative approach to achieving improved oral bioavailability.
Our prior experience in the synthesis of ionic liquids (ILs) of hydrophilic and lip-ophilic ionizable drugs with suboptimal solubility and permeability characteristics. We have extensive experience in the synthesis and characterization of ILs of hydrophilic (metformin, phenformin) and hydrophobic (anthelmintic benzimidazoles and rilpivirine) ionizable drugs ( Example_l_Scheme 1) with high crystallinity, low permeability, and poor lipid solubility.18,25 We, for the first time, showed that highly crystalline, hydrophobic, and weakly basic drugs such as anthelmintic benzimidazoles25, and an-tiretroviral drug rilpivirine (RPV) can be efficiently (yield > 90%) transformed into ILs using a pharmaceutically acceptable fatty anion sodium docusate ( Example_l_Scheme 1). We also extended this strategy to synthesize amphiphilic ILs of water soluble salt form of antidiabetic biguanides.18 We used techniques such as Fourier-transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS), and to confirm the synthesis and purity of the docusate-based ILs. Unlike pure anthelmintic benzimidazoles or antidiabetic biguanides, their docusate-based ILs showed low or no crystallinity, high thermal stability, and high solubility in organic solvents (acetone, methanol, acetonitrile, etc.) indicating improved pharmaceutical processability and significantly higher in vitro efficacy indicating improved membrane transport.18,25
Example_l_Scheme 1: Hydrophobic and hydrophilic ionizable drugs with high crystallinity and low permeability can be converted to amphiphilic ILs using docusate
Figure imgf000076_0001
■NaC-i
Metformin HCI Sodium docusate Metform i n-d ocu sate
Figure imgf000076_0002
RPV docusate
ExampIe_4A - Amphiphilic ILs can be efficiently incorporated into SoluPlus nanomicelles to further improve their in vivo efficacy. While the transformation of ionizable drugs with suboptimal solubility and permeability characteristics to amphiphilic IL(s) could improve their lipid solubility and permeability, it is necessary to package these ILs into nanoformulations to further improve the delivery, bioavailability, and efficacy of ILs. SoluPlus is a biodegradable amphiphilic copolymer, which forms nanoscale micelles upon dilution with physiological fluids and can encapsulate a variety of hydrophobic drugs leading to improved solubility, permeability, and bioavailability.24,26 We, for the first time, showed that lipophilic ILs of anthelmintic benzimidazole can be stably encapsulated in SoluPlus nanomicelles (FIG. 2).25 Furthermore, our preliminary studies show that these nanomicelles containing ILs, if necessary, can be efficiently freeze dried and can be readily reconstituted to yield nanomicelles with negligible size change. To demonstrate the ability of SoluPlus nanomicelles to improve the delivery of ILs, we developed SoluPlus na-nomicelles (size - 130 nm and PDI: < 0.2) containing oxfendazole docusate (OXF-Doc), an amphiphilic IL. Further, we evaluated the in vivo efficacy of orally delivered pure oxfendazole (OXF) in a vehicle (HPMC), OXF-Doc in a vehicle (HPMC), OXF suspended in SoluPlus nanomicelles, and SoluPlus nanomicelles containing OXF-Doc (OXF dose: 25 mg/kg oral) in a murine model of cryptococcosis. The mice were intranasally infected with Cryptococcus neoformans and the animals were treated with different OXF formulations for 14 days. After 14 days of therapy, the animals were euthanized, lungs were harvested and the C. neoformans burden in the lungs was measured (colony forming units or CFUs). Interestingly, SoluPlus nanomicelles containing OXF-Doc (OXF-Doc in SoluPlus) showed the highest in vivo efficacy compared to the pure OXF and OXF-Doc (FIG. 36) indicating the importance of packaging of ILs into SoluPlus nanomicelles or to achieve enhanced drug delivery. Thus, we propose that SoluPlus nanomicelles containing ILs of TDF/TAF will have improved oral bioavailability and in vivo antiviral efficacy.
Example_4B - Daily oral administration of oral SoluPlus nanomicelles was well tolerated by mice.
As the in vivo antiviral efficacy studies in humanized BLT mouse model of HIV infection require long-term daily oral administration of test samples, we carried out preliminary studies to establish the long-term tolerability of oral SoluPlus nanomicelles. Our preliminary studies showed that oral SoluPlus nanomicelles, at a high daily dose of 975 mg/kg did not show any gross toxicity to mice even after 25 days (FIG. 4) and studies are still ongoing.
Example_4C - Develop, characterize, and evaluate SoluPlus nanomicelles containing TDF ILs or TAF ILs.
We hypothesize that water soluble ionizable TDF/TAF can electrostatically interact with generally regarded as safe (GRAS) fatty permeation enhancers to yield lipophilic ILs with reduced water solubility and improved permeability. Subsequent incorporation of these TDF/TAF- SoluPlus nanomicelles will further improve their oral delivery leading to improved oral bioavailability and phar-macokinetics. We propose to evaluate oleic acid, capric acid, undecylenic acid, and salcaprozic acid all of which have been shown to improve gastrointestinal permeability of hydrophilic peptides/proteins.20-22
We, for the first time, show that TDF as well as TAF can readily interact with GRAS fatty permeation enhancers such as oleic acid, capric acid, undecylenic acid, and salcaprozic acid to yield ILs (Example_l_Scheme 2). While Example_l_Scheme 2 only depicts the formation of TDF oleate and TAF oleate as an IL, the other fatty acids such as capric acid, undecylenic acid, and salcaprozic acid also readily transformed TDF/TAF to TDF/TAF IL within 10 minutes. Interestingly, our LC-MS analysis showed that TDF oleate was significantly less susceptible to accelerated alkaline hydrolysis of TDF compared to pure TDF indicating the potential of ILs to attenuate premature gastrointestinal degradation of TDF. The remarkable simplicity of our process highlights the pharmaceutical viability and potential for the translation of TDF/TAF ILs for improved oral delivery.
Example_l_Scheme 2 - Synthesizing TDF/TAF ionic liquids using fatty acid permeation enhancers as a counterion. TDF/TAF oleate is readily formed within 10 minutes after mixing TDF/TAF and oleic in methanol followed by complete evaporation of ethanol. The same scheme can be used to synthesize other TDF/TAF ILs.
Figure imgf000078_0001
We have already developed TDF oleate, TDF caprate, TDF undecylenate, TDF salcaprozate, TAF oleate, TAF caprate, TAF undecylenate, and TAF salcaprozate ( Example_l_Scheme 2 and FIG. 5) all of which are lipophilic ILs. We confirmed the interaction between TDF/TAF and fatty acid permeation enhancers such as oleic acid using 1 H-NMR spectroscopy (FIG. 6A) and we will continue to characterize TDF/TAF-SoluPlus nanomicelles containing TDF/TAF-ILs and a total of 4 formulations of SoluPlus nanomicelles (2 formulations each with TDF ILs or TAF ILs) with the lowest size, polydispersity index, and surface charge and highest physicochemical and chemical stability will be selected for the PK and relative oral bioavailability studies.
To date, no PK and oral bioavailability studies have been carried out on SoluPlus nanomicelles containing amphiphilic ILs. Hence, PK studies are needed to demonstrate the ability of SoluPlus nanomicelles containing TDF/TAF ILs to improve the relative oral bioavailability of TAF, tenofovir, and its metabolites. We propose to study the pharmacokinetics of oral TDF solution, oral TAF solution, oral SoluPlus nanomicelles containing TDF IL (2 formulations), and oral SoluPlus nanomicelles containing TAF IL (2 formulations) in healthy Balb/c mice. Based on the previous reports on the pharmacokinetics of TDF and TAF in mice27, we plan to study the pharmacokinetics at a 60 mg/kg dose of TDF and 5 mg/kg of TAF. We will prepare oral solution containing TDF (10 mg/mL) or TAF (4 mg/ml) using water. The optimized SoluPlus nanomicelles containing TDF or TAF ILs will be prepared to obtain 10 mg/ml of TDF or 4 mg/ml of TAF. Briefly, six- to 8-weeks old Balb/c mice will be randomly divided into 6 groups (n > 10 mice per group; 5 male and 5 female). The mice will receive either receive 100 pL oral gavage of TDF solution, TAF solution, SoluPlus nanomicelles containing TDF IL (2 formulations), or SoluPlus nanomicelles containing TAF IL (2 formulations). To enable calculation of the pharmacokinetic parameters, we propose withdrawing blood at 8-time points viz. 0.25, 0.5, 1, 2, 3, 4, 8, and 24 h. At the final time points, the blood, lymph nodes, vaginal tissue, and brain will be collected from the eu-thanized animals for the study of tissue distribution. Blood will be centrifuged at 2000 g and 4°C for 15 min and the recovered plasma and other tissues will be stored at -20°C until further analysis. The concentration of TAF, tenofovir and tenofovir diphosphate in plasma, mesenteric lymph node, vaginal tissue, and brain homogenate will be determined using LC-MS/MS. The PK of TAF, tenofovir and tenofovir diphosphate in plasma, lymph node, vaginal tissue, and brain after delivery of oral TDF/TAF nanoformulations will be carried out at the University of Arizona Cancer Center Analytical Chemistry Shared Resource.
Example_4D - Evaluate in vivo antiviral efficacy of oral SoluPlus nanomicelles containing TDF IL.
The hu-BLT mice reconstitute all lineages of immune cells in peripheral blood, secondary lymphoid, and mucosal tissues, of which T cells have undergone ‘education’ in human thymic tissues.2829 Dr. Li’s group has over 10 years of experience in developing new hu-BLT mice models, such as double hu-BLT mice30 and hu-BLT-hIL34 mice.31 His group has also used hu- BLT mice to study HIV-1 transmission, pathogenesis, and latency, and to evaluate the efficacy of ART.30-41 In this aim, using the hu-BLT mice model of HIV- 1 infection, we will evaluate the improved bioavailability and antiviral efficacy of the best formulation of SoluPlus nanomicelles containing TDF IL (SN-TDF-IL) that is developed and selected through the experiments.
Human Fetal Tissue Research Approach: (a) Proposed characteristics, procurement, and procedures: 2 matched human fetal liver and thymic tissues (HFT) from individual human fetal donors at gestational weeks of 13-20 will be procured by Advanced Bioscience Resources, Inc. (Alameda, California) under an IRB-approved informed consent process that adheres to the requirements of federal mandate (see attached informed consent document and HFT Compliance Assureance.pdf). HFT will be shipped in ice from ABR by FedEx overnight and immediately processed at the University of Nebraska at Lincoln (UNL) into 1mm3 fragments (thymic tissues and one-third of liver tissues) and into CD34+ hematopoietic stem cells (HSC) from remaining liver tissues to generate hu-BLT-mice. Any HFT that is not transplanted into mice (including all hu- BLT-mice) at the end of the study will be disposed in biohazardous waste bags and incinerated by certified staff members at the Institutional Animal Care Program at UNL; and (b) Justification for the use of HFT: Humanized-mice are the only small animal models that are susceptible to HIV infection directly. Among the different hu-mice, human T cells in hu-HSC mice are less abundant compared to hu-BLT mice, especially in mucosal tissues. Further, human T cells are “uneducated” by the human thymus. In contrast, the hu-BLT mice reconstitute all lineages of immune cells in peripheral blood, secondary lymphatic, and mucosal tissues.29,42-45 Notably, these human T cells have undergone positive and negative selections, differentiation, and development in human thymic tissues.27,28 Thus, without HFT, the research goals of this project may not be adequately accomplished.
To evaluate the improved bioavailability and anti-HIV efficacy of the SN-TDF-IL, we will use 24 adult hu-BLT mice with good human immune system reconstitution. We will first establish a stable HIV infection according to our previously reported protocol.32 All animals will be inoculated intraperitoneally (IP) with a trans-mit/founder HIV-1 (SUMA, 5.0 x 104 TCID50), which will lead to 100% animal infection. We will bleed mice once a week to quantify pVL by using RT-qPCR which has been used in our lab for many years. When the animals reach a stable infection at 4 weeks post HIV inoculation, which will be confirmed by repeated RT-qPCR, animals will be randomly divided into 3 groups with similar levels of pVLs and body weights: SN-TDF-IL Rx, pure-TDF Rx control, and un-Rx control groups (n=8/group, 4 female and 4 male) (FIG. 7). The group size is based on previous reports on mouse models.46-49 Next, we will start SN-TDF-IL treatment. The animals in the Rx group will receive daily oral gavage of the best formulation and 60 mg/kg dose of SN-TDF-IL, while the Rx control group will receive the same dose of pure-TDF, and un-Rx control group will receive PBS only. TDF dose is selected based on a previous report on hu-BLT mice.50 We will collect blood on days 0, 2, 3, 7, 10, and 14 for quantifying plasma viral RNA load and partial plasma samples will also be used to quantify the TDF concentration. We will weigh the mice daily during the treatment period. At the end of the experiment after monitoring for 14 days post treatment, all the animals will be euthanized. Tissues of lymph nodes, spleen, and ileum gut will be harvested, of which two third will be fixed in 10% neutral formalin for vRNA quantification using HIV RNAscope as we previously reported and one third will be freshly homogenized for vRNA and TDF quantification using qPCR, RT-qPCR, and the assay. The efficacy will be indicated by the decay of pVL (vRNA copies) and infected cell frequency in lymph nodes and spleen tissues.
The following references pertain to the numerical reference citations recited in Example_4:
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10. Swedrowska M, Jamshidi S, Kumar A, Kelly C, Rahman KM, Forbes B. In Silico and in Vitro Screening for P- Glycoprotein Interaction with Tenofovir, Darunavir, and Dapivirine: An Antiretroviral Drug Combination for Topical Prevention of Colorectal HIV Transmission. Mol Pharm. 2017 Aug 7;14(8):26602669.
1 1 . van Gelder J, Deferme S, Naesens L, De Clercq E, van den Mooter G, Kinget R, Augustijns P. Intestinal absorption enhancement of the ester prodrug tenofovir disoproxil fumarate through modulation of the biochemical barrier by defined ester mixtures. Drug Metab Dispos. 2002 Aug;30(8):924-30.
12. Romano JW, Baum MM, Demkovich ZR, Diana F, Dobard C, Feldman PL, Garcia- Lerma JG, Grattoni A, Gunawardana M, Ho DK, Hope TJ, Massud I, Milad M, Moss JA, Pons-Faudoa FP, Roller S, van der Straten A, Srinivasan S, Veazey RS, Zane D. Tenofovir Alafenamide for HIV Prevention: Review of the Proceedings from the Gates Foundation Long- Acting TAF Product Development Meeting. AIDS Res Hum Retroviruses. 2021 Jun;37(6):409-420
13. Su JT, Simpson SM, Sung S, Tfaily EB, Veazey R, Marzinke M, Qiu J, Watrous D, Widanapathirana L, Pearson E, Peet MM, Karunakaran D, Grasperge B, Dobek G, Cain CM, Hope T, Kiser PF. A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques. Antimicrob Agents Chemother. 2020 Feb 21 ;64(3):e01893-19.
14. Zane D, Roller S, Shelton J, Singh R, Jain R, Wang Y, Yang B, Felx M, Alessi T, Feldman PL. A 28-Day Toxicity Study of Tenofovir Alafenamide Hemifumarate by Subcutaneous Infusion in Rats and Dogs. Microbiol Spectr. 2021 Sep 3;9(l):e0033921.
15. Massud I, Krovi A, Nishiura K, Ruone S, Li L, Holder A, Gary J, Mills P, Mitchell J, Khalil G, Pan Y, Luecke E, Gatto G, Heneine W, Garcia-Lerma JG, Johnson L, van der Straten A, Dobard C. Safety and efficacy of a biodegradable implant releasing tenofovir alafenamide for vaginal protection in a macaque model. J Antimicrob Chemother. 2022 Aug 1 :dkac252.
16. Handa M, Almalki WH, Shukla R, Afzal O, Altamimi ASA, Beg S, Rahman M. Active pharmaceutical ingredients (APIs) in ionic liquids: An effective approach for API physiochemical parameter optimization. Drug Discov Today. 2022 Sep;27(9):2415-2424.
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22. Aroda VR, Blonde L, Pratley RE. A new era for oral peptides: SNAC and the development of oral semaglutide for the treatment of type 2 diabetes. Rev Endocr Metab Disord. 2022 Jul 15. doi: 10.1007/sl 1154-022-09735-8
23. Jin X, Zhou B, Xue L, San W. Soluplus(®) micelles as a potential drug delivery system for reversal of resistant tumor. Biomed Pharmacother. 2015 Feb;69:388-95.
24. Linn M, Collnot EM, Djuric D, Hempel K, Fabian E, Kolter K, Lehr CM. Soluplus® as an effective absorption enhancer of poorly soluble drugs in vitro and in vivo. Eur J Pharm Sci. 2012 Feb 14;45(3):33643.
25. Sutar Y., Fulton S., Paul S., Mhatre S., Altamirano S., Saeed H.K., Patel P., Bhat S., Mallick S., Chauhan H., Patravale V.B., Nielsen K., Date A.A., Docusate-based ionic liquids of anthelmintic benzimidazoles show improved pharmaceutical processability, lipid solubility, and in vitro activity against Cryptococcus neoformans. ACS Infectious Diseases, 2021, 7:2637-2649.
26. Strojewski D, Krupa A. Kollidon® VA 64 and Soluplus® as modern polymeric carriers for amorphous solid dispersions. Polim Med. 2022 Jan-Jun;52(l):17-27.
27. Dyavar SR, Kumar S, Gautam N, Podany AT, Winchester LC, Weinhold JA, Mykris TM, Nallasamy P, Alnouti Y, Fletcher CV. Intramuscular and subcutaneous administration of antiretroviral drugs, compared with oral, enhances delivery to lymphoid tissues in BALB/c mice. J Antimicrob Chemother. 2021 Sep 15;76(10):2651-2658.
28. Marsden MD, Zack JA. Humanized Mouse Models for Human Immunodeficiency Virus Infection. Annual Review of Virology. 2017;4(l):393-412. doi: 10.1146/ annurev- virology- 101416-041703.
29. Victor Garcia J. Humanized mice for HIV and AIDS research. Current opinion in virology. 2016;19:56-64. Epub 2016/07/23. doi: 10.1016/j.coviro.2016.06.010. PubMed PMID: 27447446; PMCID: PMC5021593.
30. Daharsh L, Zhang J, Ramer-Tait A, Li Q. A Double Humanized BLT-mice Model Featuring a Stable Human- Like Gut Microbiome and Human Immune System. J Vis Exp. 2019(150). Epub 2019/09/17. doi: 10.3791/59773. PubMed PMID: 31524867.
31 . Zhang J, Lohani SC, Cheng Y, Wang T, Guo L, Kim W-K, Gorantla S, Li Q. Human Microglia Extensively Reconstitute in Humanized-BLT Mice With Human Interleukin-34 Transgene and Support HIV-1 Brain Infection. Frontiers in Immunology. 2021 ; 12( 1703). doi: 10.3389/fimmu.202L 672415.
32. Li Q, Tso FY, Kang G, Lu W, Li Y, Fan W, Yuan Z, Destache CJ, Wood C. Early Initiation of Antiretroviral Therapy Can Functionally Control Productive HIV-1 Infection in Humanized-BLT Mice. J Acquir Immune Defic Syndr. 2015;69(5):519-27. Epub 2015/07/15. doi: 10.1097/qai.0000000000000687. PubMed PMID: 26167617; PMCID: PMC4503390.
33. Destache CJ, Mandal S, Yuan Z, Kang G, Date AA, Lu W, Shibata A, Pham R, Bruck P, Rezich M, Zhou Y, Vivekanandan R, Fletcher CV, Li Q. Topical Tenofovir Disoproxil Fumarate Nanoparticles Prevent HIV-1 Vaginal Transmission in a Humanized Mouse Model. Antimicrob Agents Chemother. 2016;60(6):3633-9. Epub 2016/04/06. doi: 10.1128/aac.00450-16. PubMed PMID: 27044548; PMCID: PMC4879396.
34. Mandal S, Prathipati PK, Kang G, Zhao Y, Yuan Z, Fan W, Li Q, Destache CJ. Tenofovir alafenamide and elvitegravir loaded nanoparticles for long-acting prevention of HIV-1 vaginal transmission. AIDS. 2016;Publish Ahead of Print, doi: 10.1097/qad.0000000000001349. PubMed PMID: 00002030900000000-97616.
35. Sun M, Li Y, Yuan Z, Lu W, Kang G, Fan W, Li Q. VRC01 antibody protects against vaginal and rectal transmission of human immunodeficiency virus 1 in hu- BLT mice. Arch Virol. 2016; 161(9):2449-55. Epub 2016/06/28. doi: 10.1007/s00705-016-2942-4. PubMed PMID: 27343044; PMCID: PMC4988922.
36. Yuan Z, Kang G, Ma F, Lu W, Fan W, Fennessey CM, Keele BF, Li Q. Recapitulating Cross-Species Transmission of Simian Immunodeficiency Virus SIVcpz to Humans by Using Humanized BLT Mice. J Virol. 2016;90(17):7728-39. Epub 2016/06/17. doi: 10. 1128/jvi.OO86O- 16. PubMed PMID: 27307566; PMCID: PMC4988171.
37. Mandal S, Prathipati PK, Kang G, Zhou Y, Yuan Z, Fan W, Li Q, Destache CJ. Tenofovir alafenamide and elvitegravir loaded nanoparticles for long-acting prevention of HIV-1 vaginal transmission. AIDS. 2017;31(4):469-76. doi: 10.1097/qad.0000000000001349. PubMed PMID: 00002030-20170220000005.
38. Yuan Z, Kang G, Lu W, Li Q. Reactivation of HIV-1 proviruses in immune- compromised mice engrafted with human VOA-negative CD4+ T cells. J Virus Erad. 2017;3(l):61-5. Epub 2017/03/10. PubMed PMID: 28275460; PMCID: PMC5337423.
39. Mandal S, Kang G, Prathipati PK, Fan W, Li Q, Destache CJ. Long-acting parenteral combination antiretroviral loaded nano-drug delivery system to treat chronic HIV-1 infection: A humanized mouse model study. Antiviral Research. 2018;156:85-91. doi: https://doi.Org/10.1016/j.antiviral.2018.06.005. 40. Yuan Z, Kang G, Daharsh L, Fan W, Li Q. SIVcpz closely related to the ancestral HIV-1 is less or nonpathogenic to humans in a hu-BLT mouse model. Emerging Microbes & Infections. 2018;7(1):59. doi: 10.1038/s41426-018-0062-9.
41 . Mandal S, Kang G, Prathipati PK, Zhou Y, Fan W, Li Q, Destache CJ. Nanoencapsulation introduces long-acting phenomenon to tenofovir alafenamide and emtricitabine drug combination: A comparative pre-exposure prophylaxis efficacy study against HIV-1 vaginal transmission. Journal of Controlled Release. 2019;294:216-25. doi: https://doi.Org/10.1016/j.jconrel.2018.12.027.
42. Melkus MW, Estes JD, Padgett- Thomas A, Gatlin J, Denton PW, Othieno FA, Wege AK, Haase AT, Garcia JV. Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nature medicine. 2006;12(l l): 1316-22. Epub 2006/10/24. doi: 10.1038/nml431. PubMed PMID: 17057712.
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44. Olesen R, Swanson MD, Kovarova M, Nochi T, Chateau M, Honeycutt JB, Long JM, Denton PW, Hudgens MG, Richardson A, Tolstrup M, Ostergaard L, Wahl A, Garcia JV. ART influences HIV persistence in the female reproductive tract and cervicovaginal secretions. J Clin Invest. 2016;126(3):892-904. Epub 2016/02/09. doi: 10.1172/jci64212. PubMed PMID: 26854925; PMCID: PMC4767352.
45. Stoddart CA, Maidji E, Galkina SA, Kosikova G, Rivera JM, Moreno ME, Sloan B, Joshi P, Long BR. Superior human leukocyte reconstitution and susceptibility to vaginal HIV transmission in humanized NOD-scid IL-2Ry(-/-) (NSG) BLT mice. Virology. 2011 ;417(l):154-60. Epub 2011/06/21. doi: 10.1016/j.virol.2011.05.013. PubMed PMID: 21684569; PMCID: PMC3152643.
46. Shiau AL, Teo ML, Chen SY, Wang CR, Hsieh JL, Chang MY, Chang CJ, Chao J, Chao L, Wu CL, Lee CH. Inhibition of experimental lung metastasis by systemic lentiviral delivery of kallistatin. BMC Cancer. 2010;10:245. doi: 10.1186/1471-2407- 10-245. PubMed PMID: 20509975; PMCID: PMC2893111.
47. Garaulet G, Lazcano JJ, Alarcon H, de Frutos S, Martinez-Torrecuadrada JL, Rodriguez A. Display of the Albumin-Binding Domain in the Envelope Improves Lentiviral Vector Bioavailability. Hum Gene Ther Methods. 2017;28(6):340-51. doi: 10.1089/hgtb.2017.057. PubMed PMID: 29160106.
48. Cire S, Da Rocha S, Ferrand M, Collins MK, Galy A. In Vivo Gene Delivery to Lymph Node Stromal Cells Leads to Transgene-specific CD8+ T Cell Anergy in Mice. Mol Ther. 2016;24(l l):1965-73. doi: 10.1038/mt.2016.168. PubMed PMID: 27562586; PMCID: PMC5154481.
49. Palomares K, Vigant F, Van Handel B, Pemet O, Chikere K, Hong P, Sherman SP, Patterson M, An DS, Lowry WE, Mikkola HK, Morizono K, Pyle AD, Lee B. Nipah virus envelope -pseudotyped lentiviruses efficiently target ephrinB2-positive stem cell populations in vitro and bypass the liver sink when administered in vivo. J Virol. 2013;87(4):2094-108. doi: 10.1128/JVI.02032-12. PubMed PMID: 23192877; PMCID: PMC3571488.
50. Stoddart CA, Galkina SA, Joshi P, Kosikova G, Moreno ME, Rivera JM, Sloan B, Reeve AB, Sarafianos SG, Murphey-Corb M, Parniak MA. Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque. Antimicrob Agents Chemother. 2015 Jul;59(7):4190-8. Example 2. Ionic Liquid Formulations Including Rilpivirine
Globally, approximately 39 million people are infected with the human immune deficiency virus (HIV) and every year, more than 1 million individuals get infected with HIV. To date, no cure has been found for HIV infection.1, 2 The advent of combination antiretroviral therapy (cART) has remarkably improved the management of HIV infection and HIV-associated comorbidities in patients.3, 4 However, HIV-infected patients are required to take antiretroviral drugs for their lifetime. It is noteworthy that many of the antiretroviral drugs used for the management of HIV infection have low or limited oral bioavailability and show intra- and/or interindividual variability in the pharmacokinetics (PK).5-8 As HIV patients require lifelong therapy, it is critical to develop an oral delivery strategy that maximizes drug utilization and efficacy by improving oral bioavailability and minimizing inter-individual variability.
Rilpivirine (RPV) is a potent non-nucleoside reverse transcriptase inhibitor (NNRTI) prescribed for the first-line treatment of HIV-1 infection in naive patients with a viral load (VL) of less than 100,000 copies/mm.9-11 Furthermore, due to its good safety profile and dosing convenience (once daily 25 mg RPV tablet), RPV is commonly used in virologically suppressed pre-treated patients when treatment simplification is desired.12 15 Additionally, an RPV-based 2- drug regimen (Juluca) is commonly used for the long-term maintenance of virologic suppression in patients. However, several population pharmacokinetic (PK) analyses have shown that the existing oral RPV formulation shows highly variable PK and cannot yield the minimal effective concentration of RPV (50 ng/ml) in around 20-30% of the patients leading to virologic failure and increased incidence of drug resistance.8, 16, 17 It is noteworthy that no human oral bioavailability data was included in the new drug application for the RPV. Previous studies show that RPV has low aqueous solubility in the gastrointestinal fluids and high crystallinity which may be responsible for the high inter-individual variability in PK eventually leading to suboptimal efficacy.10, 13 15 Additionally, previous studies have shown that RPV has low tissue penetration ratios (< 0.1) in the brain, spleen, gut-associated lymphoid tissue (GALT), and testes compared to plasma.18, 19 The anatomical sites such as the brain, spleen, GALT, and testes are known as the HIV sanctuary sites where the replication-competent virus persists. 18, 19 20 Hence, it is essential to develop strategies that can improve the delivery of antiretroviral drugs to the HIV sanctuary sites to minimize the HIV reservoir. Oral lipid-based formulations of antiretroviral drugs such as saquinavir, ritonavir, and tipranavir were successfully translated to the clinic. 20-22 Oral lipid-based formulation technologies such as self-nanoemulsifying systems (SNES) have been successful in augmenting oral bioavailability, and efficacy of hydrophobic drugs.23, 24 However, due to the high crystallinity and poor lipid solubility of RPV (data shown later), the development of SNES was deemed impractical for RPV.
Ionic liquid (IL), is a low-melting salt containing organic/inorganic cation and anion, and depending upon the composition, IL can also be liquid at room temperature.25, 26 Over the last 15 years, pharmaceutical applications of ILs have been on the rise. Conversion of ionizable active pharmaceutical ingredients (APIs) into ILs (APLILs) using pharmaceutically acceptable counterions has emerged as a novel approach to modulate physicochemical and biopharmaceutical properties of ionizable drugs with poor solubility and/or permeability eventually leading to greater oral bioavailability.27'33 Here, we evaluate the ability of biocompatible anions with different chain lengths, extent of unsaturation, and anionic functional groups to convert RPV into amphiphilic RPV IL with high solubility in lipids used for the development of SNES. We show that only docusate sodium efficiently yields amphiphilic RPV- IL, RPV docusate (RPV-Doc) with 100- to 200-fold greater lipid solubility than RPV. Our studies show that RPV-Doc exhibits antiretroviral activity similar to RPV in vitro. Finally, we demonstrate that RPV-Doc can be converted into SNES suitable for oral delivery and that orally delivered SNES containing RPV-Doc shows significant improvement in oral bioavailability and penetration into HIV sanctuary sites indicating the promise of our approach.
Example 2A - Materials and Methods Related to Example 2
Materials. Rilpivirine (RPV) was purchased from Ambeed (Arlington Heights, IL, USA). Sodium docusate and Linoleic acid (99%) were obtained from TCI chemicals (Portland, OR, USA). Sodium lauryl sulfate (SLS) was received as a gift sample from BASF. Oleic acid and geranic acid were purchased from Thermo Fischer (MA, USA). Docosahexaenoic acid (DHA) was received from Nu-Check Prep (MN, USA). Methanol (AR grade), acetonitrile (AR grade), acetone (AR grade), and dichloromethane (AR grade) were purchased from VWR International (PA, USA). The nylon syringe filters with glass fiber prefilter (0.22 pm) were purchased from Simsii Inc. (CA, USA). Capryol 90, Labrafac MC60, Peceol and Labrasol, Plurol Oleique CC 497, Transcutol and Lauroglycol 90 (Gattefosse USA, NI, USA), Capmul MCM-C8 (Abitec Corp., OH, USA) were received as gift samples. Kolliphor ELP, Kolliphor PS80, Kolliphor PS20, Kolliphor HS15, Kollisolv PEG400, Kolliphor RH40 was received as a gift sample from BASF. All other chemicals used were of analytical grade unless otherwise indicated. Animals. C57BL/6 (B6) mice (male, 10- week-old) were obtained from breeding stocks maintained at the University of Arizona. All mice were housed under conditions of controlled temperature (22 °C) with on-off light cycle, with food and water provided ad libitum. Mice were fasted for 12h before oral administration of the drugs. All animal studies were approved by the University of Arizona Animal Care and Use Committee.
High-pressure liquid chromatography (HPLC). A previously reported RPV HPLC method was used with suitable modifications34 for the determination of the equilibrium solubility of RPV and its ILs/salts/cocrystals from different vehicles and to determine the LogP and purity of RPV-lLs/salts/cocrystals. The HPLC analysis was carried out on a Shimadzu LC2050C-3D system equipped with a PDA detector and autoinjector (Lab solution integrator software 5.87 SP1). Briefly, the stock solution of RPV (100 pg/mL), and its ILs/salts were prepared in a 10 mL volumetric flask using DMSO (HPLC grade). The working solutions were prepared by diluting the stock solution with methanol (HPLC grade) to achieve a concentration equivalent to 30 uM RPV. The chromatographic separation was achieved using Gemini (150mm x 4.6 mm, 3pm, 110 A ) C18 column (Phenomenex, Torrance, CA). The mobile phase was composed of acetonitrile and 25 mM potassium dihydrogen phosphate solution (70:30 v/v). The RPV was monitored at 290 nm at a flow rate of 0.6 mL/min and a column oven temperature of 35 °C. A standard curve was prepared by injecting 1-25 pg/mL of RPV. All the experiments were performed in triplicate. The inter-day and intra-day variability for the standard curve was always < 5%.
Determination of RPV solubility in aqueous and lipid vehicles. The saturation solubility of RPV in different aqueous and lipid vehicles such as water, pH 1.2 buffer, pH 4.5 buffer, pH 6.8 buffer, Capryol 90, Labrasol ALF, Labrafac MC60, Capmul MCM C8 and Peceol was determined in triplicate. Briefly, an excess amount of RPV (~ 5 mg) was transferred to an Eppendorf tube containing 1 ml of aqueous or lipid vehicle. The contents were mixed for 30 seconds to ensure a uniform dispersion. The dispersions were incubated in a temperature- controlled orbital shaker set at 37 °C and 50 rpm speed for 24 hours. After 24 h, the RPV- aqueous/lipid vehicle mixture was transferred to a microcentrifuge tube or Amicon-Ultra centrifuge tube, and samples were centrifuged at 10000 rpm for 15 min. The supernatant (or filtrate) was diluted with methanol and the RPV concentration was determined using the previously described HPLC method.
Synthesis of rilpivirine docusate (RPV-Doc). The equimolar quantity (0.2729 mmol) of RPV and IM hydrochloric acid solution were stirred in 15 mL of methanol at room temperature until the dissolution of RPV. To this clear methanolic solution, sodium docusate (0.2729 mmol) was added and stirred for 3 h. After 3 h of stirring, methanol was evaporated on a rotary evaporator and the residue obtained was dissolved in dichloromethane (DCM). The DCM solution was transferred to a separating funnel and washed with water until negative to a silver nitrate precipitate test. The organic layer was passed through a sodium sulfate bed and concentrated in vacuo. The resulting slurry was treated with 20 mL of acetonitrile (ACN) and stirred for 10 min. Finally, the ACN solution was microfiltered and concentrated on a rotary evaporator to obtain a viscous liquid of RPV-Doc (yield: 94%).
RPV-Doc: ' H NMR (500 MHz, DMSO) 5 10.57 (s, 1H), 8.09 (s, 1H), 7.67 (d, J= 16.4 Hz, 1H), 7.54 (s, 3H), 7.47 - 7.36 (m, 3H), 6.65 (d, J = 7.5 Hz, 1H), 6.51 (d, J = 16.8 Hz, 1H), 3.89 - 3.72 (m, 5H), 2.98 - 2.81 (m, 2H), 2.18 (s, 6H), 1.46 (d, J = 4.0 Hz, 2H), 1.29 - 1.19 (m, 16H), 0.85 - 0.77 (m, 12H). 13C NMR (500 MHz, DMSO) 5 171.13, 168.43, 162.46, 150.24, 136.20, 132.91, 127.68, 120.01, 118.99, 118.22, 105.27, 99.38, 97.17, 66.41, 66.35, 66.24, 61.63, 29.90, 29.77, 29.72, 28.51, 23.35, 23.33, 23.14, 22.57, 22.55, 18.16, 14.07, 14.05, 10.89. HRMS of RPV-Doc; HRMS m/z for C42H56N6O7S [negative]: 788.38889
Synthesis of rilpivirine lauryl sulfate (RPV-LS)/ RPV oleate. Equimolar quantities (0.2729 mmol) of RPV and IM hydrochloric acid solution were stirred in 5 mL of methanol at room temperature until the dissolution of RPV. To the methanolic solution, sodium lauryl sulfate/ sodium oleate (0.2729 mmol) was added and stirred for 3 h. After 3 hours of stirring, methanol was evaporated on a rotary evaporator, and the obtained residue was dissolved in dichloromethane (DCM). The DCM solution was transferred to the separating funnel and washed with water until negative to a silver nitrate precipitate test. The organic layer was passed through a sodium sulfate bed, microfiltered to remove undissolved residue, and concentrated in vacuo to obtain waxy RPV-LS (yield: 92%) and pale-yellow solid of RPV oleate (yield: 88%).
RPV-LS: ’H NMR (500 MHz, DMSO) 5 10.44 (s, 1H), 8.05 (s, 1H), 7.68 (d, J= 17.9 Hz, 1H), 7.54 (s, 3H), 7.49 - 7.38 (m, 3H), 6.59 (s, 1H), 6.52 (d, 7 = 16.8 Hz, 1H), 3.68 (t, 7 = 6.7 Hz, 2H), 2.18 (s, 6H), 1.47 (t, 7 = 7.3 Hz, 2H), 1.22 (s, 18H), 0.88 - 0.81 (m, 3H). 13C NMR (500 MHz, DMSO) 5 161.87, 151.63, 149.57, 141.55, 136.75, 135.54, 132.24, 127.01, 119.35, 118.32, 104.58, 98.72, 96.49, 65.06, 30.80, 28.57, 28.52, 28.27, 28.22, 25.01, 21.59, 17.50, 13.45. HRMS of RPV-LS; HRMS m/z for C34H44N6O4S [negative]: 631.30720
RPV oleate: *H NMR (500 MHz, DMSO) 5 11.96 (s, 1H), 9.60 (s, 1H), 8.93 (s, 1H), 8.02 (d, 7 = 6.3 Hz, 2H), 7.64 (d, 7 = 16.8 Hz, 3H), 7.49 (s, 2H), 7.43 (s, 1H), 6.46 (d, 7 = 16.8 Hz, 1H), 6.33 (s, 1 H), 5.32 (t, 7 = 4.6 Hz, 2H), 2.17 (s, 8H), 1.98 (q, 7 = 6.9 Hz, 4H), 1.48 (t, 7 = 7.1 Hz, 2H), 1.24 (d, 7= 5.5 Hz, 20H), 0.86 (d, 7 = 6.9 Hz, 3H). 13C NMR (500 MHz, DMSO) 5 174.61 , 161.87, 159.38, 150.49, 145.69, 136.68, 132.67, 129.80, 129.77, 127.60, 1 19.86, 1 19.15, 118.02, 101.52, 96.31, 33.81, 31.45, 29.26, 29.24, 29.00, 28.86, 28.77, 28.76, 28.72, 28.66, 26.73, 24.65, 22.26, 18.43, 14.09.
Synthesis of rilpivirine docosahexaenoate (RPV-DHA)/ RPV geranate/ RPV linoleate. The equimolar quantities (0.2729 mmol) of RPV and IM hydrochloric acid solution were stirred in 10 mL of methanol at room temperature until the dissolution of RPV. Separately, the equimolar quantities (0.2729 mmol) of DHA/ geranic acid/ linoleic acid and IM sodium hydroxide were dissolved in methanol (5 mL) under argon & dark condition. The methanolic solution of counterion was mixed with acidified methanolic RPV solution and stirred in dark for 3 h in the inert (argon) environment. Methanol was evaporated on a rotary evaporator and the obtained residue was dissolved in dichloromethane (DCM). The DCM solution was transferred to the separating funnel and washed with water until negative to a silver nitrate precipitate test. The organic layer was passed through a sodium sulfate bed, microfiltered to remove undissolved residues, and concentrated in vacuo to obtain a yellowish opaque paste of RPV-DHA (yield: 98%), pale yellow solid of RPV geranate (yield: 86%) and RPV linoleate (yield: 87%).
RPV-DHA: :H NMR (500 MHz, DMSO) 5 12.07 (s, 1H), 9.60 (s, 1H), 8.93 (s, 1H), 8.01 (d, J = 6.0 Hz, 2H), 7.63 (d, J = 16.5 Hz, 3H), 7.48 (s, 2H), 7.43 (s, 1H), 6.44 (s, 1H), 6.34 (s, 1H), 5.46 - 5.07 (m, 12H), 2.86 - 2.74 (m, 8H), 2.25 (s, 2H), 2.17 (s, 10H), 2.05 - 1.99 (m, 2H), 0.91 (t, 7 = 7.6 Hz, 3H). 13C NMR (500 MHz, DMSO) 5 173.87, 161.71, 159.16, 150.31, 145.50, 136.50, 132.52, 131.54, 128.50, 128.39, 128.11, 127.99, 127.89, 127.87, 127.83, 127.78, 127.68, 119.68, 118.99, 117.87, 101.38, 96.12, 33.62, 25.20, 25.16, 25.11, 22.40, 20.03, 18.26, 14.10.
RPV geranate: !H NMR (500 MHz, DMSO) 8 11.89 (s, 1H), 9.61 (s, 1H), 8.94 (s, 1H), 8.01 (d, J = 5.6 Hz, 2H), 7.63 (d, J = 16.6 Hz, 3H), 7.48 (s, 2H), 7.43 (s, 1H), 6.45 (d, J = 16.8 Hz, 1H), 6.33 (s, 1H), 5.59 (d, 7= 7.5 Hz, 1H), 5.12 - 5.01 (m, 1H), 2.17 (s, 6H), 2.13 - 2.04 (m, 6H), 1.84 (d, 7 = 1.4 Hz, 1H), 1.64 (s, 3H), 1.57 (d, 7 = 1.5 Hz, 3H). 13C NMR (500 MHz, DMSO) 5 167.60, 161.88, 158.55, 150.49, 145.69, 136.67, 132.69, 131.72, 127.61, 123.40, 119.86, 119.16, 118.03, 116.31, 101.54, 96.35, 32.80, 25.72, 25.63, 18.43, 18.32, 17.71.
RPV linoleate: ’H NMR (500 MHz, DMSO) 5 11.98 (s, 1H), 9.60 (s, 1H), 8.93 (s, 1H), 8.01 (d, 7 = 5.9 Hz, 1H), 7.64 (d, 7 = 16.6 Hz, 3H), 7.48 (s, 2H), 7.43 (s, 1H), 6.46 (d, 7 = 16.6 Hz, 1H), 6.33 (s, 1H), 5.41 - 5.19 (m, 3H), 2.73 (t, 7 = 6.6 Hz, 1H), 2.16 (s, 7H), 2.00 (t, 7 = 6.8 Hz, 3H), 1.48 (t, 7 = 7.3 Hz, 4H), 1.34 - 1.12 (m, 16H), 0.86 (d, 7 = 6.9 Hz, 3H). 13C NMR (500 MHz, DMSO) 5 174.12, 161.37, 158.87, 149.98, 145.18, 136.17, 132.17, 129.38, 127.42, 1 19.36, 118.65, 1 17.52, 101.03, 95.76, 33.31 , 30.56, 28.66, 28.39, 28.26, 26.27, 24.87, 24.14, 21.75, 17.93, 13.57.
Spectroscopic characterization of RPV-IL/lipophilic salts. The developed RPV- IL/lipophilic salts were characterized using FTIR spectroscopy and NMR spectroscopy using the procedures described in our recently published papers.32, 33
Thermal characterization of RPV-IL/lipophilic salts. The thermal stability of pure RPV, anions, and RPV ILs/salts were analyzed using TA Instruments Discovery TGA 5500 (TA instruments, New Castle, DE, USA). The DSC analysis of pure RPV and RPV ILs/salts was carried out on TA Instruments DSC 2500 RCS40 cooling unit (TA instruments, New Castle, DE, USA). The thermal characterization protocols were akin to the procedures described in our recently published papers.32, 33
Evaluation using the powder X-ray diffraction (PXRD). RPV and all ILs/salts were analyzed for crystallinity using a Philips PANalytical X’Pert PRO MPD (Malvern Panalytical, Malvern, UK) equipped with copper X-ray source (Ka radiation with /. = 1 .5406 A). Samples were mounted on a 20 x 20 mm, 0.2 mm deep pocket sample holder. The diffraction patterns of the samples were collected at room temperature scanning between 3.992° and 90.0° (29) at a rate of 1.55° per second. Samples were run 24 hours after synthesis.
Determination of the octanol-water partition coefficient (LogP). The octanol-water partition coefficient of RPV, and its ILs/salts was determined using the previously described method.32 Before beginning the experiment, water and 1 -octanol were equilibrated for 24 hours using an orbital shaker. Octanol (0.5 ml) and 10 mg of RPV or RPV ILs/salts were mixed in an Eppendorf tube and equilibrated for 24 hours at room temperature. Octanol-saturated water (0.5 ml) was added to each Eppendorf tube containing RPV/RPV-IL/salts and octanol mixture and both phases were allowed to equilibrate for 24 hours. The phases were allowed to settle before aliquots were taken from each phase. Aliquots of the aqueous phase were taken using a pipette, expelling air as the tip moved through the organic phase to ensure only water would be uptaken. The concentration of RPV in each phase was determined via HPLC using the method detailed previously and the apparent LogP value was calculated as previously reported.
Solubility of RPV-ILs/salts/cocrystals in aqueous vehicles. The saturation solubility of RPV-ILs/salts/cocrystals in ultra-pure water, pH 1.2, pH 4.8, and pH 6.8 buffer was determined using previously reported procedures.
In vitro antiretroviral activity evaluation. The purpose of this study was to determine whether the transformation of RPV to RPV-IL/salts affected the inherent antiviral activity of RPV. Hence, the in vitro antiviral activity of RPV or RPV-IL/salts was evaluated only at one concentration (1 nM RPV). Briefly, TZM-bl cells (5000 cells/well) were cultured in a 96-well plate in cDMEM and incubated overnight at 37°C, 5% CO2. After overnight incubation, each well was infected with 2 ng HIV- IBAL and simultaneously treated with the indicated RPV (1 nM), RPV-IL/salt (equivalent to 1 nM RPV) or vehicle control and left for 48 hours. After 48 h, each well was lysed with IX Passive Lysis Buffer for 10-15 mins at 37°C, 5% CO2, and the lysate was collected. The luciferase assay was performed using a Berthhold Detection Systems Sirius Luminometer. To each assay tube, 50 pl of luciferase substrate (Promega) was added followed by 20 pl cell lysate. The mix was immediately vortexed and assessed by the luminometer. All experiments were carried out in triplicate.
Determination of the kinetic solubility of RPV and RPV ILs/salts in lipids. The solubility of the RPV and its IL/salt in the bioavailability -enhancing lipid vehicles like Labrasol ALF (capryl macrogol glyceride), Capryol 90 (propylene glycol monocaprylate), Labrafac MC 60 (glycerol monocaprylocaprate), Peceol (glycerol monooleate) and Capmul MCM C8 (glyceryl monocaprylate) was determined using a previously reported method.32, 33 Briefly, 0.5 mL of lipid vehicle was dissolved in 1 mL of ethanol. Separately, RPV or RPV ILs/salts stock was prepared in ethanol: acetone (1: 1; RPV concentration: 5 mg/mL), and 1 mL of this solution was added to the previously prepared ethanolic solution of lipid vehicle. The mixture was vortexed, evaporated on a rotary evaporator, and dried in a vacuum oven overnight to remove traces of organic solvents. The vacuum-dried lipid solution was inspected visually for the solubilization/precipitation of RPV or RPV ILs/salts. The addition of RPV or RPV ILs/salts in the lipid vehicle was continued in 5 mg increments until precipitation was observed.
Development and characterization of the SNES containing Labrafac MC60 and RPV-Doc (RPV-Doc-MC60SNES). Due to the highest kinetic solubility of RPV-Doc in Labrafac MC60, we focused on the development of SNES containing Labrafac MC60 and RPV- Doc. Due to the unavailability of any previous SNES containing Labrafac MC60, we focused on the systematic development of SNES containing Labrafac MC60 and RPV-Doc.
Screening of surfactants. We used our previously reported method 35 to evaluate the ability of various pharmaceutically acceptable surfactants to emulsify a mixture of Labrafac MC60 and RPV-Doc. The surfactants used for this study were Kolliphor ELP, Kolliphor PS20, Kolliphor PS80, Kolliphor HS 15, and Kolliphor RH40. Briefly, 100 mg of RPV-Doc:Labrafac MC60 (1 :2 w/w) mixture was combined with 100 mg of surfactant and the blend was heated at 70-80°C until a homogenous mixture was obtained. A portion of this isotropic mixture (10 mg) was weighed and diluted with 10 mL of distilled water to form a fine emulsion. After 2 h, the transmittance of the emulsion was measured at 638.2 nm using a UV-visible spectrophotometer with distilled water as the reference.
Screening of cosurfactants. A range of co-surfactants were evaluated for their ability to facilitate the formation of nanoemulsion. Briefly, 100 mg of RPV-Doc:Labrafac MC60 (1 :2 w/w) mixture, 50 mg of surfactant (selected from the previous screening), and 50 mg of each cosurfactant underwent homogenization through heating at 70-80°C. A portion of this isotropic mixture (10 mg) was weighed and diluted with 10 mL of distilled water to form a fine emulsion. Following a 2 h settling period, its transmittance was measured at 638.2 nm using a UV-visible spectrophotometer with distilled water as a reference.
Construction of the ternary phase diagram. We used our previously reported method 35 to construct the ternary phase diagram of RPV-Doc:Labrafac MC60 (oil phase), Kolliphor ELP (surfactant), and Transcutol HP (cosurfactant). As described previously, 48 compositions containing various ratios of oil, surfactant, and cosurfactant were generated. The weight percentage of the oil phase ranged from 25-75%, while that of surfactant and co- surfactant varied from 30-75% and 0-30% respectively. For each composition generated for the phase diagram, a portion (10 mg) was diluted with 10 mL of distilled water, and the globule size of the resulting dispersions was determined using dynamic light scattering (Litesizer 500 particle analyzer, Anton-Paar USA, Inc., Torrance, CA). The compositions that yielded nanoemulsion with an average globule size < 250 nm and uniform size distribution were identified. A ternary phase diagram highlighting the optimal RPV-Doc-MC60SNES compositions was generated using Origin Pro software.
Evaluation of the effect of pH of gastrointestinal fluids on the nanoemulsification of shortlisted SNES compositions. The ternary phase diagram identified seven optimal formulations suitable for further evaluation. To identify the SNES composition(s) that can withstand the variation in the gastrointestinal pH values, shortlisted compositions (10 mg each) were diluted with 10 ml of water, pH 1.2 buffer, pH 4.5 buffer, and pH 6.8 buffer. The globule size of the resulting dispersions was determined using dynamic light scattering (Litesizer 500 particle analyzer, Anton-Paar USA, Inc., Torrance, CA), and the composition that had the highest amount of oil phase and yielded a nanoemulsion in all dilution mediums irrespective of their pH value was selected for the PK and biodistribution studies.
Development and characterization of the SNES containing Capryol 90 and RPV- Doc (RPV-Doc-Cap90SNES). We evaluated the ability of previously reported SNES compositions containing Capryol 90 32, 35 to incorporate RPV-Doc and yield nanoemulsion irrespective of the pH of the dilution medium. Briefly, 428 mg of RPV-Doc and 1000 mg of Capryol 90 were dissolved in 1 mL of acetone which was followed by the evaporation of acetone using rotary evaporator and drying in a vacuum oven (Fisher Scientific, Waltham, MA) for 24 h. For the preparation of RPV-Doc-Cap90SNES, RPV-Doc-Capryol 90 mixture (125 mg), Kolliphor ELP (93.7 mg), and Capmul MCM (31.5 mg) were weighed in a microcentrifuge tube and homogenized with heating (70-75 °C). The RPV-Doc-Cap90SNES (50 mg) was diluted to 50 mL with distilled water, pH 1.2 buffer, pH 4.5 buffer, pH 6.8 buffer, or pH 7.4 buffer and vortexed at 1500 rpm for 1 min to yield nanoemulsion. The globule size and polydispersity index (PDI) of the nanoemulsion were evaluated using a Litesizer 500 particle analyzer (Anton-Paar USA, Inc., Torrance, CA). All batches were carried out in triplicate.
Size and morphology evaluation using transmission electron microscopy (TEM).
RPV-Doc-MC60SNES, or RPV-Doc-Cap90SNES were diluted with ultrapure Millipore water (5x and 2x respectively) and the diluted nanoemulsion (7.5 pl) was applied to copper grids (Electron Microscopy Sciences, PA, USA) and left to dry at room temperature overnight. Images were obtained at the Microscopy Core facility at the University of Arizona using a FEI Tecnai G2 Spirit BT TEM (FEI, Hilsboro, OR, USA) at an accelerating voltage of lOOkV. TIFF images were captured using an AMT XR41 side-mounted camera at various magnifications to obtain suitable TEM images. All samples were imaged within 24 hours of rehydration.
Pharmacokinetics and biodistribution studies. For the PK and biodistribution studies, uniform RPV suspension (RPV concentration: 2 mg/ml) was prepared using 1% hydroxypropyl methylcellulose (HPMC) dispersion. RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES were diluted with ultrapure water to obtain a nanoemulsion containing RPV-Doc equivalent to 2 mg/mL. of RPV. Briefly, B6 male mice were randomly divided into 3 groups (n > 4 mice per group). The mice received either 100 pL oral gavage of RPV suspension, RPV-Doc - MC60SNES, or RPV-Doc-Cap90SNES (RPV dose: 10 mg/kg). The blood samples were collected through the tail vein using heparinized capillary tubes at 0, 0.5, 1, 3, 6, 9, and 12 h. The collected blood was centrifuged at 10000 g and 4°C for 15 min and the recovered plasma samples were stored at -20°C until further analysis.
For the biodistribution studies, male mice were randomly divided into 6 groups (n > 3 mice per group). The mice received either 100 pL oral gavage of RPV suspension, RPV-Doc- MC60SNES, or RPV-Doc-Cap90SNES (RPV dose: 10 mg/kg). Tire mice were euthanized using carbon dioxide at 1 h or 6 h after drug administration. The blood was first collected from the heart, and various tissues such as the hrain, mesenteric lymph node (MLN), and testis were collected. All samples were stored at -80 °C until further analysis. The brain and testis were homogenized in 4x volumes of PBS buffer (pH=7.2) using a Polytron Bio-Gen Series PRO200 homogenizer (speed level: 3, duration: 5 seconds; 2 homogenization cycles). MLNs were homogenized in 100 pl PBS with a glass homogenizer by hand. The plasma samples (5 pl each) or tissue homogenates (10 pl each) were mixed with 50 pl methanol and 10 pl of internal standard (IS, d6-rilpivirine 100 ng/ml). The mixture was vortexed for 30 seconds, diluted with 950 pL Milli Q water, and loaded onto an OASIS® HLB cartridge (Cl 8, 1 ml/30mg, Waters Corporation, Milford, MA) pre-conditioned with 1 mL methanol followed by 1 mL water. The cartridge was then washed with 1 mL water and the analytes were finally eluted from the cartridges with 1 ml methanol for LC-MS/MS analysis.
LC-MS/MS analysis of RPV. The LC-MS system consisted of an Agilent 1290 UPLC system (Agilent Technologies, Santa Clara, CA) and a Sciex Qtrap6500+ Mass Spectrometer (AB SCIEX, Framingham, MA). Analytes were separated on an EclipsePlus Cl 8 column (2.1x50 mm, 1.8 pm, Agilent) at a temperature of 35 °C, with mobile phase A containing 0.1% formic acid (v/v) in water and mobile phase B containing 0.1% formic acid (v/v) in acetonitrile. Elution was at a flow rate of 0.2 mL/min as follows: 10% B (0-0.2 min), 10% B^95% B (0.2-5 min), 95% B (5-7 min), 95% B^10% B (7-7.1 min), 10% B (7.1-10 min). The MS was operated in the positive ion mode, using electrospray ionization. The ion spray voltage and temperature were set at 5000 V and 500°C, respectively. Curtain gas, ion source gas 1, and ion source gas 2 were set at 25, 50, 50 psi, respectively. RPV and the IS were detected using Multiple Reaction Monitoring (MRM), with a dwell time of 200 msec per transition, at m/z 367.1/224.1 (Collision Energy 38eV) and 373.1/230.1 (Collision Energy 40eV), respectively. Retention times for RPV and the IS were 3.53 min. For quantitative analysis of rilpivirine, the standards (10 to 10000 ng/mL in 5 pl methanol), ppi methanol), along with 10 pl IS (at 100 ng/mL in methanol), were added to 5 pl of blank mouse plasma to construct the calibration curve. The correlation coefficient of RPV in plasma was >0.99 across a range of 10-10,000 ng/ml. The limit of quantification (LOQ) was established at 10 ng/ml with a signal to noise ratio >10:1. The recovery was more than 85% under the current sample preparation method.
Data analysis. The pharmacokinetic parameters were calculated using PK solver (Microsoft, Redmond, WA) by assuming a noncompartmental model. The statistical analyses were carried out using GraphPad Prism 10 and the differences were deemed significant at P < 0.05. Example 2B - RPV showed extremely low solubility in aqueous vehicles and low solubility in lipid vehicles.
The saturation solubility studies showed that RPV was poorly soluble (< 1.5 pg/ml) in various aqueous vehicles (water and buffers) irrespective of the pH of the vehicle (Example_2_Table 1). RPV showed much greater solubility in lipid vehicles (< 3 mg/ml) compared to aqueous vehicles, but it was still insufficient to enable the development of oral lipid-based formulations.
Example_2_Table 1: Equilibrium solubility of RPV in water, different pH buffers, and oral bioavailability enhancing lipid vehicles. Data expressed as mean ± S.D.; n = 3; BLQ: Below the limit of quantification (50 ng/ml).
Figure imgf000095_0001
Example 2C - Only sodium docusate could convert RPV into amphiphilic IL, RPV docusate (RPV-Doc).
Pharmaceutically acceptable six anions; sodium docusate, sodium oleate, sodium lauryl sulfate, DHA, geranic acid, and linoleic acid (FIG. 8) were utilized to investigate the formation of RPV IL/salt/cocrystal. We force-ionized RPV in situ to produce RPV hydrochloride and followed our previously reported metathesis reaction protocol 32, 33 for the synthesis of RPV- docusate. For the synthesis of RPV lauryl sulfate (RPV-LS) and RPV oleate, we slightly modified the workup procedure whereas, for RPV-DHA, RPV geranate, and RPV linoleate synthesis, we first generated sodium salt of DHA, geranate and linoleate in situ and reacted the sodium salt with in-situ generated RPV hydrochloride under inert conditions in the dark to prevent potential oxidation of unsaturated fatty acids during the reaction. Following the screening of various organic solvents such as methanol, acetone, ethanol, dichloromethane, and chloroform; methanol was identified as the appropriate solvent for the metathesis reaction (data not shown). To remove sodium chloride formed during the metathesis reaction alongside unreacted water-soluble counterions, we conducted DCM/water extraction treatment. However, for RPV-docusate, additional ACN treatment was performed to eliminate unreacted sodium docusate. Among all the anions that were screened, only sodium docusate successfully converted RPV into viscous IL, RPV-Doc. Interaction between SLS and RPV resulted in a slightly waxy mass, while DHA produced a yellow turbid pasty mass. Sodium oleate, geranic acid, and linoleic acid, upon interaction with RPV, yielded a crystalline solid.
Example 2D - Spectroscopic studies on the developed RPV ILs/salts/cocrystals confirmed significant interactions between RPV and biocompatible anions.
We performed various spectroscopic analyses (FT-IR spectroscopy, and ]H & 13C NMR spectroscopy) to confirm the interaction between RPV and biocompatible anions (FIG. 9, FIG. 10, and FIG. 18-29). The purity of the synthesized RPV ILs/salts/cocrystals was confirmed by the HPLC (FIG. 30) and HR-MS (FIG. 31) analyses.
The FT-IR spectra of DHA, linoleic acid, oleic acid, and geranic acid displayed characteristic peaks of the carboxylic acid (-C=O) stretch in the range of 1732-1673 cm 1 and O- H (acid) bend in the range of 1457-1419 cm'1 whereas the spectrum of sodium docusate showed prominent peaks at 1209 to 1046 cm'1 corresponding to the -S=O stretch of the sulfonate group. The bands at 1216 & 1081 cm'1 in the SLS spectrum corresponded to the -S=O stretch of the sulfate group. The significant peak at 3315 cm'1 in the RPV spectrum was attributed to the -NH stretch of the secondary amine (FIG. 9). The FT-IR spectra of synthesized RPV ILs/salts/cocrystals exhibited noticeable shifts in the -NH stretch of the secondary amine of the RPV. Additionally, significant shifts and reduction in peak intensities for the carboxylic acid (- C=O) stretch and disappearance of O-H (acid) bending peak of DHA, linoleic acid, oleic acid, and geranic acid were observed in the FT-IR spectra of their corresponding RPV ILs/salts/cocrystals. Similarly, RPV-docusate and RPV-LS displayed significant shifts and reduced peak intensities for the -S=O stretch of docusate (sulfonate) and lauryl sulfate (sulfate), respectively. These changes in the FT-IR spectra of RPV ILs/salt/cocrystals indicate the electrostatic interactions between RPV and their respective counterions (FIG. 9).
The JH and 13C NMR spectra of RPV, RPV-docusate, RPV-oleate, RPV-LS, RPV geranate, RPV-DHA, and RPV-linoleate were analyzed to validate the interaction between RPV and anions (FIG. 10 and FIG. 18-29). The displacement in characteristic proton signals of RPV and counterions in their respective ILs/salts/cocrystals confirmed the interaction between RPV and counterions (FIG. 10). The NMR spectra of RPV-doc and RPV-LS revealed significant displacements in the -CH proton signal of the pyrimidine ring (adjacent to the tertiary amine [proton: a]) and displacement of the secondary amine proton signal (proton: b) of RPV (FIG. 10) whereas, RPV-DHA, RPV oleate, RPV geranate, and RPV-linoleate exhibited very slight displacements in the -CH proton signal of the pyrimidine ring (FIG. 10). RPV-DHA, RPV linoleate, and RPV oleate ]H NMR spectra showed a disappearance of the carboxylic acid proton signal whereas the RPV geranate showed a notable shift in the carboxylic acid proton signal (FIG. 10). The observed changes in proton signals particularly in the heterocyclic region, secondary amine of RPV, and carboxylic acid of counterions, strongly suggest the involvement of electrostatic interactions between RPV and its respective counterions.
The purity of the synthesized RPV-ILs/salts/cocrystals was confirmed using HPLC (FIG. 30). All RPV-ILs/salts/cocrystals showed HPLC chromatograms with only characteristic RPV peak indicating their purity. The HR-MS data further confirmed the purity of RPV- ILs/salts/cocrystals (FIG. 31 and FIG. 32).
Example 2E - Thermal characterization techniques and X-ray diffractometry studies confirmed that RPV-Doc is an ionic liquid.
Thermogravimetric analysis was carried out to determine the impact of the counterions on the thermal stability of RPV-ILs/salts/cocrystals (FIG. 11A-B). The 5% weight loss of RPV occurred at 328.34 °C (FIG. 11A; Example_2_Table SI). Among all RPV ILs/salts/cocrystals, RPV-Doc exhibited the highest thermal stability (Ts%=283.86°C), followed by RPV-LS, RPV- DHA, RPV-oleate, RPV-linoleate, and RPV geranate in the descending order (FIG. 11B). These findings from TGA data demonstrated the differential thermal stabilities of the synthesized RPV ILs/salts/cocrystals, compared to pure RPV and their corresponding counterions.
Example_2_Table SI: Globule Size and Polydispersity Index of RPV-Doc:Labrafac MC60 containing SNES at different pH conditions*
Figure imgf000097_0001
*32.5 mg of isotropic mixture of RPV-Doc :Labrafac MC60, Kolliphor ELP & Transcutol (46:39: 15) was diluted to 1.163 mL water and different buffer systems and vortexed on vortex mixer at 1500 rpm for 1 min. The concertation of RPV in the formed emulsion was 2 mg/mL. The formed emulsions were diluted 15 folds with corresponding solvents to check globule size, (data expressed as mean + S.D.; n = 3). The differential scanning calorimeter (DSC) thermograms of RPV and its ILs/salts/cocrystals are shown in FIG. 11C. The DSC thermogram of RPV exhibited a sharp melting endotherm at 229.3 °C, confirming its crystalline nature. However, in the DSC thermogram of various RPV ILs/salts/cocrystals, the disappearance or shift in the melting endotherm of RPV was seen indicating the alterations in its crystal lattice due to the interaction between RPV and anions. RPV-Doc displayed the complete amorphization of RPV, as evidenced by the absence of the RPV melting endotherm in their respective DSC thermograms. Interestingly, RPV-LS exhibited a Tg at 64.72 °C, a small melting endotherm at 116.64 °C (FIG. 33), and a sharp endotherm at 222.4 °C, corresponding to the degradation of the RPV which was consistent with the TGA data of RPV-LS (FIG. 11 A-B). In RPV-DHA, a noticeable endotherm was observed at 100.6°C followed by an exothermic event likely attributed to the initiation of degradation. The DSC thermograms of RPV linoleate, and RPV oleate showed melting endotherms at 107.3°C & 140.9°C, respectively. RPV geranate, on the other hand, exhibited a degradation peak at 137.7°C. The alteration in melting endotherms compared to pure RPV suggests the distortion of the crystalline lattice of RPV and the formation of partially amorphous salts or cocrystals (discussed later). Additionally, the changes in the baseline after the melting endotherm indicate the degradation occurring within these samples. Next, we determined the glass transition temperature (Tg) of RPV-Doc using modulated DSC. The Tg of RPV-Doc was observed at 17.74°C (FIG. 1 ID).
The X-ray diffractogram of RPV and its ILs/salts/cocrystals are shown in FIG. 12. The sharp diffraction peaks at 20 values of 17.5°, 20.85°, and 27.75° in the RPV diffractogram confirmed its crystalline behavior. The absence of diffraction peaks in the RPV-Doc diffractogram signified the successful conversion of RPV into amorphous IL by sodium docusate. Similarly, the RPV-LS diffractogram showed a distinct and altered diffraction pattern compared to pure RPV, indicating changes in its crystal lattice. Finally, RPV-DHA, RPV linoleate, RPV oleate, and RPV geranate displayed reduced peak intensities compared to RPV, suggesting their partial amorphization.
Example 2F - RPV ILs/salts/cocrystals showed greater LogP and improved solubility characteristics compared to pure RPV.
The experimental LogP values of the RPV and its ILs/salts/cocrystals are presented in Example_2_Table 2. The RPV-Doc showed the highest LogP value (~2.4-fold higher LogP value compared to RPV) followed by RPV geranate, RPV linoleate, RPV-DHA, RPV-LS, and RPV oleate in the descending order (Example_2_Table 2).
Example_2_Table 2: Experimental LogP values of RPV, and its ILs/salts/cocrystals.
Figure imgf000099_0001
We further evaluated the impact of counterions used for the development of RPV- ILs/salts/cocrystals on the solubility characteristics of RPV using aqueous media (Example_2_Table 3). All RPV-ILs/salts/cocrystals improved the water solubility of RPV with RPV geranate showing the highest solubility increase.
Example_2_Table 3: Equilibrium solubility of RPV and its ILs/salts in water and different buffers.
Drug/IL/salt/ Solubility (pig/mL; Data expressed as mean ± S.D.; n = 3) cocrystal . Water . Buffer pH L2. Buffer ph 4.5. Buffer'pH 6.8.
RPV-LS 0.84 ± 0.4 17.49 ± 0.21 0.13 ± 0.11 1.21 ± 0.02
RPV linoleate . 0.20 ± 0^27.14.63 ± 0d 3. 6.09 ± 0. 045 ± 6 01.
RPV-DHA 0.54 ± 0.08 67.95 ± 0.41 0.29 ± 0 6.74 ± 0.08
RPV docusate 0.13 ± 0.1 4.34 ± 0.07 0.10 ± 0.03 0.08 ± 0.01
RPV oleate 676'6 "± 6.01 51.56 ± 2.45 0.06 + 0.01. BLQ
RPV geranate . 5724 + 6.12.8.22 ± 0709. 2.04 "±0.6'6. BLQ .
BLQ: Below the limit of quantification (50 ng/ml)
The kinetic solubility of RPV-ILs/salts/cocrystals in the bioavailability-enhancing lipid vehicles is shown in Example_2_Table 4. Compared to pure RPV (Example_2_Table 1), all RPV ILs/salts/cocrystals exhibited improved solubility in lipid vehicles used for SNES development. Among the synthesized RPV ILs/salts/cocrystals, compared to RPV (Example_2_Table 1), RPV-Doc showed the highest solubility in lipid vehicles, with ~100-fold improvement in Capryol 90, and Peceol, and an impressive ~250-fold enhancement in Labrafac MC60 & Capmul MCM Cs. RPV-DHA, RPV linoleate, RPV geranate, and RPV oleate also displayed higher lipid solubility. RPV-LS showed the lowest solubility in all the selected lipid vehicles (<50 mg/mL).
Example 2 Table 4: Kinetic solubility RPV ILs/salts/cocrystals in lipid Vehicles (data presented as an average of 3 independent solubilization experiments)
Figure imgf000100_0001
Example 2G - RPV ILs/salts/cocrystals showed no changes in the antiretroviral activity of RPV in vitro.
We evaluated the impact of transforming RPV into RPV ILs/salts/cocrystals on the inherent antiretroviral activity of RPV using TZM-bl assay. As shown in FIG. 13, the transformation of RPV into RPV ILs/salts/cocrystals did not impact the antiretroviral activity in vitro.
Example 2H - SNES containing RPV-Doc and Labrafac MC60 could be developed.
Our studies showed that RPV-Doc has the highest solubility in Labrafac MC60 (glycerol monocaprylocaprate; HLB:5). We used our previously developed protocol to develop SNES Labrafac MC60 and RPV-Doc. The surfactant screening studies indicated that Kolliphor ELP (PEG-35-castor oil) is most suitable for the emulsification of Labrafac MC60:RPV-Doc mixture (FIG. 14A). During the assessment of different cosurfactants, PEG400, Capmul MCM Cs, Labrafil M 1944 CS, Lauroglycol FCC, and Transcutol HP yielded dispersions with the highest % transmittance values indicating their suitability as a cosurfactant. (FIG. 14B). We chose Transcutol HP as the cosurfactant for further development. As described in our previous paper 35, we developed a ternary phase diagram (FIG. 14C) using Labrafac MC60: RPV-Doc mixture as an oily phase, Kolliphor ELP as a surfactant, and Transcutol HP as a cosurfactant to identify optimal compositions that will yield nanoemulsion of globule size < 250 nm and acceptable homogeneity. The red region in the ternary phase diagram (FIG. 14C) corresponds to the compositions that were deemed suitable for further development. From this region, we selected 7 different compositions (FIG. 14D) to further evaluate their ability to yield nanoemulsion irrespective of the pH of the dilution medium. As anticipated, all compositions yielded a stable nanoemulsion (< 250 nm) in water (FIG. 14E) but depending on the SNES composition, the pH of the dilution medium showed a differential impact on the globule size of the nanoemulsion (FIG. 14E). Based on this study, composition R3 which yielded nanoemulsions with acceptable size and low polydispersity index irrespective of the pH of the dilution medium was selected for further evaluation.
Example 21 - RPV-Doc could be successfully incorporated into the previously reported SNES composition containing Capryol 90.
We evaluated the ability of our previously reported SNES composition containing Capryol 90, Kolliphor ELP, and Capmul MCM 32, 35 to incorporate RPV-Doc. Due to the appreciable solubility of RPV-Doc in Capryol 90, it could be easily incorporated into our previously reported SNES composition (RPV-Doc-Cap90SNES). The RPV-Doc-Cap90SNES formed a nanoemulsion (size <100 nm) when mixed with water or various buffers with different pH values (Example_2_Table 5). The pH of the dilution medium impacted the size of the nanoemulsions but the resultant nanoemulsions were found to be optimal for further evaluation.
Example_2_Table 5: Globule Size and Polydispersity Index (PDI) of RPV-Doc- Cap90SNES when diluted with different media (data expressed as mean ± S.D.; n = 3).
°T!Ol“tlOn Water Buffer pH 1.2 Buffer pH 4.8 Buffer pH 6.8 medium r r r
Globule size (nm) 54.11 ± 1.52 97.2 ± 3.32 68 ± 1.1 23.37 ± 0.1
PDI 0.27 ± 0.01 0.18 ± 0.02 0.24 ± 0.01 0.14 ± 0.01 Example 2J - TEM analysis confirmed the formation of lipid nanoemulsion after dilution of RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES. We wanted to confirm the formation of lipid nanoemulsion upon dilution of RPV-Doc-Cap90SNES as well as RPV-Doc-MC60SNES with water. The TEM images (FIG. 15A-B) corroborated the globule size data obtained from the dynamic light scattering and showed the spherical morphology of the nanoemulsions.
Example 2K - Oral administration of RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES significantly increased the oral bioavailability of RPV and its delivery to HIV sanctuary sites such as mesenteric lymph nodes (MLNs), brain, and testis.
Based on the previous reports on the oral pharmacokinetics of RPV I K 19, we decided to evaluate the PK and biodistribution of RPV when administered at a dose of 10 mg/kg. To achieve this dose in 0.1 ml of aqueous vehicle to facilitate oral administration to B6 mice, the composition of RPV-Doc-MC60SNES had to be slightly modified from composition R3 (FIG. 14D). The RPV-Doc-MC60SNES used for the PK and biodistribution studies contained RPV- Doc:Labrafac MC60:Kolliphor ELP:Transcutol HP (46:39: 15% w/w ratio). Before proceeding to the PK and biodistribution studies, we ensured that this composition yielded nanoemulsion with optimal properties irrespective of the pH of the dilution medium (Supplementary Table SI). No such adjustments were necessary for the RPV-Doc-Cap90SNES composition. Interestingly, RPV-Doc-MC60SNES as well as RPV-Doc-Cap90SNES showed quick absorption of RPV with (Tmax) at 1.5 h and 0.67 h, respectively, and significantly higher (~ 6-fold) plasma RPV concentration (Cmax) compared to RPV suspension (FIG. 16, Example_2_Table 6). Both SNES formulations also resulted in significantly higher AUC values (~ 5 to 6-fold) compared to RPV suspension (Example_2_Table 6) indicating the advantage of our approach. We noted some differences in the PK of RPV delivered via RPV-Doc-MC60SNES or RPV-Doc-Cap90SNES but the differences were not statistically significant.
Example_2_Table 6: Pharmacokinetic parameters obtained after oral administration of RPV suspension, RPV-Doc-MC60SNES, or RPV Doc-Cap90SNES to B6 mice (data expressed as mean ± S.D.; n >5).
_ Mean±SD _ Parameter Unit RPV RPV-Doc- RPV-Doc- suspension MC60SNES Cap90SNES Cmax ng/ml 1 ,062±496 6,582±2,070 6,490±1 ,716
Figure imgf000103_0001
We also evaluated the ability of RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES to improve the delivery of RPV to the HIV sanctuary sites such as the MLNs, brain, and testis. Interestingly, both RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES showed significantly higher (approximately 7 to 10-fold) RPV levels in the MLNs and brain at a 1-hour time point compared to RPV suspension (FIG. 17). The RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES maintained considerably higher RPV concentration in the MLNs and brain even at 6 hours but the differences were significant only for RPV concentrations in the brain at 6 h for RPV-Doc- Cap90SNES (FIG. 17). RPV-Doc-MC60SNES and RPV-Doc-Cap90SNES also yielded considerably higher RPV concentrations in testis at 1 hour and 6 hours compared to RPV suspension but the differences were not significant. Although not statistically significant, RPV- Doc-Cap90SNES appeared to maintain higher RPV concentrations in different tissues at 6 hours compared to RPV-Doc-MC60SNES.
Example 2L - Discussion of Examples 2A-2K
Given the need for lifelong antiretroviral therapy for HIV management, irrespective of the route of administration of antiretroviral drugs, it is critical to achieve a maximum antiretroviral effect in the plasma and HIV sanctuary sites at a lower therapeutic dose to minimize the healthcare cost and drug- and/or dose-related long-term side effects. For example, it is estimated that a 33% improvement in oral bioavailability of tenofovir disoproxil fumarate (therapeutic dose reduction from 300 mg to 225 mg) will save $50-75 million in healthcare costs annually.36 It is noteworthy that several existing antiretroviral drugs including RPV have low aqueous solubility and/or permeability which result in low and/or variable oral bioavailability and penetration into HIV sanctuary sites.5 Hence, developing new oral delivery strategies to optimize the utilization of antiretroviral drugs is highly desirable.
RPV is a potent NNRTI that is now commonly used in combination with dolutegravir as a part of the 2-drug regimen (Juluca®). Additionally, RPV can also be used as a part of the first- line antiretroviral therapy for HIV-infected patients with plasma HIV RNA less than 100,000 copies/ml. While the low therapeutic dose (25 mg) and once-daily administration highlight the advantages of RPV-based regimens, current oral RPV formulations result in highly variable PK and suboptimal therapeutic RPV levels in 20-30% of patients eventually leading to treatment failure.8, 16, 17 It is noteworthy that the absolute oral bioavailability of RPV in humans is unknown. Moreover, previous studies have also shown that RPV has limited penetration into various HIV sanctuary sites such as MLNs, and the brain.18, 19 A few studies have examined the potential of cyclodextrin nanosponges to improve the oral bioavailability of RPV 37 ’ 38 but these systems were not evaluated for the delivery to the HIV sanctuary sites. Our studies show that RPV has very low solubility in water and buffers representing pH in the gastrointestinal environment. Previous studies show that fatty meals can improve oral bioavailability of RPV 39 indicating the potential of oral lipid-based formulations. However, our studies showed that RPV has low solubility in lipids which precludes the development of oral lipid-based formulations. RPV is a classic example of crystalline hydrophobic ionizable drugs with low solubility in water and lipids.
Transformation of hydrophobic ionizable active pharmaceutical ingredients (APIs) into amphiphilic API-based ionic liquids (API-ILs) with high lipid solubility and subsequent incorporation of these amphiphilic API-ILs into translational oral lipid-based formulations such as self-nanoemulsifying systems (SNES) is an emerging delivery strategy to improve oral delivery of drugs. We and others have successfully used this strategy for improving oral delivery of various hydrophobic ionizable drugs such as itraconazole, lumefantrine, anthelmintic benzimidazoles, and investigational antiviral compounds such as BX795.29-33 However, to date, the ability of SNES containing API-ILs to improve the tissue penetration of drugs has not been demonstrated. Hence, in this investigation, we focused on developing SNES containing RPV- IL(s) and evaluated the ability of this innovative approach to improving the oral bioavailability of RPV and its delivery to the HIV sanctuary sites (a measure of tissue penetration capability).
In the experiments described in Example 2 (Examples 2A-2K), we wanted to evaluate the ability of several anionic bulky counterions with different chemical structures, pKa values, and anionic functional groups to form ionic liquids (ILs) with RPV, highly crystalline hydrophobic weak base (pKa: 5.6). We selected sodium docusate (pKa: -0.75) and sodium lauryl sulfate (pKa: -1.5) as strongly acidic anions with sulf onate/sulf ate functional groups for IL formation. Additionally, we evaluated the potential of anionic unsaturated fatty carboxylic acids such as oleic acid (pKa: 5), linoleic acid (pKa: 4.77), docosahexaenoic acid (DHA; pKa: 4.89) and geranic acid (pKa: 5.26) for the formation of ILs. While geranic acid-based ILs such as choline geranate has been widely used for drug delivery 40-42, no reports exist on the development of API-ILs containing geranic acid as a counterion. In this investigation, we evaluated the ability of geranic acid to form ILs with a weakly basic drug.
Similar to our previous reports on ILs of weakly basic drugs 32, 33 , to maximize the ionic interaction between RPV and bulky anions, we generated RPV hydrochloride salt in situ and used sodium salt of fatty anions. The geranic acid, DHA, and linoleic acid, were converted to sodium salt in situ and then interacted with RPV hydrochloride. Using our modified salt metathesis reaction, RPV-Doc was obtained as a viscous liquid, RPV-LS and RPV-DHA were obtained as soft solids, and RPV oleate, RPV geranate, and RPV linoleate were obtained as solids. The spectroscopic studies confirmed the ionic interaction between RPV and various bulky anions. Notably, docusate and lauryl sulfate, due to their bulkier nature and strong acidity, led to a pronounced displacement of the -CH proton signal of the pyrimidine ring of RPV as well as the secondary amine proton signals of RPV in the 1 H NMR spectra of RPV-Doc and RPV-LS.
However, based on the DSC and XRD data, only RPV-Doc proved to be an ionic liquid. RPV-LS showed partial crystallinity in the XRD and due to the ApKa > 2 between RPV and lauryl sulfate, RPV-LS could be regarded as partially amorphous lipophilic salt. The PXRD spectra of RPV oleate, RPV geranate, RPV-DHA, and RPV linoleate showed crystalline or partially crystalline nature and the DSC thermograms showed melting endotherms that were distinctly different than RPV. However, the ApKa between RPV and DHA/oleic acid/linoleic acid/geranic acid are considerably lower than 1. Hence, in our opinion, RPV oleate, RPV linoleate and RPV geranate and RPV-DHA should be considered as lipid cocrystals. Previously, fatty acids, under right conditions, have been shown to form lipid cocrystals with weakly basic drugs such as itraconazole.43- 44
Our previous work showed that the counterions used for the synthesis of ILs/lipophilic salts could impact the physicochemical and biological properties of API-ILs.31-33 As anticipated, RPV-IL/lipophilic salt/lipid cocrystals showed considerably different aqueous solubility and apparent LogP values compared to pure RPV. Furthermore, compared to pure RPV, RPV- IL/lipophilic salt/lipid cocrystals also showed considerably higher kinetic solubility in the lipids used for the SNES development with RPV-Doc showing solubility as high as -500 mg/g. While the high apparent LogP value (~ 2.9) could be the reason for the highest lipid solubility, this correlation was not applicable to the other RPV lipophilic salt/lipid cocrystals. The PXRD studies showed that RPV-Doc was amorphous whereas RPV-LS, RPV-DHA, RPV linoleate, RPV oleate and RPV geranate were completely or partially crystalline and this may he the reason for their low kinetic solubility in the lipids. The chemical structure and molecular volume of the lipids also showed impact on the kinetic solubility of RPV-IL/lipophilic salt/lipid cocrystals. The lipids with higher molecular volume and long-chain fatty acid esters such as glyceryl monooleate (Peceol) were not efficient in solubilizing RPV-IL/lipophilic salt/lipid cocrystals compared to lipids containing esters of short-chain fatty acids (Capryol 90, Labrafac MC60, Labrasol, and Campul MCMCs). Indeed, the high lipid solubility of RPV-Doc opened the possibility of developing oral lipid-based formulations such as SNES. Finally, we confirmed that the transformation of RPV into RPV-IL/lipophilic salt/lipid cocrystals did not alter the inherent antiretroviral activity of RPV in vitro.
Oral lipid-based formulation strategies such as self-nanoemulsifying systems have shown significant promise in improving the oral delivery of hydrophobic drugs such as cyclosporine A (Sandimmune® Neoral, Gengraf®,), saquinavir (Fortovase®) and tipranavir (Aptivus®). SNES is an anhydrous isotropic mixture containing oil, surfactant(s), co-surfactants, and drug in a suitable proportion which can be incorporated into hard gelatin or soft gelatin capsules for oral delivery. SNES composition is optimized to form an oil-in-water nanoemulsion upon dilution with the aqueous phase upon gentle agitation. Upon oral consumption, the motility of the gastrointestinal tract provides the necessary agitation for transforming SNES into nanoemulsions.23’ 24 Previous studies have shown that oral absorption and the pharmacokinetic parameters of drug encapsulated into the lipid nanoemulsion will be impacted by the composition and globule size of the oral nanoemulsion.45"47 Hence, we decided to develop two SNES compositions containing RPV-Doc that have different compositions and yield nanoemulsion of different sizes. To develop two different SNES compositions, we chose Capryol 90 (propylene glycol monocaprylate) or Labrafac MC60 (glyceryl mono and dicaprylocaprate) as the lipid phase of the SNES. Capryol 90 and Labrafac MC60 are both known oral bioavailability enhancers and showed the ability to solubilize a high amount of RPV- Doc.
We used the previously reported SNES composition containing Capryol 90 with suitable modifications to develop SNES containing RPV-Doc (RPV-Doc-Cap90SNES) and the RPV- Doc-Cap90SNES yielded nanoemulsion less than 100 nm irrespective of the pH of the dilution medium. Thus far, no SNES compositions have been reported for Labrafac MC60. Hence, we focused on the systematic development of SNES compositions containing Labrafac MC60 and RPV-Doc. We previously reported a simple % transmittance-based method to screen surfactants and cosurfactants 35 for the development of SNES containing oral bioavailability-enhancing lipid and drug. We used the same method to identify components of the SNES containing Labrafac MC60 and RPV-Doc. Our previous studies showed that incorporation of drug into the SNES compositions significantly affects the phase behavior and size of the nanoemulsions. Hence, we used Labrafac MC60 containing solubilized RPV-Doc as a lipid phase to screen surfactants and cosurfactants for the SNES development. Our systematic % transmittance-based screening studies identified Kolliphor ELP (PEG-35-castor oil) and Transcutol HP (diethyleneglycol monoethyl ether) as surfactant and cosurfactant respectively. Kolliphor ELP and Transcutol HP are well-known pharmaceutical excipients which are used in FDA-approved pharmaceutical products. We constructed a ternary phase diagram to identify SNES compositions containing RPV-Doc that yield uniform nanoemulsions of size < 250 nm and several compositions met our preliminary screening criteria. However, we selected a composition that 1) contained a higher amount of oral bioavailability enhancing Labrafac MC60 and 2) yielded uniform and stable nanoemulsion in buffers representing different pH values of the gastrointestinal tract. The optimized RPV-Doc-MC60SNES yielded nanoemulsion with size < 250 nm irrespective of the pH of the dilution medium.
Further, we evaluated the pharmacokinetics and biodistribution of RPV-Doc - Cap90SNES and RPV-Doc-MC60SNES in mice to evaluate the promise of our unique approach and to evaluate the impact of the SNES composition on the PK and biodistribution of RPV. Interestingly, RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES both yielded rapid and significantly higher oral absorption and bioavailability of RPV (~ 6-fold higher Cmax and AUC) compared to pure RPV suspension (FIG. 16) indicating the promise of our approach. The Cmax and AUC values achieved with RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES were not significantly different. However, RPV-Doc-Cap90SNES resulted in considerably quicker absorption (tmaX: ~ 0.7 h) compared to RPV-Doc-MC60SNES and RPV suspension (Example_2_Table 6). This difference could be due to the lower globule size (< 100 nm) of nanoemulsion obtained upon the dilution of RPV-Doc-Cap90SNES compared to RPV-Doc- MC60SNES which yielded nanoemulsion with size < 250 nm. The greater oral bioavailability seen for RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES is likely due to the uniform and gastrointestinal pH-independent solubilization of and delivery of RPV across the gastrointestinal tract. Additionally, due to the modification of RPV into RPV-IL and its subsequent incorporation into SNES, the RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES likely showed considerably lower clearance compared to pure RPV suspension.
Several studies have demonstrated that within the first few weeks after HIV infection, the virus disseminates throughout the body including the various cellular and anatomical HIV reservoir sites such as CD4+ T cells, macrophages, central nervous system (CNS), male and female reproductive tracts, gut- associated lymphoid tissue (GALT), secondary and lymph nodes.48-51 While the antiretroviral (ARV) drugs and their delivery systems have evolved remarkably to yield undetectable plasma viral load in patients, they are unable to attain therapeutic ARV concentrations in the tissues that have the potential to harbor the virus.48-51 Hence, the replication-competent virus is persistently seen in the GALT, CNS, and reproductive organs in patients on suppressive ART therapy and upon interruption in the ART regimen, viral rebound is observed. There is a dire need to develop strategies that can augment the penetration of ARV drugs into HIV reservoirs/sanctuary sites to improve the treatment outcomes in the patients. Previous studies have shown that RPV has suboptimal penetration into MLNs and the brain when delivered orally.18' 19 Hence, in this investigation, we also evaluated the ability of RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES to improve the delivery of RPV to various HIV sanctuary sites.
Interestingly, both RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES were able to considerably increase the delivery of RPV to HIV sanctuary sites such as MLNs, brain, and testis, and the higher levels were retained for at least 6 hours (FIG. 17). At 1-hour time point, RPV-Doc-Cap90SNES and RPV-Doc-MC60SNES resulted in significantly greater RPV levels (~ 7 to 10-fold) in the MLNs and brain compared to pure RPV suspension, and the higher RPV levels were maintained up to 6 hours (FIG. 17). The RPV-Doc-Cap90SNES and RPV-Doc- MC60SNES both augmented the delivery of RPV to testis compared to RPV suspension but the differences were not significant for both time points. The amount of RPV delivered to MLNs, brain, and testis was not significantly different between RPV-Doc-Cap90SNES and RPV-Doc- MC60SNES although higher variability was observed for RPV-Doc-MC60SNES. The ability of RPV-Doc-Cap90SNES as well as RPV-Doc-MC60SNES to significantly enhance RPV levels in the MLNs indicates that intestinal lymphatic transport may have played a significant role in improving oral bioavailability of RPV and its delivery to the HIV sanctuary sites. Additionally, RPV-Doc, due to its greater amphiphilicity may have greater tissue penetration compared to pure RPV. However, the detailed mechanism responsible for greater bioavailability and tissue penetration of RPV seen with RPV-Doc-Cap90SNES as well as RPV-Doc-MC60SNES needs further exploration. Our future efforts will focus on exploring the absorption mechanisms of orally delivered APLILs with and without SNES.
Hydrophobic ionizable drugs with poor water and lipid solubility such as RPV can be successfully transformed into amphiphilic IL with remarkably high lipid solubility with the help of sodium docusate, a strongly acidic amphiphilic anion with sulfonate functional group. RPV docusate could be efficiently incorporated into self-nanoemulsifying systems that contained bioavailability-enhancing lipids. The SNES containing RPV docusate showed dramatic improvement in the oral bioavailability of RPV and its delivery to the HIV sanctuary sites. This unique delivery strategy will eventually result in a reduction in the pharmacokinetic variability and the therapeutic dose of RPV leading to optimal management of HIV infection.
The following references pertain to the numerical reference citations recited in Example 2:
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Figure imgf000109_0001
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Example 3 - Ionic Liquid Formulations including Adefovir Dipivoxil
HSV can cause recurrent and lifelong ocular infection leading to HSV keratitis and ocular HSV infection is the most common cause of infection-associated vision loss worldwide.1’ 4 Ocular HSV infections are estimated to affect ~ 50000 individuals every year in the United States.5 While HSV can infect different regions in the eye leading to a variety of complications, HSV epithelial keratitis is the most common herpetic eye disease worldwide including the US and it is characterized by the active viral infection in the corneal epithelium.1’7
Although oral therapy with nucleoside analogs such as acyclovir, valacyclovir, penciclovir, and famciclovir is effective for the treatment of ocular HSV- 1 infection, the emergence of drug resistance and severe nephrotoxicity leading to acute renal failure in some cases make them less than ideal.8’14 Topical ocular therapy with 0.15% ganciclovir gel and 1% trifluridine eye drops is also effective for HSV epithelial keratitis but these ocular formulations need to be delivered 5- to 9-times daily for the treatment of ocular HSV-1 infections, which significantly affect patient compliance and treatment outcome.14 16 Additionally, the emergence of drug-resistant strains further limits the use of these existing antiviral drugs.17 Hence, there is a dire need to develop new therapies for the treatment of ocular herpes infections.
There is a resurgence in the repurposing of FDA-approved acyclic nucleoside phosphonates (ANPs) for the prevention and/or treatment of HSV infections. ANPs are a class of broad-spectrum antiviral drugs that are active against a variety of DNA viruses including HSV and retroviruses. ANPs contain a phosphonate (FIG. 34) stably attached to the acyclic nucleoside.18-23 As the chemical structure of ANPs mimics the monophosphorylated nucleosides, the ANPs bypass the critical step of viral kinase-mediated activation and the host cellular kinases transform ANPs into triphosphate mimicking ANPs eventually leading to the inhibition of viral DNA polymerase.18-23 Currently, ANPs such as cidofovir, adefovir, and tenofovir or their prodrugs, adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are used for the treatment of various viral infections but not for HSV infections. The repurposing of FDA-approved ANPs for the prevention/therapy of HSV infections gained momentum when 1 % tenofovir vaginal gel unexpectedly showed -51% prevention of the acquisition of HSV-2 in CAPRISA-004 clinical trial but limited therapeutic efficacy in the patients with recurrent GH in another clinical trial.24,25 It is well documented that tenofovir has low potency against HSV-1 and would not be useful for ocular herpes therapy. However, adefovir has at least 10-fold higher potency against HSV compared to tenofovir26 but like tenofovir, its extreme hydrophilicity and low tissue permeability27-30 can further limit its potential for ocular delivery.
The pivalic acid ester prodrug is used for ocular delivery. Dipivefrin, a pivalic acid ester prodrug (FIG. 35) was developed to improve the ocular tissue permeability of epinephrine, a polar molecule with limited permeability31 and this prodrug is commercially available as Propine® eye drops indicating the potential of pivalic acid ester prodrugs in ocular delivery. ADV, is an FDA-approved pivalic acid prodrug which is currently approved for the treatment of chronic hepatitis B infection. Our preliminary studies showed that ADV has remarkably higher in vitro antiviral activity (FIG. 36) compared to adefovir (AFV) and acyclovir (ACV). Hence, ADV can be repurposed for ocular herpes therapy.
Poloxamer 407-coated mucus-penetrating ocular nanosuspensions have been translated to the clinic. The presence of the mucin layer in the tear film poses a significant barrier to the ocular delivery of various drugs. The mucin layer, due to its complex mesh-like structure, can trap foreign molecules including drugs and particles due to adhesive interactions; thus, preventing effective delivery to the corneal epithelium. Poloxamer 407 is an FDA-approved amphiphilic polymeric surfactant approved for oral, parenteral, and topical (vaginal, dermal, and ocular) routes.32,33 Our previously published studies showed that Poloxamer 407 coated nanosuspensions (NS) of size ~ 200 nm can penetrate through the mucin layer present on various mucosal surfaces including the eye.34 39 A previous study showed that the transformation of loteprednol etabonate to mucus-penetrating loteprednol etabonate NS resulted in a reduction in therapeutic dose as well as dosing frequency.40 This mucus-penetrating strategy culminated in the translation and FDA approval of two eye drop products (EYsuVIS® and INVELTYS®) containing Poloxamer 407-coated loteprednol etabonate NS for the treatment of dry eye symptoms and/or ocular inflammation.40,41 Thus, mucus-penetrating NS technology has tremendous potential for ocular drug delivery of hydrophobic drugs, but it is difficult to apply this technology to improve ocular delivery of water soluble drugs such as ADV.
Transformation of hydrophilic ionizable drugs into lipophilic salts or ionic liquids (ILs) is a pharmaceutically viable strategy to improve their transformation into nanoformulations and delivery. Many pharmaceutical drugs including those present in the clinically approved topical eye drops are available as water-soluble salts42-45 and in the context of ocular delivery, these water soluble salts often show increased systemic absorption and rapid drug elimination.44,45 Water soluble drug salts are also difficult to formulate into nanoparticles or other sustained release drug delivery systems. Conversion of water soluble salts into water insoluble lipophilic salts using pharmaceutically acceptable bulky counterions is a clinically validated approach to sustain the drug release which led to the approval of Zyprexa® Relprevv (olanzapine pamoate).46-48 ILs are organic salts with a melting point of < 100°C and depending upon the cations and anions involved in the formation, ILs can even be liquid at room temperature. Transformation of highly water soluble salts such as metformin hydrochloride into metformin docusate, a lipophilic IL (FIG. 37) using pharmaceutically acceptable fatty anions is a novel and pharmaceutically viable strategy to improve their permeability, bioavailability, and efficacy.49,50 Furthermore, a transdermal anesthetic patch containing IL of lidocaine has successfully completed the Phase III clinical trial51 and is waiting for approval indicating the pharmaceutical viability of the IL approach.
Experiments were conducted with the proposal to develop lipophilic salt(s) and lipophilic IL(s) of ADV with pharmaceutically acceptable fatty anions to reduce water solubility and prolong the release of ADV and subsequent transformation of ADV lipophilic salt/ ADV lipophilic IL into mucus-penetrating nanoformulations to achieve enhanced ocular pharmacokinetics (PK) and prolonged ocular herpes therapy. Example 3A - Adefovir dipivoxil (ADV) showed ocular safety and higher activity against HSV compared to other acyclic nucleoside phosphonates (ANPs), their prodrugs, and ACV.
We first evaluated the cytocompatibility of ADV to human corneal epithelial cells (HCE) and our in vitro studies showed that ADV was well tolerated by the HCE even at 100 pM (FIG. 38) indicating the potential of ADV for ocular delivery.
Our preliminary studies, carried out at the multiplicity of infection (MOI) of 0.1, showed that ADV has significantly higher in vitro antiviral activity against HSV-2 in HeLa cells compared to adefovir and ACV (FIG. 36). We further evaluated the in vitro antiviral activity of ADV, other ANPs, their prodrugs, and ACV in HCE at a much higher viral titer of HSV-1 (MOI of 1) to identify the most active candidate. Interestingly, even at such a level of viral infection, ADV showed 1- to 1.5 log-fold lower intracellular HSV-1 (FIG. 39A) compared to adefovir, other ANPs, their prodrugs, and ACV indicating its promise as an antiviral drug for the treatment of ocular HSV infection. In a separate study, we used a recombinant HSV-1 (KOS)tkl2 at 0.1 MOI to infect HCE and the cells were treated with various concentrations of ANPs, prodrugs, and ACV. Interestingly, only ADV, even at a very low concentration of 0.39 uM showed a > 65% reduction in viral titer compared to untreated control (FIG. 39B). Taken together, among ANPs and their prodrugs, only ADV is the most effective antiviral agent that has the potential for the topical treatment of ocular HSV infections.
Example 3B - Topical administration of 1 % w/v ADV solution showed robust antiviral efficacy in a mouse model of ocular herpes infection.
Our preliminary data show that topical (ocular) delivery of 1 % w/v ADV solution (3 times a day) showed significant antiviral activity on days 2- and 4-post HSV-1 infection in a murine model of ocular HSV-1 infection (FIG. 40). Thus, ADV can be repurposed for topical therapy of ocular HSV-1 infection. However, strategies are needed to alter the high water solubility of ADV to yield long-acting ocular formulations to achieve once-daily therapy of ocular HSV infections.
Example 3C - Moxifloxacin hydrochloride (MOX), the water soluble antibiotic salt, can be converted to a lipophilic salt, MOX pamoate (MOX-PAM) which can be further transformed into a mucus-penetrating nanosuspension (NS).
Commercial MOX eye drops (Vigamox®) must be administered at least 3 times daily to be effective. MOX, being a water soluble salt, is rapidly drained away which results in a higher systemic exposure and rapid decline in ocular levels necessitating multiple administrations per day.37 We screened various generally regarded as safe (GRAS) fatty anions for their ability to form lipophilic salt of MOX with high yield. We found that disodium pamoate (PAM) resulted in a near 100% transformation of MOX into lipophilic MOX-PAM salt (FIG. 41 A) and this salt was further processed using freeze drying. We used our previously developed bench-top nanomilling method (FIG. 41B) which uses appropriately sized inert zirconium beads and high mechanical energy to break hydrophobic drugs/lipophilic salts (MOX-PAM) down to mucus-penetrating NS stabilized by Poloxamer 407 (MOX-PAM NS). The developed MOX-PAM NS showed particle size ~ 200 nm, low polydispersity index (a sign of homogeneity), and acceptable surface charge indicating suitability for further evaluation.37
Example 3D - Topical administration of MOX-PAM NS showed improved ocular pharmacokinetics (PK) compared to Vigamox®.
We evaluated ocular PK of topically applied 0.5 w/v Vigamox® and MOX-PAM NS (equivalent to 0.5% w/v MOX) in healthy rats. Interestingly, compared to Vigamox®, MOX- PAM NS showed higher MOX levels in aqueous humor, similar levels in the cornea, and significantly higher AUCO-24 in aqueous humor and cornea indicating enhanced and prolonged delivery compared to water soluble MOX eye drops.37
Example 3E - Once daily topical administration of MOX-PAM NS was more or equally effective compared to thrice daily topical Vigamox eyedrops in the rat model of Staphylococcus aureus infection.
Due to the encouraging ocular PK data, we evaluated the ability of MOX-PAM NS to reduce the frequency of administration of the MOX without compromising its therapeutic efficacy. We studied the comparative efficacy of topical Vigamox administered i) thrice daily (3x/day) as per the clinical indication or ii) once daily to obtain a head-to-head comparison with MOX-PAM NS administered once a day. Interestingly, in our rat model of S. aureus infection, once daily MOX-PAM NS treatment was significantly more effective than once daily Vigamox and equal and more effective compared to thrice daily Vigamox (FIG. 42).37 Thus, we envisage that transformation of water soluble and ionizable ADV into lipophilic salt followed by its transformation into mucus-penetrating NS could be an effective way to improve and prolong ocular delivery of ADV potentially leading to once-daily topical therapy of ocular HSV infections. Our prior experience in the synthesis of ionic liquids (ILs) of hydrophilic and lipophilic ionizable drugs with suboptimal solubility and permeability characteristics. We have extensive experience in the synthesis and characterization of ILs of hydrophilic (metformin, phenformin) and hydrophobic (anthelmintic benzimidazoles) ionizable drugs (Example_3_Scheme 1) with high crystallinity, low permeability, and poor lipid solubility.49,50 We, for the first time, showed that highly crystalline, hydrophobic, and weakly basic drugs such as anthelmintic benzimidazoles49, can be efficiently (yield > 90%) transformed into ILs using a pharmaceutically acceptable fatty anion sodium docusate (Example_3_Scheme 1). We also extended this strategy to synthesize lipophilic ILs of water soluble salt form of antidiabetic biguanides.50 We used techniques such as Fourier- transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS), and to confirm the synthesis and purity of the docusate-based ILs. Unlike pure anthelmintic benzimidazoles or antidiabetic biguanides, their docusate-based ILs showed low or no crystallinity, high thermal stability, and high solubility in organic solvents (acetone, methanol, acetonitrile, etc.) indicating improved pharmaceutical processability and significantly higher in vitro efficacy indicating improved membrane transport.49,50
Example_3_Scheme 1. Synthesis of lipophilic docusate-based ILs of anthelmintic benzimidazoles and antidiabetic biguanide.
Figure imgf000118_0001
Example 3F - Lipophilic ILs can be efficiently incorporated into SoluPlus nanomicelles to further improve their in vivo efficacy.
While the transformation of ionizable drugs with suboptimal solubility and permeability characteristics to lipophilic IL(s) could improve their lipid solubility and permeability, it is necessary to package these TLs into nanoformulations to further improve the delivery, bioavailability, and efficacy of ILs. SoluPlus is a biodegradable amphiphilic copolymer, which forms nano-scale micelles upon dilution with physiological fluids and can encapsulate a variety of hydrophobic drugs leading to improved solubility, permeability, and bioavailability.52 We, for the first time, showed that lipophilic ILs of anthelmintic benzimidazole can be stably encapsulated in SoluPlus nanomicelles (FIG. 43).49 Furthermore, our preliminary studies show that these nanomicelles containing ILs, if necessary, can be efficiently freeze dried and can be readily reconstituted to yield nanomicelles with negligible size change. To demonstrate the ability of SoluPlus nanomicelles to improve the delivery of ILs, we developed SoluPlus nanomicelles (size - 130 nm and PDI: < 0.2) containing oxfendazole docusate (OXF-Doc), a lipophilic IL.
Further, we evaluated the in vivo efficacy of orally delivered pure oxfendazole (OXF) in a vehicle (HPMC), OXF-Doc in a vehicle (HPMC), OXF suspended in SoluPlus nanomicelles, and SoluPlus nanomicelles containing OXF-Doc (OXF dose: 25 mg/kg oral) in a murine model of cryptococcosis. The mice were intranasally infected with Cryptococcus neoformans and the animals were treated with different OXF formulations for 14 days after 14 days of therapy, the animals were euthanized, lungs were harvested and the C. neoformans burden in the lungs was measured (colony forming units or CFUs). Interestingly, SoluPlus nanomicelles containing OXF-Doc (OXF-Doc in SoluPlus) showed the highest in vivo efficacy compared to the pure OXF and OXF-Doc (FIG. 44) indicating the importance of packaging of ILs into SoluPlus nanomicelles or to achieve enhanced drug delivery.
Previous studies by other groups showed that SoluPlus nanomicelles can be used for improving the topical (ocular) delivery and in vivo efficacy of hydrophobic/lipophilic drugs.53’56 Hence, we envisage that our preliminary data on SoluPlus nanomicelles containing ILs can potentially be extrapolated to improve ocular drug delivery. Importantly, due to the presence of muco-inert polyethylene glycol (PEG) as a part of the polymer backbone, SoluPlus nanomicelles have near neutral surface charge and low size (< 200 nm) and we anticipate that these characteristics should enable efficient transport of drug/drug-based IL packaged in SoluPlus nanomicelles through ocular mucin barrier. Thus, we propose that SoluPlus nanomicelles containing ILs of ADV will have promise for the topical therapy of ocular HSV infections.
Example 3G - Develop, characterize, and evaluate topical nanosuspension of ADV lipophilic salt for the treatment of ocular HSV infection Our preliminary data showed that 1 % w/v solution of ADV, a water soluble and ionizable prodrug, showed robust antiviral efficacy after thrice daily administration in a mouse model of ocular herpes infection. Hence, in order, to achieve once-daily ocular herpes therapy, conversion of ADV to a suitable lipophilic salt with low water solubility followed by its transformation to a mucus-penetrating nanosuspension (NS) using a lab-scale wet milling method could be a pharmaceutically viable approach to achieve once daily ocular herpes therapy. Unlike moxifloxacin hydrochloride, ADV is not available as a water soluble salt. However, even without salt formation, ADV has high water solubility (~ 10 mg/ml) and its amino group can be ionized to interact with pharmaceutically acceptable fatty anions such as pamoic acid. To achieve the in-situ ionization of ADV and pamoic acid, we slightly modified the previous procedure (FIG. 45 A) to obtain ADV pamoate (ADV-PAM) as a lipophilic salt. The 1 H-NMR spectrum of the obtained ADV-PAM (FIG. 45B) confirmed the molecular interaction between ADV and PAM. Further, we processed the ADV-PAM using vacuum drying and freeze-drying to remove traces of water. The freeze- dried ADV-PAM was then subjected to our lab-scale wet milling process (FIG. 45C) to obtain Poloxamer 407 coated mucus-penetrating ADV-PAM NS suitable for ocular delivery. After 6-8 hours of wet milling, a prototype ADV-PAM NS was obtained (FIG. 45D).
We will continue to optimize the process parameters involved in ADV-PAM synthesis to avoid/minimize the use of DMF and steps to obtain solid ADV-PAM. We will also continue optimizing the parameters of the wet milling process to obtain ADV-PAM NS with optimal characteristics. Numerous formulation variables and operating parameters such as drug concentrations, concentration, type of stabilizers, bead types, and quantity, nanomilling duration can be evaluated39 to obtain ADV-PAM NS with optimal characteristics. If necessary, we will also evaluate previously reported57 mucus-penetrating stabilizers such as Poloxamer Pl 03, and Poloxamer P123 at various concentrations to obtain mucus -penetrating ADV-PAM NS. We will evaluate the physicochemical properties of ADV-PAM NS using standard techniques and the chemical stability of ADV using a previously developed HPLC method to ensure that the preparation method does not compromise the quality and potency of ADV. The colloidal stability of ADV-PAM NS will be evaluated at room temperature and 4°C for at least 3 months. Various formulation variables and characterization methods are summarized in Example_3_Table 1.
Example 3 Table 1: Summary of formulation variables and characterization methods for ADV-PAM NS
Figure imgf000121_0002
Although our previous studies demonstrate that pamoate is safe for ocular delivery and that pamoate salt/complex does not compromise the inherent efficacy of the drug, it is still necessary to evaluate the impact of transforming ADV into ADV-PAM and its subsequent conversion to mucus-penetrating ADV-PAM NS on the in vitro cytocompatibility and in vitro antiviral activity.
Our previous studies with ocular formulations containing pamoate did not show any signs of tolerability issues associated with pamoate. We also showed that disodium pamoate is very well tolerated by primary mouse retinal ganglion cells and finally, our preliminary data show that ADV is well tolerated by HCE.
We will evaluate the in vitro cytocompatibility of ADV, pamoic acid, ADV-PAM, and ADV-PAM NS using HCE. Various concentrations of the test substances will be evaluated to determine the concentration that causes 50% cytotoxicity (CC50). The summary of in vitro antiviral experiments is presented in Scheme 2. The antiviral efficacy assays will be performed on vaginal epithelial cells maintained in Keratinocyte serum-free medium (KSFM) (Gibco/BRL, Carlsbad, CA, USA) supplemented with epidermal growth factor (EGF), bovine pituitary extract (BPE), and 1% penicillin/streptomycin. The requisite number of cells will be plated and incubated at 37°C in a humidified 5% CO2 incubator overnight to allow the formation of confluent monolayers before infection with a GFP tagged HSV-1 at an MOI (multiplicity of infection) of 0.1. Dilutions of test compounds (ACV solution, ganciclovir solution, ADV solution, ADV-PAM solution, and ADV- PAM NS) in media will be freshly prepared in a series of 5-fold dilutions in duplicate wells to yield final concentrations that range from 10 to 0.01 pM depending on the efficacy of the compounds. These dilutions will then be added to infected cell monolayers 2 hours postinfection to initiate treatment for 24 hours.58 Using a Lionheart LX automated cell imager, all the requisite wells will be imaged and the percentage of GFP positive (infected) cells will be automatically analyzed. To confirm the results from the imaging system, plaque assays (on Vero cells) using intracellular and extracellular virus from all the treatment groups will be titrated.
Example_3_Scheme 2: Summary of the in vitro antiviral activity experiments
Figure imgf000121_0001
Herpes simplex virus (HSV) keratitis is a recurrent and lifelong infection that results in visual morbidity, and it is the leading cause of infection-associated corneal blindness worldwide. Depending upon the anatomical location, HSV keratitis can be categorized into epithelial, stromal, and endothelial keratitis. HSV epithelial keratitis involves active HSV infection in the corneal epithelium and antiviral therapy is needed to diminish discomfort, minimize vision loss, and reduce the recurrence rate. Topical eye drops of nucleoside analogs such as ganciclovir and trifluridine are FDA-approved for HSV epithelial keratitis therapy. However, these eye drops need to be administered 5- to 9-times a day to manage the ocular HSV infection which affects patient compliance and thus, treatment efficacy. Our long-term goal is to develop once daily antiviral eye drop formulation for the treatment of HSV epithelial keratitis.
It is essential to evaluate the ability of ADV-PAM NS to offer improved and prolonged ocular delivery of ADV using PK studies which will eventually lead to long-lasting antiviral efficacy. We have already shown the efficacy of 1% w/v ADV solution in the murine model of ocular HSV-1 infection. For the ocular PK studies, we will use ADV solution, and ADV-PAM NS at a dose equivalent to 1% w/v of ADV.
A summary of PK experiments is provided in Example_3_Table 2.
Example 3 Table 2: Summary of ocular PK experiments for ADV solution and ADV-
PAM NS
Figure imgf000122_0001
The ocular PK studies and systemic exposure will be carried out in healthy C57BL/6 mice. We propose to evaluate the ocular PK parameters and systemic exposure of ADV, and its metabolites adefovir and adefovir diphosphate on topical (ocular) administration of 10 pL of 1% ADV solution, or ADV-PAM NS equivalent to 1% w/v ADV. Briefly, six- to 8-weeks old C57BL/6 mice will be randomly divided into 2 groups [n > 6 (3 males and 3 females) per time point; (n > 48 per group)]. The mice will receive a 10 pL topical dose of 10 pL of ADV solution, or ADV-PAM NS. Blood will be withdrawn, and the ocular tissues (aqueous humor, vitreous, and cornea) and the blood will be harvested at 0.25, 0.5, 0.75, 1, 3, 6, 10, and 24 h. Blood will be centrifuged at 2000 g and 4°C for 15 min and the recovered plasma, and ocular tissues (aqueous humor, vitreous, and cornea) will be stored at -20°C until further analysis. The concentration of ADV, and its metabolites adefovir and adefovir diphosphate in plasma, and ocular tissue homogenate will be determined using LC-MS/MS. We will utilize the services of Dr. Michelle Rudek, Johns Hopkins Mass Spectroscopy Core for the LC-MS/MS analysis of ADV and its metabolites. We previously collaborated with Dr. Rudek for the PK studies of ANPs such as tenofovir and TDF. The pharmacokinetics of ADV, adefovir, and adefovir diphosphate in plasma and ocular tissues will be calculated using WinNonlin or suitable pharmacokinetic data analysis software.
Eight- week-old male C57BL6 mice (n = 10 per group; 5 males and 5 females) will be used. Approximately 10 pL volume of 1% w/v ADV solution, ADV-PAM NS (equivalent to 1% w/v ADV) or PBS will be placed on the murine eye and the mice will be held for a period of 30 seconds. This will be repeated 3 times a day for ADV solution and once daily for ADV-PAM NS for a period of 1 month. Weekly assessments of ocular health will help establish the ocular safety profile. Ocular health will be assessed using the protocols mentioned below.
A 30-mm-long phenol-red impregnated thread with the 3-mm bent end will be placed in the lower fornix of the mouse eye for 15 seconds. When the phenol red comes in contact with alkaline tears, it will change color from yellow to red. The thread will be removed after 15 seconds, and the length of the red portion will be measured using a ruler. The results will be interpreted as follows: wet length <2 mm as severe dry eye, <5 mm as borderline dry eye, and >10 mm as normal tear production. Both treated and non-treated eyes will be evaluated while the non-treated eye will serve as an internal control.
Anesthetized mice will be placed on a sturdy mice holder and held in the right position such that the entire frame of the cornea will be visible. Multiple images at 10X magnification will be taken in quick succession to avoid drying of the eye using a slit-lamp biomicroscope (Haag- Streit AG, Koeniz, Switzerland). Fluorescein (1%) dye will be added in a total volume of 5 pL to each eye followed by manual blinking of the eye 5 times before images will be captured in the GFP channel. The cornea will be divided into 4 quadrants and each quadrant will be scored in a double-blinded fashion to give a dryness score.
OCT images will be taken on a Micron IV (Phoenix Technology group, CA, USA) with an OCT probe to specifically look at corneal thickness. Anesthetized mice will be placed on a holder, and the OCT probe will be brought closer to the eye. The light intensity, focus, angle, and contrast will be standardized on the first week of measurement. The same standard values will be used throughout the entire course of the study. Once the images will be captured, the epithelial, stromal, and total thickness of the cornea will be measured for all the mice to evaluate the presence of inflammation. The results will be interpreted as follows: <5% variation in thickness as normal, >5% and <10% as moderate inflammation, >10% as severe inflammation.
Comeal sensitivity of the mice eyes will be measured by a manual Esthesiometer (12/100 mm, LUNEAU SAS, France). Corneal sensitivity measurements as a function of blink response will be conducted on wake mice. The nylon fiber of the esthesiometer will be extended to the full length (6 cm), and the tip of the nylon thread will be gently impressed to the center of the cornea. In a normal cornea, this should inflict a blink response which corresponds to a sensitivity score. If the mouse does not blink, the length of the nylon fiber will be reduced by 0.5 cm, and the exercise will be repeated until a blink response will be observed. The results of this study will be interpreted as follows: Score of 5.5 to 6 as normal, 5.5 to 3.5 as moderate loss of sensitivity, and <3.5 as severe loss of comeal sensitivity.
The intraocular pressure (IOP) of mice will be measured using a hand-held tonometer device which has been calibrated to measure the IOP specifically for murine models. The measurement of IOP will be initially performed both on wake and anesthetized mice to understand the variation in values. A new tip will be loaded into the tonometer for every sample group and calibrated before measurement. Each measurement will be performed in triplicates to account for variability.
The summary of in vivo experiment to determine the efficacy of topical ADV solution and ADV-PAM NS is given in Example_3_Scheme 3. Eight- week-old male C57BL6 mice (n = 10 per group; 5 males and 5 females) will be infected with 1x10s PFU HSV-1 (McKrae) on the comeal surface on day 0. Starting 1-day post-infection (1 dpi), mice will be topically treated with 10 pL of : 1) 1% ADV solution (once daily), 2) 1% ADV solution (thrice daily), 3) 0.15% ADV-PAM NS (once daily), 4) 1% ADV-PAM NS (once daily), 5) 0.15% ganciclovir eye drops (5 times daily) and 6) PBS until 7 dpi. Animal weights will be recorded, and eye health (disease score) will be monitored in a blinded fashion every alternate day during the treatment period.59,60 Stereoscopic images and ocular swabs will be taken before drug administration and plaque assays will be conducted using the swabs on alternate days to record the extent of viral shedding.
ExampIe_3_Scheme 3 Summary of the in vivo experiments to determine the efficacy of ADV-PAM NS.
Figure imgf000125_0001
The animals will be monitored daily for up to 30 days by a slit lamp photograph biomicroscope (FS2; Topcon Corp., Tokyo, Japan). Clinical scores for neovascularization, stromal opacification, and blepharitis will be determined and are discussed below. Corneal swabs will be collected from the left eye once before inoculation of the virus and on days 2, 5, 7, 8, 10, 12, 14, 21, and 30 after infection. The swabs will be used for determining virus shedding estimated by plaque assay on Vero cells. Starting 3 days post-infection and every 3 days thereafter until d30, mouse corneas will be evaluated for neovascularization and corneal opacity according to adopted scoring indices. Briefly, the cornea will be divided into four quadrants, and the length of the longest neovessel in each quadrant will be graded between 0 and 4 in increments of 0.4 mm (the radius of the cornea is about 1.5 mm). The angiogenesis score for each cornea will be the sum of the scores for the four quadrants, ranging from 0 to 16. Corneal opacity, a clinical correlate of the degree of inflammation, will be scored as follows by slit-lamp examination: 0, normal; 1, mild haze; 2, moderate opacity or scarring; 3, severe opacity but iris visible; 4, opaque with corneal ulcer; 5, corneal rupture and necrotizing stromal keratitis. Blepharitis will also be scored using a range from 0 to 2 where 0 is normal eyelid morphology, 1 mild, and 2 is necrotizing dermatitis.
To study whether ADV and ADV-PAM NS reduce the viral spread in the cornea and establishment of latency, HSV-1 infected treated with ADV solution, ADV-PAM NS, ganciclovir, or the vehicle will be euthanized at 2, 5, 7, 14 and 30 dpi (discussed above) and the eyes, eyelids, and TG will be aseptically removed. The replication and spread of HSV-1 in these tissues will be tested by (i) detecting the infectious virus in homogenized tissue by plaque assay, (ii) measuring HSV-1 DNA present by quantitative real-time (RT-) PCR, (iii) detecting HSV-1 protein expression in tissues by immunohistochemistry using a polyclonal HSV-1 antibody, and (iv) detecting and estimating LAT expression by in situ hybridization.
Example 3H - Develop, characterize, and evaluate topical nanomicelles containing ADV- fatty acid IL for the treatment of ocular HSV infection
Previous studies have shown that SoluPlus nanomicelles are well tolerated upon ocular delivery and they can improve ocular permeability and retention of various hydrophobic drugs.53-56 A recent study also showed that linoleic acid can be beneficial for the tear stability and docosahexaenoic acid has been reported to be beneficial in ocular pathologies including inflammation associated with HSV-1 infection.61-64 We hypothesize that water soluble ionizable ADV can electrostatically interact with generally regarded as safe (GRAS) mono- or polyunsaturated fatty acids to yield lipophilic ADV ILs with low water solubility and improved permeability. Subsequent incorporation of these ADV-ILs into SoluPlus nanomicelles will further improve their topical (ocular) delivery and prolong the release of ADV from ILs leading to long- lasting ocular herpes therapy.
Our preliminary data, for the first time, show that ADV can readily interact with mono- and polyunsaturated fatty acids such as oleic acid, linoleic acid, and docosahexaenoic acid to yield ILs (Example_3_Scheme 4). While Scheme 4 only depicts the formation of ADV oleate as an IL, the other fatty acids such as linoleic and docosahexaenoic acid also readily transformed ADV to ADV IL in less than 10 minutes. The remarkable simplicity of our process highlights the pharmaceutical viability and potential for translation of ADV ILs for improved drug delivery.
Example_3_Scheme 4 Scheme for synthesizing ADV-fatty acid ionic liquids. ADV oleate is readily formed within 10 minutes after mixing ADV and oleic in ethanol followed by complete evaporation of ethanol.
Figure imgf000126_0001
We have already developed ADV oleate, ADV linoleate, and ADV docosahexaenoate (Scheme 4 and FIG. 46A) all of which are lipophilic ILs. We confirmed the interaction between ADV and fatty acids such as linoleic acid using 1 H-NMR spectroscopy (FIG. 46B) and we will continue to characterize ADV-ILs using UV-Vis spectroscopy, fluorescence spectroscopy, FT-IR spectroscopy, 1 H NMR, 13C NMR, MS, HPLC, DSC, and powder X-ray diffraction (PXRD) as described previously.49 We will monitor the long-term physical stability of ADV ILs at a previously developed HPLC method. Using our previously reported method, we already developed SoluPlus nanomicelles containing ADV ILs (FIG. 46C) all of which have size <150 nm, polydispersity index < 0.25, and slightly negative surface charge. We will continue to optimize SoluPlus nanomicelles by ADV-IL to SoluPlus to obtain nanomicelles with optimal size (preferably < 100 nm), and colloidal stability (at least 3 months), and high chemical stability of ADV.
We will evaluate the physicochemical stability and ADV chemical stability of SoluPlus nanomicelles containing ADV-ILs and only one SoluPlus nanomicellar formulation with the lowest size, polydispersity index, and surface charge and highest colloidal stability and ADV chemical stability (at least 3 months) will be selected for the further studies.
We will evaluate the in vitro cytocompatibility and antiviral activity of blank SoluPlus nanomicelles, ADV-IL, and SoluPlus nanomicelles containing ADV-IL.
The summary of the PK experiment for SoluPlus nanomicelles containing ADV IL is given in Example_3_Table 3.
Example_3_Table 3 Summary of ocular PK experiments for ADV solution and SoluPlus nanomicelles containing ADV IL.
Figure imgf000127_0001
We will carry out the ocular PK of ADV solution and SoluPlus nanomicelles containing ADV-IL.
We will evaluate the ocular safety of blank SoluPlus nanomicelles, SoluPlus nanomicelles containing ADV-IL (equivalent to 1% w/v ADV) ADV solution and PBS.
We will evaluate the in vivo antiviral efficacy of 1 % w/v ADV solution (once daily application), 1% ADV solution (thrice daily application), 0.15% ganciclovir eye drops (5 times daily) and SoluPlus nanomicelles containing ADV IL (equivalent to 1% ADV or 0.15% ADV; applied once daily) and PBS in a mouse model of ocular herpes infection. The methods will be used for the in vivo antiviral efficacy studies. We will also evaluate the effect of the aforementioned formulations on viral spread and latency.
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Example 4 - LogP and pKa Information for Various Anions
Example_4_Table 1 - Anions: Saturated fatty acids
Figure imgf000131_0001
Example_4_Table 3 - Anions: Aromatic acids
Figure imgf000131_0002
Example_4_Table 4 - Anions: Sulfonate
Figure imgf000131_0003
Figure imgf000132_0001
Example_4_Table 5 - Anions: Sulfates
Figure imgf000132_0002
Example_4_Table 6 - Anions: Phosphate
Figure imgf000132_0003
ExampIe_4_Table 7 - Anions: Sulfamate
Figure imgf000132_0004
Example 5. Example_5_Table 1: Meglumine & dicarboxylic acid-based ILs self-assembly in water at various concentrations.
Figure imgf000132_0005
Example_5_Table 2 - Various drugs solubility in Meglumine & bile acid-based ILs.
Figure imgf000132_0006
Figure imgf000133_0001
Example_5_Table 3 - Niclosamide dissolved in Meglumine Cholate IL self-assemble in water at various dilutions.
Figure imgf000133_0002
* 15 mg niclosamide dissolved in 100 mg of Meglumine cholate IL. 10 mg from the mixture was reconstituted with 1 mL of water.
Example 6.
This example describes self-assembling meglumine containing ionic liquids and their applications to drug delivery.
The self-assembling ionic liquid, meglumine vitamin E succinate (Meg-VES-IL) was dissolved in water at a concentration > 1.5 mg/ml to yield self-assembled nanostructures (see, Example_6_Figure 1).
Example 6A:
The drug-loaded Meg-VES self-assembling nanostructures were prepared by codissolving drug and Meg-VES in organic solvents such as methanol/ethanol and adding this mixture to the water followed by removal of organic solvent to yield nanostructures (Example_6_Table 1).
Example_6_Table 1: Size, and polydispersity index (PDI) of blank and drug-loaded meglumine-tocopherol succinate (Meg-VES) self-assembling IL prepared in water.
Figure imgf000133_0003
Figure imgf000134_0001
Example 2B:
This example describes the preparation of self-assembling meglumine ionic liquids using stabilizers such as PEGylated phospholipid (DSPE-PEG2000) and polysorbate 80.
Meglumine-based ionic liquids can be interacted with stabilizers such as PEGylated phospholipids and polysorbate 80 to yield nanostructures and these nanostructures can also encapsulate drugs (see, Example_6_Table 2).
Example_6_Table 2: Size, and poly dispersity index (PDI) and surface charge of selfassembling meglumine containing ILs prepared with additional stabilizers.
Figure imgf000134_0002
Example 7.
This example describes the development of carnitine-fatty acid amphiphilic ionic liquids and their transformation into self-nanoemulsifying systems to improve oral bioavailability of hydrophobic drugs.
Carnitine is a GRAS amino acid used as a nutraceutical in adults and infant formula. The development of amphiphilic ILs using carnitine and anionic fatty permeation enhancers such as is a hitherto unexplored approach to improving solubility and delivery of hydrophobic drugs.
Example 7A:
This example describes the synthesis of carnitine-fatty acid ionic liquids. We dissolved carnitine with various fatty acids in methanol ( 1 : 1 molar ratio) and interacted them at room temperature for 30 min to 3 hours. (Example_7_Scheme 1) The solvent was evaporated and the ionic liquid was thoroughly dried using a vacuum oven.
Example_7_Scheme 1: Scheme for the synthesis of carnitine fatty acid ionic liquids
Figure imgf000135_0001
Example 7B:
This example describes the kinetic solubility of various hydrophobic drugs in carnitine-fatty acid ionic liquids.
The kinetic solubility of various drugs with low solubility and/or low permeability in the carnitine linoleate was determined. Briefly, 100 mg of carnitine salcaprozate and carnitine linoleate was dissolved in ethanol/acetone/methanol or a mixture of these solvents, and 5 mg of drug of interest was dissolved in this organic solution containing IL. The solvent was then evaporated using rotary evaporator followed by drying in the vacuum oven to further remove the solvent trace. If the drug did not precipitate after extensive drying, then the drug was added to the IL in 5 mg increments to continue the solubility study. The solubility study was halted when the drug precipitation was observed after solvent evaporation or on standing for at least 1 week. FIG. 48 and Example_7_Table 1 shows the solubilization of various drugs in carnitine linoleate IL and table
Example_7_Table 1: Solubility of various drugs in carnitine salcaprozate
Figure imgf000135_0002
Figure imgf000136_0001
Example 7C:
This example describes the improvement of oral bioavailability of hydrophobic drugs when packaged into carnitine fatty acid ionic liquid self-nanoemulsifying systems.
We developed self-nanoemulsifying systems containing carnitine linoleate and carnitine salcaprozate which could solubilize hydrophobic drugs such as sorafenib and venetoclax. These self-nanoemulsifying systems yielded nanoemulsions of size < 250 nm in water and other buffers. The carnitine ionic liquid self-nanoemulsifying systems significantly improved the oral bioavailability of venetoclax and sorafenib (FIG. 49 and FIG. 50).
INCORPORATION BY REFERENCE
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. The following references are herein incorporated by reference in their entireties:
EQUIVALENTS
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

What Is Claimed Is:
1. A composition comprising an ionic liquid formulation comprising a cationic component and an anionic component.
2. The composition of claim 1, wherein the cationic component is selected from a cationic or ionized therapeutic agent, a cationic or ionized amino acid, a cationic nutraceutical, a cationic agrochemical molecule, a cationic functional food, a cationic excipient, and a pharmaceutically acceptable cation.
3. The composition of claim 1 , wherein the cationic component is a protonated form or cationic derivative of one of the following:
Amino su ars t-Carnitine
Figure imgf000137_0001
Nucleoside analogue Carbamimidoylphenyl analogue
Figure imgf000137_0002
4. The composition of claim 1 , wherein the cationic component is a protonated form or cationic derivative of one of the following: D-glucamine, n-methyl-D-glucamine, N-ethyl-D- glucamine, N-octyl-d-glucamine, N-oxodecyl meglumine, D-glucosamine, D-mannosamine, D- galactosamine, L-camitine, D-carnitine, Acetyl-L-camitine, Propionyl-L-carnitine, O-hexanoyl- L-carnitine, Phosphatidyl-camitine, Isovaleryl-L-camitine, Propionyl-L-carnitine, Palmitoyl-L- carnitine, Sarcosine, Sarcosine methyl ester, Sarcosine ethyl ester, N-lauroyl arginine ethyl ester, N-lauroyl lysine Albendazole, mebendazole, flubendazole, triclabendazole, Alexidine, Chlorhexidine, Picloxydine Metformin, Phenformin, Buformin, Proguanil, Moroxydine, BX- 795, Rilpivirine, etravirine, nucleotide analogues (antiviral agents) (e.g., Tenofovir, Tenofovir disoproxil, Tenofovir alafenamide, Adefovir, Adefovir dipivoxil Acyclovir, Ganciclovir, Penciclovir, Lamivudine, abacavir, Emtricitabine, Zalcitabine, and Cytidine monophosphate), Imiquimod, Resiquimod, Gardiquimod, Pentamidine, Furamidine, nafamostat Pafuramidine, Diminazene, indoximod, rasagiline, ropinirole, venetoclax, navitoclax, obatoclax, moxifloxacin, levodopa, imeglimin, cyloguanil, clofazimine, bedaquiline, dabrafenib, vemurafenib, trametinib, sorafenib, and hexaminolevulinate hydrochloride.
5. The composition of claim 1, wherein the cationic component is a cationic molecule (e.g., linear, branched or cyclic) containing a primary, a secondary, a tertiary, or a quaternary ammonium group.
6. The composition of claim 1, wherein the cationic component is a cationic carnitine derivative of the following formula:
Figure imgf000138_0001
; wherein Rl, R2, and R3 are each independently selected from hydrogen, C1-C6 alkyl group, a C1-C3 alkyl group, a C1-C6 alkyl group, and a methyl group; wherein R is selected from hydrogen, CH3C=O, CH3 (CH2)MC=0, and a -(0=0- C1-C15 alkyl group.
7. The composition of claim 1 , wherein the cationic component is a protonated form or cationic derivative (e.g., 1°, 2°, or 3° ammonium salt or imine salt) selected from: N-methyl-D-glucamine L-CarnitinG
Figure imgf000139_0001
Figure imgf000139_0002
Figure imgf000140_0001
Figure imgf000140_0002
8. The composition of claim 1, wherein the cationic component is selected from:
Figure imgf000140_0003
Figure imgf000141_0003
R: alkyl group or alkyl alcohol or alkyl acid or heteroalkyl
Rj to R ncompassed within the following: 15: -H or halides or alkyl or -NO e 2
10. The composition of claim 1 , wherein the cationic component is selected from mitoquinone, mitoquinone mesylate, and SKQ1.
11. The composition of claim 1 , wherein the cationic component is selected from.
Figure imgf000141_0001
12. The composition of claim 1 , wherein the cationic component is a protonated form of a benzimidazole compound (e.g., anthelmintic benzimidazoles such as albendazole, mebendazole, flubendazole and triclabendazole) not including oxfendazole; wherein the benzimidazole
Figure imgf000141_0002
compound is encompassed within the following: wherein
Rl, R2 are each independently selected from hydrogen, alkyl, aryl, heteroalkyl and heteroaryl groups.
13. The composition of claim 1, wherein the anionic component is selected from an anionic therapeutic agent, an anionic amino acid, an anionic nutraceutical, an anionic agrochemical molecule, an anionic functional food, an anionic excipient, and a pharmaceutically acceptable anion.
14. The composition of claim 1, wherein the anionic component is an anionic carboxylate, an anionic sulfonate, an anionic sulfate, an anionic phosphate, an anionic phosphonate, an anionic sulfamate, or a chemical moiety having negatively charged functional group.
15. The composition of claim 1, wherein the anionic component is
Figure imgf000142_0001
16. The composition of claim 1, wherein the anionic component is a bile acid selected from cholic acid, chenodeoxy cholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, and tauroursodeoxycholic acid.
17. The composition of claim 1, wherein the anionic component is selected from:
Figure imgf000142_0002
18. The composition of claim 1, wherein the anionic component is a negatively charged functional group (e.g. carboxylate) selected from one of the following: HOOC-R
HOOC-R
R = saturated or mono/di/poly unsaturated aliphatic R - alky or aryl group
Figure imgf000143_0001
R = -CH- or -O- or -NH-; R' = -OH or -O-alkyi or
R = -CH2- or -NH-; R1 = alkyl and/or aryl group -O-acyt or -O-carbamoyl or -O-carbonyl
Figure imgf000143_0002
R = alkyl and/or aryl group R = alky or aryl group R = alkyl or aryl group anj
Figure imgf000143_0003
19. The composition of claim 1 , wherein the anionic component is a molecule with negatively charged functional group selected from: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, undecylenic acid, oleic acid, linoleic acid, linolenic acid, myristoleic acid, ricinoleic acid, elaidic acid, N-decanoyl sarcosine, Lauryl sarcosine, docosahexaenoic acid, biotin, lactobionic acid, eicosapentaenoic acid, nervonic acid, Vitamin E succinate, 4-phenylbutyric acid, pamoic acid, a-lipoic acid, ibuprofen, naproxen, squalene acid, cholesterol hemisuccinate, capric acid, salcaprozic acid, docusic acid, cholic acid, glycocholic acid, taurocholic acid, tauroursodeoxycholic acid and other anionic bile acids, taurine, camphor sulfonic acid lauryl sulfate, cholesterol sulfate, dioleoyl phosphatidic acid (DOPA), vitamin E phosphate, thiamine phosphate, saccharine sodium, acesulfame potassium, cyclamate sodium, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, gallic acid, vanillic acid, and phthalic acid.
20. The composition of claim 1 , wherein the anionic component is an anionic carboxylate molecule selected from one of the following: HOOC-R
Caproic acid: R = CgHn
Caprylic acid: R = C7H15
Lauric acid: R - C14H23
Oleic acid: R ~ C17H33 HOOC-R
Docosahexaenoic acid: R ~ C24H31 a-tocopherol succinate: R « C2H4COO-vit E
Nervonic acid: R = C23H4S 4-pheny I butyric acid: R = C3H6(C6H5)
Figure imgf000144_0001
21. The composition of claim 1 , wherein the anionic component is selected from a saturated fatty acid derivative moiety (carboxylate), an unsaturated fatty acid derivative moiety, an aromatic acid derivative moiety, a sulfonate derivative moiety, a sulfate derivative moiety, a phosphate derivative moiety, and a sulfamate derivative moiety.
22. The composition of claim 1 , wherein the anionic component is selected from a negatively charged functional group of saturated fatty acids selected from butyric acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid)lauric acid (dodecanoic acid), palmitic acid (hexadecenoic acid), and cholic acid.
23. The composition of claim 1 , wherein the anionic component is selected from a negatively charged functional group of unsaturated fatty acids selected from: undecylenic acid, oleic acid, linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, nervonic acid, myristoleic acid, elaidic acid, and ricinoleic acid.
24. The composition of claim 1 , wherein the anionic component is selected from a negatively charged functional group of aromatic acids selected from: salcaprozic acid, a- tocopherol succinate, 4-phenyl butyric acid, Ibuprofen, Naproxen, pamoic acid, Dolutegravir, Cabotegravir, and Bictegravir.
25. The composition of claim 1 , wherein the anionic component is selected from a negatively charged functional group of sulfonate anions selected from: docusic acid, camphor sulfonic acid, taurocholic acid, tauroursodeoxycholic acid, and taurine.
Figure imgf000145_0001
Taurocholic acid
26. The composition of claim 1, wherein the anionic component is selected from a negatively charged functional group of sulfate anions selected from: lauryl sulfate
Figure imgf000145_0002
Figure imgf000145_0003
27. The composition of claim 1, wherein the anionic component is selected from a negatively charged functional group of a phosphate anion selected from: a-tocopherol phosphate, l,2-dioleoyl-sn-glycero-3-phosphate (DOPA;
Figure imgf000145_0004
28. The composition of claim 1 , wherein the anionic component is selected from a negatively charged functional group of sulfamate anions selected from: acesulfame
Figure imgf000146_0001
o Acesulfame ), saccharin, and cyclamate. 29. The composition of claim 1, wherein the anionic component is selected from:
Figure imgf000146_0002
30. The composition of claim 1, wherein the cationic components and anionic components are present in a ratio in the range of about 5:1 to about 1 :5.
31. The composition of claim 1 , wherein the ionic liquid formulations comprise one of more
Figure imgf000147_0002
Figure imgf000147_0001
Alexidine oleate Metformin docusate
Figure imgf000147_0003
Meglumine-tocopherol phosphate Meglumine-salcaprozate
Figure imgf000148_0001
Meglumine-oleate Meglumine-oleate (1:2)
Figure imgf000148_0002
hexaenoate
Figure imgf000148_0003
Meglumine-cholesterol sulfate , and Imiquimod oleate 32. The composition of claim 1 , wherein the ionic liquid formulations comprise one or more nucleotide analogues (antiviral agents) selected from:
Figure imgf000149_0001
ADV Docosahexanoate an j Adefovir dipivoxil Oleate
33. The composition of claim 1, wherein ionic liquid formulations comprise of:
Figure imgf000149_0002
34. The composition of claim 1 , wherein ionic liquid formulations comprise a carnitine or carnitine derivative, and an anionic component recited in claims 13-29; wherein the carnitine or carnitine derivative is encompassed within the following formula: wherein Rl, R2, and R3 are each independently
Figure imgf000150_0004
selected from hydrogen, C1-C6 alkyl group, a C1-C3 alkyl group, a C1-C6 alkyl group, and a methyl group; wherein R is selected from hydrogen, CH3C=0, CH3 (CH2)i4C=O, and a -(O=C)- C1-C15 alkyl group.
35. The composition of claim 34, wherein ionic liquid formulations comprise of carnitine and fatty anions:
Figure imgf000150_0001
Carnitine nervonate Carnitine linoleate Carnitine laurate
Figure imgf000150_0002
Carnitine docoshexaenoate Carnitine a-toeopherol phosphate
Figure imgf000150_0003
Carnitine tocopherol succinate
36. The composition of claim 34, wherein ionic liquid formulations comprise of carnitine as a cation and bile acids an anion:
Figure imgf000151_0001
37. The composition of claim 34, wherein ionic liquid formulations comprise of carnitine as a cation and dicarboxylic acids as an anion:
Figure imgf000151_0002
Carnitine azelate (2:1) Carnitine azelate (1:1)
Figure imgf000151_0003
Figure imgf000152_0001
38. The composition of claim 1, wherein the ionic liquid formulations are associated with (e.g., encapsulated within) nanoformulations prepared from polymers, peptides, lipids and/or inorganic materials.
39. The composition of claim 1, wherein the ionic liquid formulations are associated with (e.g., encapsulated within) polymeric nanoformulations such as but not limited to polylactide-co- glycolide (PLGA) nanoparticles, polymethylmethacrylate nanoparticles.
40. The composition of claim 1, wherein the ionic liquid formulations are associated with (e.g., encapsulated within) micelles prepared using micelle- forming agents such as but not limited to polyvinyl caprolactam-poly vinyl acetate-polyethylene glycol graft co-polymer (SoluPlus), polyethylene glycol-phospholipid conjugate, polyethylene glycol-polylactide, poly- lipoic acid or a PEG-poly-lipoic acid.
41. The composition of claim 1, wherein the ionic liquid formulations are associated with a self-emulsifying composition comprising a mixture of surfactant (such as polysorbate and poloxamer), cosurfactant (such as alcohol, glycol ether), lipids (such as triglycerides, mono- or diglycerides, fatty acids, and their esters), and ionic liquid according to any of the preceding claims that yields emulsion or nanoemulsion after dilution with water, buffer, 5% dextrose or other physiological fluids.
42. The composition of claim 1, wherein ionic liquid formulations undergo self-assembly to form nanostructures of size less than 900 nm (or less than 500 nm) over a period of 48 hours or less.
43. The composition of claim 42 wherein self-assembling ionic liquids contain cationic meglumine derivative of Formula II or Ila and one or more anionic molecules (e.g. carboxylates, phosphates, sulfate, sulfonates, sulfamate, etc) listed in claims 13-29:
Figure imgf000153_0003
Formula Ila wherein R”, Rl, R2, and R3 are independently a C1-C6 alkyl group, a C1-C3 alkyl group, or a methyl group.
44. The self-assembling ionic liquid of claim 43, wherein R1=R2=H and R3 = C1-C6 alkyl group, a C1-C3 alkyl group, or a methyl group.
45. The self-assembling ionic liquid of claim 42, wherein said ionic liquid is one of:
Figure imgf000153_0001
Meglumine-tocopherol succinate Meglumine-tocopherol phosphate
Figure imgf000153_0002
Meglumine-salcaprozate Meglumine-oleate
Figure imgf000154_0005
46. The self-assembling ionic liquid of claim 42, wherein said ionic liquid is one of:
Figure imgf000154_0001
Figure imgf000154_0002
Meglumine-glycocholate Meglumine-lithocholate Mcgln m in c-ta u rocholatc
Figure imgf000154_0003
Figure imgf000154_0004
Meglumine-ursodeoxycholate
47. The self-assembling ionic liquid of claim 42, wherein said ionic liquid is one of:
Figure imgf000155_0001
Meglumine azelate (2:1) Meglumine azelate (1 :1)
Figure imgf000155_0002
Meglumine suberate (2:1) Meglumine suberate (1 :1) 48. The self-assembling ionic liquid formulation from claims 42-47 wherein the concentration of self-assembling IL is between about 0.01 to about 10 mg/ml in water, buffer or other aqueous vehicles.
49. The ionic liquid of any of the preceding claims that can solubilize drugs, natural products or nutraceuticals with low solubility (less than 1 mg/ml) and/or low permeability (< 1* 106 cm/s) such as but not limited to cyclosporin A, docetaxel, paclitaxel, cabizataxel, dabrafenib, trametinib, sorafenib, venetoclax, coenzyme Q10, idebenone, triclabendazole, olaparib, urolithin A, myricetin, quercetin, resveratrol, genistein, pterostilbene, gefitinib, dolutegravir, cabotegravir, bictegravir, tenofovir alafenamide, adefovir dipivoxil, ivermectin, fluconazole, ibuprofen, niclosamide, idebenone, niclosamide oleate, niclosamide lipoate, at least at a concentration of 25 mg/ml of ionic liquid.
50. A pharmaceutical composition (e.g. cream, emulsion, self-nanoemulsifying system, transdermal patch, liquid-filled capsule, solid dispersion, hydrogel, oleogel, aerosol, powder, microneedles, foam, film, aqueous solution, etc.) comprising an effective amount of a ionic liquid composition of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
51. The pharmaceutical composition of claim 50, wherein the composition further comprises at least one additional therapeutic agent.
52. The pharmaceutical composition of claim 51, wherein the at least one additional therapeutic agent comprises any type or kind of therapeutic agent capable of inhibiting fungal activity.
53. The pharmaceutical composition of claim 52, wherein the at least one additional therapeutic agent is selected from the following: a polyene, imidazole, triazole, thiazole, allylamine, echinocandin, among others. Examples include Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, econazole, fenticonazole, isoconazole, kentoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, polygodial, tolnaftate, undecylenic acid, and crystal violet.
54. The pharmaceutical composition of claim 53, wherein the at least one additional therapeutic agent comprises any type or kind of agent capable of inhibiting reverse-transcriptase (RT) activity.
55. The pharmaceutical composition of claim 53, wherein the at least one additional therapeutic agent comprises any type or kind of anti- HIV agent or an anti-viral agent.
56. The pharmaceutical composition of claim 55, wherein the at least one additional therapeutic agent comprises any type or kind of anti- HIV agent selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, lenacapavir, and a combination thereof.
57. The pharmaceutical composition of claim 53, wherein the at least one additional therapeutic agent comprises any type or kind of anti-viral agent.
58. The pharmaceutical composition of claim 53, wherein the at least one additional therapeutic agent comprises any type or kind of anti-viral agent selected from helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, or ritonavir.
59. A method of killing or inhibiting the growth of a fungus comprising contacting the fungus with a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions recited in Claims 1-58.
60. The method of claim 59. wherein the fungus is selected from Aspergillus spp., Fusarium spp., Malassezia spp., Candida spp., or Cryptococcus spp..
61. The method of claim 59, wherein the fungus is selected from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Fusarium moniliforme, Fusarium proliferatum. Malassezia pachydermatis, Candida albicans, Candida glabrata infection, Candida tropicalis, Candida krusei, Candida auris, Cryptococcus neoformans, Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis, Sarcopodium oculorum, M. circinelloides, Rhizopus delemar, Rhizopus oryzae, and Lichtheimia corymbifera.
62. Use of a compositions or pharmaceutical compositions of any one of claims 1-584, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a fungal infection.
63. The use of claim 62. wherein the fungal infection is related to one or more of: Aspergillus spp., Fusarium spp., Malassezia spp., Candida spp., and Cryptococcus spp..
64. Hie use of claim 62, wherein the fungal infection is related to one or more of: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Fusarium moniliforme, Fusarium proliferation, Malassezia pachydermatis, Candida albicans, Candida glabrata infection, Candida tropicalis, Candida krusei, Candida auris, Cryptococcus neoformans, Chrysosporium parvum, Metarhizium anisopliae, Phaeoisaria clematidis, Sarcopodium oculorum, M. circinelloides, Rhizopus delemar, Rhizopus oryzae, and Lichtheimia corymbifera.
65. A method for treating or preventing herpes simplex virus (HSV) infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions recited in Claims 1-58.
66. The method of claim 65, wherein the ionic liquid formulation comprises adefovir dipivoxil (ADV).
67. The method of claim 65, wherein the method further comprises administering to the mammal one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir.
68. The method of claim 65, wherein the ionic liquid formulation is configured for topical administration, transdermal, ocular, systemic administration, and/or oral administration.
69. A method for treating or preventing epithelial keratitis related to HSV infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions recited in Claims 1-58.
70. The method of claim 69, wherein the ionic liquid formulation comprises adefovir dipivoxil (ADV).
71. The method of claim 69, wherein the method further comprises administering to the mammal one or more anti-HSV agents selected from a helicase-primase inhibitor, tenofovir, emtricitabine, lamivudine, interfereon, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, ledipasvir, tenofovir, ombitasvir, paritaprevir, pritelivir, brincidofovir, cidofovir, ganciclovir, valganciclovir, and ritonavir.
72. The method of claim 71 , wherein the ionic liquid formulation is configured for topical administration, optical administration, and/or oral administration.
73. A method for treating or preventing HIV infection in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions recited in Claims 1-58.
74. The method of claim 73, wherein the ionic liquid formulation comprises tenofovir disoproxil, tenofovir alafenamide, and/or adefovir dipivoxil.
75. The method of claim 73, wherein the method further comprises administering to the mammal one or more anti-HIV agents selected from atazanavir, atazanavir sulfate, bictegravir, cabotegravir, dolutegravir, doravirine, efavirenz, tenofovir disoproxil fumarate, tenofovir alafenamide, elvitegravir, etravirine, darunavir, a combination of darunavir and cobicistat, rilpivirine, doravirine, lenacapavir, and a combination thereof.
76. The method of claim 73, wherein the ionic liquid formulation is configured for topical administration, optical administration, and/or oral administration.
77. A method for treating cancers such as melanoma, breast cancer, non-small cell lung cancer, hematological cancers, renal cancer, liver cancer and brain cancers in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions recited in Claims 1-58.
78. A method of treating or preventing a parasitic infection in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions recited in Claims 1-58.
79. The method of claim 78, wherein the mammal is a human being.
80. The method of claim 78, wherein the mammal is a human being suffering from or at risk of suffering from a parasitic infection.
81. The method of claim 78, wherein the parasitic infection is selected from African trypanosomiasis, amoebiasis, ascariasis, babesiosis, Chagas disease, cryptosporidiosis, cutaneous larva migrans, dirofilariasis, echinococcosis, fasciolosis, filariasis, lymphatic filariasis, giardiasis, helminthiasis, hookworm infection, leishmaniasis, visceral leishmaniasis, malaria, neurocysticercosis, onchocerciasis, protozoan infection, schistosomiasis, taeniasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinosis, and zoonosis.
82. The method of Claim 78, further comprising administering to the mammal an antiparasitic agent.
83. The method of Claim 82, wherein the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal.
84. The method of Claim 82, wherein the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole.
85. A method of treating or preventing neurocysticercosis in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of one or more of the compositions or pharmaceutical compositions recited in Claims 1 -58.
86. The method of claim 85, wherein the mammal is a human being.
87. The method of claim 85, wherein the mammal is a human being suffering from or at risk of suffering from neurocysticercosis.
88. The method of Claim 85, further comprising administering to the mammal an antiparasitic agent.
89. The method of Claim 88, wherein the antiparasitic agent is an antiprotozoal, an antihelminthic, an antinematode, an anticestode, an antitrematode, an antiamoebic, or an antifungal.
90. The method of Claim 88, wherein the antiparasitic agent is selected from albendazole, amphotericin B, benznidazole, bephenium, diethylcarbamzine, eflornithine, flubendazole, ivermectin, mebendazole, meglumine antimonite, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nitazoxanide, pentavalent antimony, praziquantel, pyrantel, pyrvinium, sodium stibogluconate, thiabendazole, and tinidazole.
91. A method for treating parasitic infections such as malaria, human filariasis, cysticercosis, and human parasitosis in a mammal comprising administering to the mammal in need thereof one or more of the compositions or pharmaceutical compositions recited in Claims 1-58.
92. The method of claim 91 wherein said pharmaceutical composition containing ionic liquid is administered via oral, parenteral, transdermal, skin, bladder, nasal, comea/general ophthalmic, intraocular, pulmonary, mucosal, transrectal/enemas, or vaginal route of administration.
93. The method of claims 59-92, wherein the mammal is a human or agricultural or companion animal.
PCT/US2024/035153 2023-06-22 2024-06-22 Bioavailability enhancing ionic liquid formulations and uses thereof Pending WO2024264013A1 (en)

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