WO2022265880A1 - Improved methods and compositions for drug delivery relating to ionic liquids - Google Patents

Improved methods and compositions for drug delivery relating to ionic liquids Download PDF

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WO2022265880A1
WO2022265880A1 PCT/US2022/032439 US2022032439W WO2022265880A1 WO 2022265880 A1 WO2022265880 A1 WO 2022265880A1 US 2022032439 W US2022032439 W US 2022032439W WO 2022265880 A1 WO2022265880 A1 WO 2022265880A1
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composition
aspects
concentration
drug
active agent
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Samir Mitragotri
Jayoung Kim
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President And Fellows Of Harvard College
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12Carboxylic acids; Salts or anhydrides thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0048Eye, e.g. artificial tears
    • A61K9/0051Ocular inserts, ocular implants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/107Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic 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
    • A61K31/47Quinolines; Isoquinolines
    • A61K31/4738Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid

Definitions

  • the technology described herein relates to ionic liquids for stabilization and delivery of active agents.
  • ionic liquids e.g., those with an oleic acid anion
  • these compositions permit preparation of higher concentrations of drugs than older approaches such as DMSO.
  • Such compositions when comprising a chemotherapeutic drug, successfully demonstrate efficient cellular uptake as well as retention, tumor spheroid penetration, and tumor accumulation in vivo.
  • Systemic administration of the formulation significantly inhibits primary tumor growth and metastasis in vivo.
  • these compositions display a surprising ability to overcome multiple biological barriers to achieve successful cancer therapy in vivo. Accordingly, these ionic liquids are described herein as biocompatible agents for dissolving poorly water-soluble drugs.
  • compositions comprising: a) an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion; and b) an active agent.
  • the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the aspects, the active agent comprises at least one drug with a water solubility of less than 10 mg/mL.
  • the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da.
  • the drug has a water solubility of less than 1 mg/mL.
  • the at least one drug has a water solubility of less than 1 mg/mL and a molecular weight of less than 1000 Da.
  • the at least one drug is selected from a photosensitizer, an anthracylcine, ataxane, or camptothecin and camptothecin analogs.
  • the at least one drug is selected from verteporfm, gemcitabine, doxorubicin, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, doxorubicin, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, doxorubicin, and paclitaxel. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug comprises verteporfm.
  • the active agent is present at a concentration of at least 1 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 2 mg/mL. In some embodiments of any of the aspects, the ionic liquid is at a concentration of at least 20 mg/mL in water. In some embodiments of any of the aspects, the ionic liquid is at a concentration of 20-40 mg/mL in water. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 1-lOOx the concentration of the agent. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 5 -5 Ox the concentration of the agent. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 10- 3 Ox the concentration of the agent. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 15-20x the concentration of the agent.
  • the cation is choline. In some embodiments of any of the aspects, the molar ratio of cation to anion is 2: 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.5: 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1 : 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 2: 1 to 1:1, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.7: 1 to 2: 1, inclusive.
  • the molar ratio of cation to anion is 1.87: 1.
  • the ionic liquid and active agent are present together in nanocomplexes.
  • a method of treating cancer in a subject in need thereof comprising administering a composition as described herein to the subject.
  • a composition as described herein for use in a method of treating cancer in a subject in need thereof.
  • the cancer is epithelial cancer, e.g., breast cancer.
  • the composition is administered intravenously.
  • a method of treating macular degeneration in a subject in need thereof comprising administering a composition as described herein to the subject.
  • a composition as described herein for use in a method of treating macular degeneration in a subject in need thereof.
  • the at least one drug comprises verteporfm.
  • the composition is administered intraocularly.
  • a method of drug delivery comprising administering a composition as described herein to a subject in need of the active agent.
  • a composition as described herein for use in a method of drug delivery.
  • the administration and/or drug delivery is intravenous, parenteral, or intraocular.
  • Figs. 1A-1C depict Choline and Oleic acid Deep Eutectic Solvent (CODES).
  • Fig. 1A Synthesis of CODES via metathesis reaction between choline bicarbonate and oleic acid
  • Fig. IB 'H NMR spectrum of CODES indicating 1.87: 1 ratio of choline to oleate
  • Fig. 1C photographs of CODES at room temperature (top) and 100 °C (bottom).
  • FIGs. 2A-2E depict the characterization of COVERT40.
  • Fig. 2D x-ray diffraction of verteporfm powder, COVERT40, and VPDMSO
  • Fig. 2A-2E depict the characterization of COVERT40.
  • Fig. 2B transmission electron microscopy image of C
  • Figs. 3A-3C depict the in vitro cell killing efficacy in 4T1 murine breast cancer cells.
  • Fig. 3A Cytotoxicity of COVERT and VPDMSO formulations with 3 hr treatment followed by 24 hr incubation in fresh media
  • Fig. 3B IC50 values
  • Figs. 4A-4C depict in vitro cellular uptake and retention in 4T1 murine breast cancer cells. Fluorescence intensity of verteporfm per cell was detected by flow cytometry.
  • Fig. 3A-3C depict the in vitro cell killing efficacy in 4T1 murine breast cancer cells.
  • Fig. 3A Cytotoxicity of COVERT and VPDMSO formulations with 3 hr treatment followed by 24 hr incubation in fresh media
  • Fig. 3B IC50 values
  • Figs. 5A-5B depict three-dimensional multicellular tumor spheroid penetration.
  • Figs. 6A-6E depict the in vivo biodistribution and efficacy on orthotopic 4T1 breast tumor.
  • Fig. 6A Biodistribution of COVERT40 and VPDMSO in major organs and tumor at 3 hr post intravenous injection of 5 mg/kg drug dose and
  • Fig. 6C primary tumor growth of 4T1 inoculated in mammary fat pad with four tail-vein injections of therapeutics 3 days apart (n>5, one-way ANOVA with Sidak multiple comparison test)
  • Fig. 6D body weight
  • Fig. 6E number of metastatic nodules from lungs harvested at the termination of tumor growth inhibition study on day 26 (n>5, one-way ANOVA with Tukey multiple comparison test). All data are presented as mean ⁇ sd.
  • Figs. 7A-7B depict the characterization of CODES.
  • Fig. 7B photograph of paste-like CODES at 37 °C.
  • Figs. 10A-10D depict the in vitro cellular uptake and retention of COVERTso in 4T1 murine breast cancer cells. Fluorescence intensity of verteporfm per cell was detected by flow cytometry.
  • Figs. 1 lA-1 IB depict three-dimensional multicellular tumor spheroid penetration. Fig.
  • FIG. 11A Histogram plot of Hoescht staining of tumor spheroid by flow cytometry used to separate the spheroid into inner core and outer perimeter layers
  • FIGs. 12A-12E depict the in vivo biodistribution, efficacy, and safety on orthotopic 4T1 breast tumor.
  • Fig. 12C photographs of primary 4T1 tumor tissue excised from the mammary fat pad at the termination of the tumor growth inhibition study on day 26, Fig.
  • FIG. 12D representative H&E-stained sections of the major organs harvested following four intravenous injections of the formulations
  • Fig. 12E metastatic nodules visualized from histological sections of the lungs harvested at the termination of tumor growth inhibition study on day 26 (black arrows indicate metastatic nodules).
  • compositions comprising certain ionic liquids, e.g., those with an oleic acid anion, and an active agent, e.g., a small hydrophic drug, can provide solubilized drugs at significantly higher concentrations than prior art approaches. Additionally, these compositions display a surprising ability to overcome multiple biological barriers to achieve successful therapy in vivo, providing effective and superior parenteral (e.g., intravenous) delivery.
  • certain ionic liquids e.g., those with an oleic acid anion
  • an active agent e.g., a small hydrophic drug
  • composition comprising: a) an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion (CODES); and b) an active agent.
  • an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion (CODES); and b) an active agent.
  • composition comprising: a) an ionic liquid comprising i) a choline cation and ii) an oleate and/or oleic acid anion (CODES); and b) an active agent.
  • an ionic liquid comprising i) a choline cation and ii) an oleate and/or oleic acid anion (CODES); and b) an active agent.
  • CODES oleate and/or oleic acid anion
  • ionic liquids refers to organic salts or mixtures of organic salts which are in liquid state at room temperature. This class of solvents has 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 at least one anionic and at least one cationic component may be present in any molar ratio.
  • Exemplary molar ratios include but are not limited to 1 : 1, 1:2, 2: 1, 1 :3, 3: 1, 2:3, 3:2, and ranges between these ratios.
  • ionic liquids see, e.g., Hough, et ah , "The third evolution of ionic liquids: active pharmaceutical ingredients", New Journal of Chemistry, 31 : 1429 (2007) and Xu, et al., "Ionic Liquids: Ion Mobilities, Glass Temperatures, and Fragilities", Journal of Physical Chemistry B, 107(25): 6170-6178 (2003); each of which is incorporated by reference herein in its entirety.
  • the ionic liquid or solvent exists as a liquid below 100 °C.
  • the ionic liquid or solvent exists as a liquid at room temperature.
  • the cation of an IL described herein can be a cation comprising a quaternary ammonium.
  • a quaternary ammonium is a positively charged polyatomic ion of the structure NR_f. each R independently being an alkyl group or an aryl group.
  • the general term “quaternary ammonium” relates to any compound that can be regarded as derived from ammonium hydroxide or an ammonium salt by replacement of all four hydrogen atoms of the NFL t + ion by organic groups.
  • the quaternary ammonium has the structure of NRi + , where each R is independently selected from hydroxyl, optionally substituted Ci-Cioalkyl, optionally substituted C2-Cioalkenyl, optionally substituted C2-Cioalkynyl, optionally substituted aryl, or optionally substituted heteroaryl.
  • the cation has a molar mass equal to or greater than choline, e.g., a molar mass equal to or greater than 104.1708 g/mol. In some embodiments of any of the aspects, the cation has a molar mass greater than choline, e.g., a molar mass equal greater than 104.1708 g/mol.
  • each R group of the quaternary ammonium independently comprises an alkyl, alkane, alkene, or aryl. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl, alkane, or alkene. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkane or alkene. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 10 carbon atoms in length, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms in length.
  • each R group of the quaternary ammonium independently comprises a carbon chain of no more than 12 carbon atoms in length. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 15 carbon atoms in length. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 20 carbon atoms in length.
  • each R group of the quaternary ammonium independently comprises a carbon chain of no more than 10 carbon atoms, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 12 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 15 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 20 carbon atoms.
  • each R group of the quaternary ammonium independently comprises an alkyl group of no more than 10 carbon atoms, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl group of no more than 12 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl group of no more than 15 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl group of no more than 20 carbon atoms.
  • each R group of the quaternary ammonium independently comprises an alkane, alkene, aryl, heteroaryl, alkyl, or heteroalkyl. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an unsubstituted alkane, unsubstituted alkene, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkyl, or unsubstituted heteroalkyl. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an unsubstituted alkane.
  • each R group of the quaternary ammonium independently comprises an unsubstituted alkene. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises one or more substituent groups.
  • At least one R group of the quaternary ammonium comprises a hydroxy group. In some embodiments of any of the aspects, one R group of the quaternary ammonium comprises a hydroxy group. In some embodiments of any of the aspects, only one R group of the quaternary ammonium comprises a hydroxy group.
  • Exemplary, non-limiting cations can include choline, and any of the cations designated C1-C7 which are defined by structure below.
  • cations include the following: 1 -(hydroxymethyl)- 1 -methylpyrrolidin- 1 -ium 1 -(2-hydroxy ethyl) - 1 -methylpyrrolidin- 1 -ium 1 -ethyl- 1 -(3 -hydroxypropyl)pyrrolidin- 1 -ium 1 -(3-hydroxypropyl)- 1 -methylpyrrolidin- 1 -ium 1 -(4-hydroxybutyl)- 1 -methylpyrrolidin- 1 -ium 1 -ethyl- 1 -(4-hydroxybutyl)pyrrolidin- 1 -ium 1 -(4-hydroxybutyl)- 1 -propylpyrrolidin- 1 -ium 1 -(5-hydroxypentyl)- 1 -propylpyrrolidin- 1 -ium 1 -ethyl- 1 -(5-hydroxypentyl)pyrrolidin- 1 - 1 -ethyl- 1
  • the cation is choline, Cl, C6, and/or C7. In some embodiments of any of the aspects, the cation is Cl, C6, and/or C7. In some embodiments of any of the aspects, the cation is choline.
  • the anion of an ionic liquid described herein can comprise oleate and/or oleic acid.
  • the structure of oleic acid is:
  • Salts and esters of oleic acids are known as oleates.
  • a first and second ionic liquid have the same cation, e.g., choline.
  • a first and second ionic liquid have different anions.
  • the IL is at a concentration of at least 0.01% w/v. In some embodiments of any of the aspects, the IL is at a concentration of at least 0.05% w/v.
  • the IL is at a concentration of at least 0.1% w/v. In some embodiments of any of the aspects, the IL is at a concentration of at least 0.2% w/v, at least 0.3% w/v, at least 0.4% w/v, at least 0.5% w/v, at least 1% w/v or greater. In some embodiments of any of the aspects, the IL is at a concentration of from about 0.01% w/v to about 1% w/v. In some embodiments of any of the aspects, the IL is at a concentration of from 0.01% w/v to 1% w/v.
  • the IL is at a concentration of from about 0.05% w/v to about 0.5% w/v. In some embodiments of any of the aspects, the IL is at a concentration of from 0.05% w/v to 0.5% w/v. [0039] In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in water. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in saline or a physiologically compatible buffer.
  • the IL is at a concentration of from about 5% w/w to about 75% w/w. In some embodiments of any of the aspects, the IL is at a concentration of from 5% w/w to 75% w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 5% w/w to about 75% w/w in water, saline or a physiologically compatible buffer. In some embodiments of any of the aspects, the IL is at a concentration of from 5% w/w to 75% w/w in water, saline or a physiologically compatible buffer.
  • the IL is at a concentration of at least about 0.1 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of at least 0.1 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 10 % w/w to about 70 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from 10 % w/w to 70 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 30 % w/w to about 50 % w/w.
  • the IL is at a concentration of from 30 % w/w to 40 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 30 % w/w to about 50 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from 30 % w/w to 40 % w/w.
  • the % w/w concentration of the IL is % w/w concentration in water, saline, or a physiologically compatible buffer.
  • the IL is 100% by w/w or w/v.
  • the IL is an anhydrous salt, e.g., an ionic liquid not diluted or dissolved in water.
  • the IL is provided as an aqueous solution.
  • the IL is at a concentration of at least 25% w/w and has a ratio of catiomanion of at least 1:3. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in water and has a ratio of catiomanion of at least 1:3. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w and has a ratio of catiomanion of 1:3 or 1:4. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in water and has a ratio of cation: anion of 1:3 or 1:4. In some embodiments of any of the aspects, the IL is a gel, or a shear-thining Newtonian gel.
  • the IL has a ratio of cation: anion of from about 10: 1 to about 1: 10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 10: 1 to 1 : 10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 5: 1 to about 1:5. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 5: 1 to 1:5. In some embodiments of any of the aspects, the IL has a ratio of cation:anion of from about 2: 1 to about 1:4.
  • the IL has a ratio of catiomanion of from 2: 1 to 1 :4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 2: 1 to about 1:10. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from about 2: 1 to about 1 : 1. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 2: 1 to 1:10. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from 2: 1 to 1 : 1.
  • the IL has a ratio of catiomanion such that there is a greater amount of anion, e.g., a ratio of less than 1:1. In some embodiments of any of the aspects, the IL has a ratio of catiomanion such that there is an excess of anion. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 1: 1 to about 1:10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 1 : 1 to 1 : 10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 1: 1 to about 1:4.
  • the IL has a ratio of catiomanion of from 1:1 to 1:4. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from about 1: 1 to about 1:3. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 1 : 1 to 1:3. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from about 1 : 1 to about 1 :2. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from 1:1 to 1:2.
  • the IL has a ratio of catiomanion of about 1:1, 1:2, 1:3, or 1:4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of 1 : 1, 1:2, 1 :3, or 1:4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion less than about of 1 : 1. In some embodiments of any of the aspects, the IL has a ratio of catiomanion less than 1:1. Without wishing to be constrained by theory, compositions with higher amounts of anion relative to cation display greater hydrophobicity. [0047] In some embodiments of any of the aspects, the IL has a catiomanion ratio with an excess of cation.
  • the molar ratio of cation to anion is 2: 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.5:1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1 : 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 2: 1 to 1:1, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is
  • the molar ratio of cation to anion is 1.87:1.
  • the IL is at a concentration of at least 20 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least about 20 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least 25 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least about 25 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least 50 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least about 50 mM.
  • the IL is at a concentration of at least 100 mM, 500 mM, 1 M, 2 M, 3 M or greater. In some embodiments of any of the aspects, the IL is at a concentration of at least about 100 mM, 500 mM, 1 M, 2 M, 3 M or greater.
  • the IL is at a concentration of from about 50 mM to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 50 mM to 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from about 500 mM to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 500 mM to 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from about 1 M to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 1 M to 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from about 2 M to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 2 M to 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 2 M to 4 M.
  • the IL concentration in the composition or formulation is about 0.1 mM to 20 mM. In some embodiments of any of the aspects, the IL concentration in the composition or formulation is about 0.5 mM to 20 mM, 0.5 mM to 18 mM, 0.5 mM to 16 mM, 0.5 mM to 14 mM, 0.5 mM to 12 mM, 0.5 mM to 10 mM, 0.5 mM to 8 mM, 1 mM to 20 mM, 1 mM to 18 mM, 1 mM to 16 mM, 1 mM to 14 mM, ImM to 12 mM, 1 mM to 10 mM, 1 mM to 8 mM, 2 mM to 20 mM, 2 mM to 18 mM, 2 mM to 16 mM, 2 mM to 14 mM, 2 mM to 12 mM, 2 mM
  • the IL concentration in the composition or formulation is about ImM, about 2 mM, about 3mM, about 4mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM or about 20 mM.
  • a composition or combination described herein can comprise one, two, three, or more of any of the types of components described herein.
  • a composition can comprise a mixture, solution, combination, or emulsion of two or more different ionic liquids (e.g., different ionic liquids described herein), and/or a mixture, solution, combination, or emulsion of two or more different active agents.
  • the one or more ILs can be in combination with at least one active agent.
  • the active agent(s) can be comprised by a superstructure, e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like, said superstructure is which in solution, mixture, admixture, suspension, etc., with the IL.
  • an “active compound” or “active agent” is any agent which will exert an effect on a target cell or organism.
  • the terms “compound” and “agent” refer to any entity which is normally not present or not present at the levels being administered and/or provided to a cell, tissue or subject.
  • An agent can be selected from a group comprising: chemicals; small organic or inorganic molecules; signaling molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; enzymes; aptamers; peptidomimetic, peptide derivative, peptide analogs, antibodies; intrabodies; biological macromolecules, extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions or functional fragments thereof.
  • the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities.
  • Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • Non-limiting examples of active agents contemplated for use in the methods described herein include small molecules, drugs, polypeptides, nucleic acids, chemotherapies/chemotherapeutic compounds, antibodies, and antibody reagents.
  • the active agent can be a therapeutic compound or drug, e.g., an agent or compound which is therapeutically effective for the treatment of at least one condition in a subject.
  • Therapeutic compounds are known in the art for a variety of conditions, see, e.g., the database available on the world wide web at drugs.com or the catalog of FDA-approved compounds available on the world wide web at catalog.data.gov/dataset/drugsfda- database; each of which is incorporated by reference herein in its entirety.
  • the active agent comprises, consists of, consists essentially of, or is at least one drug. In some embodiments of any of the aspects, the active agent comprises, consists of, consists essentially of, or is one drug. In some embodiments of any of the aspects, the active agent comprises, consists of, consists essentially of, or is a combination of two or more drugs.
  • the drug is a photosensitizer.
  • photosensitizers include 5-Aminolaevulinic acid, verteporfm, purlytin, foscan, lutex, ATMPn, zinc phthalocyanine, naphthalocyanines, expanded metallo-porphyrins, metallochlorins/bacteriochlorins, metallo-phthalocyanines, metallo-naphthocyaninesulfobenzo- porphyrazines (M-NSBP), and metallo-naphthalocyanines.
  • Drugs include chemotherapeutic agents.
  • chemotherapeutic agent refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation.
  • a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor.
  • chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells.
  • a chemotherapeutic agent is a radioactive molecule.
  • the chemotherapeutic agent is a taxane (e.g., paclitaxel, docetaxel, cabazitaxel, and taxine).
  • a taxane e.g., paclitaxel, docetaxel, cabazitaxel, and taxine.
  • the chemotherapeutic is a topoisomerase inhibitor.
  • the topoisomerase inhibitor is camptothecin or a camptothecin analog (e.g., topotecan, irinotecan, belotecan, and tastuzumab deruxtecan).
  • the chemotherapeutic is an anthracycline (e.g., doxorubicin, daunorubicin, epirubicin, and idarubicin).
  • anthracycline e.g., doxorubicin, daunorubicin, epirubicin, and idarubicin.
  • the active agent e.g., the drug
  • the term “small molecule” refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • the active agent has a molecular weight of less than 1000 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 900 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 800 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 700 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 600 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 500 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 400 Da.
  • the active agent has a molecular weight of less than 300 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 200 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 100 Da.
  • the active agent e.g., the drug
  • the active agent is poorly water soluble.
  • the active agent, e.g., the drug has a water solubility of less than 10 mg/mL.
  • the active agent, e.g., the drug has a water solubility of less than 9 mg/mL.
  • the active agent, e.g., the drug has a water solubility of less than 8 mg/mL.
  • the active agent, e.g., the drug has a water solubility of less than 7 mg/mL.
  • the active agent e.g., the drug
  • the active agent e.g., the drug
  • the water solubility is the water solubility at a physiological pH.
  • the water solubility is the water solubility over the physiological pH range (e.g., 7.35-7.45).
  • the water solubility is the water solubility at pH 7.4.
  • the water solubility for several small molecule drugs are:
  • a drug described herein has a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the asepcts, a drug described herein has a water solubility of less than 5 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the asepcts, a drug described herein has a water solubility of less than 1 mg/mL and a molecular weight of less than 1000 Da.
  • Non-limiting examples of drugs include verteporfm, gemcitabine, doxorubicin, paclitaxel, and camptothecin.
  • the at least one drug is selected from verteporfm, doxorubicin, paclitaxel, and camptothecin.
  • the at least one drug is selected from the at least one drug is selected from verteporfm, doxorubicin, and paclitaxel.
  • the at least one drug is selected from the at least one drug is selected from verteporfm, paclitaxel, and camptothecin.
  • the at least one drug comprises, consists of, or consists essentially of paclitaxel. In some embodiments of any of the aspects, the at least one drug comprises, consists of, or consists essentially of camptothecin. In some embodiments of any of the aspects, the at least one drug comprises, consists of, or consists essentially of verteporfm.
  • the active agent is present at a concentration of at least 1 mg/mL, e.g, at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 20 mg/mL, at least 30 mg/mL, at least 40 mg/mL, or more.
  • the active agent is present at a concentration of at least 2 mg/mL.
  • the active agent is present at a concentration of at least 5 mg/mL.
  • the active agent is present at a concentration of at least 10 mg/mL.
  • the active agent is present at a concentration of at least 20 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 30 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 40 mg/mL.
  • the active agent is present at a concentration of 1-100 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-50 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-40 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-100 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-50 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-40 mg/mL.
  • the active agent is present at a concentration of 20-100 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-50 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-40 mg/mL.
  • the active agent is present at a concentration of at least 1 mg/mL, e.g, at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 20 mg/mL, at least 30 mg/mL, at least 40 mg/mL, or more, in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 2 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 5 mg/mL in water.
  • the active agent is present at a concentration of at least 10 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 20 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 30 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 40 mg/mL in water.
  • the active agent is present at a concentration of 1-100 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-50 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-40 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-100 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-50 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-40 mg/mL in water.
  • the active agent is present at a concentration of 20-100 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-50 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-40 mg/mL in water.
  • the the ionic liquid is at a concentration which is 1-lOOx the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 5-50x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 10-3 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 15 -2 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL.
  • the the ionic liquid is at a concentration which is at least 5x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least lOx the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least 15x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL.
  • the the ionic liquid is at a concentration which is no greater than lOOx the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 5 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 3 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 200x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL.
  • the the ionic liquid is at a concentration which is 1-lOOx the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 5 -5 Ox the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 10-30x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL.
  • the the ionic liquid is at a concentration which is 15 -2 Ox the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. [0079] In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least 5x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least lOx the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL.
  • the the ionic liquid is at a concentration which is at least 15x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than lOOx the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 5 Ox the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL.
  • the the ionic liquid is at a concentration which is no greater than 30x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 200x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL.
  • composition refers to any IL, combination of ILs, or combination of one or more ILs and one or more active agents described herein, unless further specified.
  • a composition or combination as described herein, comprising at least one IL and optionally an active agent can be formulated as an oral, subcutaneous, transdermal, intratumoral, intravenous, intradermal, or parenteral formulation.
  • the composition or combination as described herein can be formulated for delivery to a mucus membrane, e.g., to a nasal, oral, or vaginal membrane.
  • an oral formulation can be a degradable capsule comprising the composition comprising the at least one IL and optionally, an active agent.
  • composition or combination as described herein can be formulated for intravenous or intraocular delivery. In some embodiments of any of the aspects, the composition or combination as described herein can be formulated for intravenous delivery. In some embodiments of any of the aspects, the composition or combination as described herein can be formulated for in intraocular delivery.
  • compositions comprising at least one IL as described herein and at least one active agent. In some embodiments of any of the aspects, described herein is a composition consisting essentially of at least one IL as described herein and at least one active agent. In some embodiments of any of the aspects, described herein is a composition consisting of at least one IL as described herein and at least one active agent. In some embodiments of any of the aspects, the composition comprising at least one IL as described herein and at least one active agent is administered as a monotherapy, e.g., another treatment for the condition is not administered to the subject.
  • a pharmaceutical composition comprising at least one active agent in combination with at least one IL as described herein.
  • the pharmaceutical composition comprises the at least one IL and the one or more active agents as described herein.
  • the pharmaceutical composition consists essentially of the at least one IL and the one or more active agents as described herein.
  • the pharmaceutical composition consists of the at least one IL and the one or more active agents as described herein.
  • the pharmaceutical composition consists essentially of an aqueous solution of the at least one IL and the one or more active agents as described herein.
  • the pharmaceutical composition consists of an aqueous solution of the at least one IL and the one or more active agents as described herein.
  • compositions, formulations, and combinations described herein can comprise at least one IL as described herein, e.g., one IL, two ILs, three ILs, or more.
  • a composition, formulation, or combination as described herein can comprise at least one IL as described herein and CAGE (Choline And GEranate).
  • the at least one active agent and the at least one ionic liquid are further in combination with at least one non-ionic surfactant.
  • non-ionic surfactant refers to a surfactant that lacks a net ionic charge and does not dissociate to an appreciable extent in aqueous media.
  • the properties of non-ionic surfactants are largely dependent upon the proportions of the hydrophilic and hydrophobic groups in the molecule. Hydrophilic groups include the oxyethylene group (— OCH2 CH2 — ) and the hydroxy group.
  • hydrophobic molecule such as a fatty acid
  • substances are obtained which range from strongly hydrophobic and water insoluble compounds, such as glyceryl monostearate, to strongly hydrophilic and water-soluble compounds, such as the macrogols.
  • strongly hydrophobic and water insoluble compounds such as glyceryl monostearate
  • strongly hydrophilic and water-soluble compounds such as the macrogols.
  • Suitable non ionic surfactants may be found in Martindale, The Extra Pharmacopoeia, 28th Edition, 1982, The Pharmaceutical Press, London, Great Britain, pp. 370 to 379.
  • Non-limiting examples of non-ionic surfactants include polysorbates, a TweenTM, block copolymers of ethylene oxide and propylene oxide, glycol and glyceryl esters of fatty acids and their derivatives, polyoxyethylene esters of fatty acids (macrogol esters), polyoxyethylene ethers of fatty acids and their derivatives (macrogol ethers), polyvinyl alcohols, and sorbitan esters, sorbitan monoesters, ethers formed from fatty alcohols and polyethylene glycol, polyoxyethylene-polypropylene glycol, alkyl polyglycoside, Cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, decyl polyglucose, glycerol monostearate, IGEPAL CA-630, isoceteth-20, lauryl glucoside, maltosides, monolaurin, mycosubt
  • polysorbate refers to a surfactant derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Common brand names for polysorbates include ScatticsTM, AlkestTM, CanarcelTM, and TweenTM. Exemplary polysorbates include polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 0.1% to about 50% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of 0.1% to 50% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 1% to about 5% w/v.
  • the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of 1% to 5% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 3% to about 10% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of 3% to 10% w/v.
  • the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of less than about 5% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of less than 5% w/v.
  • the combination of the at least one active agent and at least one IL as described herein is provided in one or more nanoparticles.
  • the combination of the at least one active agent and at least one IL as described herein comprises nanoparticles comprising the active agent, the nanoparticles in solution or suspension in a composition comprising at least one IL as described herein.
  • a composition as described herein e.g., a composition comprising at least one IL and an active agent
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier
  • pharmaceutically acceptable physiologically tolerable
  • grammatical variations thereof as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • a pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
  • the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition of the present disclosure can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field of art.
  • a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
  • carrier in the context of a pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations, and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). The formulation should suit the mode of administration.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media.
  • the use of such carriers and diluents is well known in the art.
  • Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean
  • polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non toxic compatible substances employed in pharmaceutical formulations.
  • PEG polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • agar such as a
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent.
  • pharmaceutically acceptable carrier excludes tissue culture medium.
  • a composition as described herein e.g., a composition comprising at least one IL as described herein and an active agent
  • a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent can be formulated as an intraocular, subcutaneous, intravenous, intradermal, or parenteral formulation.
  • a composition as described herein e.g., a composition comprising at least one IL as described herein and an active agent
  • a composition as described herein can be formulated as a subcutaneous, intravenous, intradermal, or parenteral formulation.
  • a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent can be formulated as a intravenous or parenteral formulation.
  • a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent can be formulated as an intravenous formulation.
  • a composition as described herein e.g., a composition comprising at least one IL as described herein and an active agent
  • an oral formulation can be formulated as an intraocular formulation.
  • an oral formulation can be a degradable capsule comprising the composition described herein, e.g., a composition comprising at least one IL as described herein and an active agent.
  • an oral formulation can be a capsule comprising a degradable coating or enteric coating, and further comprising the composition described herein, e.g., a composition comprising at least one IL as described herein and an active agent.
  • the biological activity of the active agent is improved or stabilized as compared to the activity in the absence of the at least one IL.
  • the IL greatly enhances permeation of the active agent across the skin compared to a control where the at least one IL is absent.
  • the composition or combination described herein is for a method of administering or delivering at least one active agent, e.g., for the treatment of a disease.
  • described herein is a method of administering at least one active agent, the method comprising administering the active agent in combination with at least one IL as described herein.
  • described herein is a method of treating a disease by administering at least one active agent, the method comprising administering the active agent in combination with at least one IL as described herein.
  • the disease treated by the methods described herein can be, e.g., cancer (breast cancer, leukemia, lymphoma, B-cell chronic lymphocytic leukemia, glioblastoma, carcinoma, urothelial carcinoma, lung cancer, colorectal cancer, lymphoblastic leukemia, lymphocytic leukemia, sarcoma, melanoma, prostate cancer, myeloma, multiple myeloma, Non-Hodgkin’s lymphoma), neuroblastoma, macular degeneration, and the like.
  • cancer breast cancer, leukemia, lymphoma, B-cell chronic lymphocytic leukemia, glioblastoma, carcinoma, urothelial carcinoma, lung cancer, colorectal cancer, lymphoblastic leukemia, lymphocytic leukemia, sarcoma, melanoma, prostate cancer, myeloma, multiple myeloma, Non-Hod
  • the methods described herein relate to treating a subject having or diagnosed as having a condition with a composition as described herein, e.g., a comprising at least one IL and an active agent.
  • a condition e.g., cancer
  • Subjects having a condition can be identified by a physician using current methods of diagnosing cancer.
  • Symptoms and/or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, for example, a lump/mass/tumor, swelling, or pain. Tests that may aid in a diagnosis of, e.g.
  • cancers include, but are not limited to, x-rays, MRI, ultrasound, a biopsy, or tests for the function/activity of affected organs or systems.
  • a family history of cancer or exposure to risk factors for cancer e.g. smoke, radiation, pollutants, mutation, etc. can increase the risk of a subject having cancer.
  • compositions and methods described herein can be administered to a subject having or diagnosed as having a condition described herein.
  • the methods described herein comprise administering an effective amount of compositions described herein, e.g. a composition comprising at least one IL as described herein and an active agent, to a subject in order to alleviate a symptom of a condition described herein.
  • "alleviating a symptom” is ameliorating any marker or symptom associated with a condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • compositions described herein can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration can be local or systemic.
  • the administration is intravenous. In some embodiments of any of the aspects, the administration is intraocular. In some embodiments of any of the aspects, the administration is transdermal. In some embodiments of any of the aspects, the administration is transdermal, to a mucus membrane (e.g., to a nasal, oral, or vaginal membrane), oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous.
  • a mucus membrane e.g., to a nasal, oral, or vaginal membrane
  • Oral administration can comprise providing tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion.
  • Oral formulations can comprise discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion.
  • Such compositions contain a predetermined amount of the IL and the at least one active agent, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
  • intraocular, subcutaneous, intradermal or intravenous administration comprises administration via injection, catheter, port, or the like.
  • parenteral delivery of at least one active agent comprising parenterally administering the active agent in combination with at least one IL as described herein.
  • the parenteral administration comprises delivery to a tumor, e.g., a cancer tumor.
  • the composition or combination described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.
  • parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS ® -type dosage forms and dose-dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms of a composition comprising at least one IL (e.g., CODES) in combination with at least one active agent as disclosed within are well known to those skilled in the art.
  • aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection
  • water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol
  • non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • compositions as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • compositions comprising the at least one IL in combination with at least one active agent can obviate certain reasons for using a controlled-release formulation
  • the methods and compositions can be utilized in controlled-release formulations in some embodiments.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • the composition comprising the at least one IL in combination with at least one active agent can be administered in a sustained release formulation.
  • Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated herein by reference.
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
  • active ingredients for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
  • OROS ® Alza Corporation, Mountain View, Calif. USA
  • the term “effective amount” as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the term "therapeutically effective amount” therefore refers to an amount of a composition that is sufficient to provide a particular effect when administered to a typical subject.
  • An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • a treatment for cancer is effective when the rate of growth of a tumor or cancer cell population is slowed. In some embodiments of any of the aspects, a treatment for cancer is effective when the growth of a tumor or cancer cell population is stopped. In some embodiments of any of the aspects, a treatment for cancer is effective when a tumor or cancer cell population is reduced in size. In some embodiments of any of the aspects, a treatment for cancer is effective when the rate of metastasis is slowed. In some embodiments of any of the aspects, a treatment for cancer is effective when the rate of metastasis is slowed. In some embodiments of any of the aspects, a treatment for cancer is effective when the rate of cell death in a tumor or cancer cell population is increased.
  • a treatment for macular degeneration is effective when the rate of growth of one or more blood vessels under the macular is slowed. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the growth of one or more blood vessels under the macular is stopped. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of blood or fluid leakage into the retina is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of blood or fluid leakage into the retina is slowed.
  • a treatment for macular degeneration is effective when the rate of retinal pigment epithelium thinning or depigrmentation is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when retinal pigment epithelium thinning or depigrmentation is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when retinal pigment epithelium thinning or depigrmentation is stopped. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of photoreceptor death is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when photoreceptor death is stopped.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e..
  • the concentration of the active agent which achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor cell growth, among others.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the active agent can be a chemotherapeutic agent or agent effective for the treatment of cancer.
  • described herein is a method of treating cancer, the method comprising administering a composition described herein, comprising at least one IL and at least one active agent, to the subject.
  • the at least one active agent is a chemotherapeutic agent or agent effective for the treatment of cancer.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems.
  • Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.
  • Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
  • Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
  • the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e.. intrusion on and destruction of adjacent tissues), and metastasis (i.e. , spread to other locations in the body via lymph or blood).
  • metastasize refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre -malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • neoplasm refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues.
  • a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastases. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma
  • a “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material.
  • transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.
  • Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
  • the cancer is an epithelial cancer. In some embodiments of any of the aspects, the cancer is breast cancer.
  • the composition as described herein e.g., a composition comprising at least one IL as described herein in combination with at least one active agent, is administered as a monotherapy, e.g., another treatment for the condition is not administered to the subject.
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy, either in the composition described herein, e.g., a composition comprising at least one IL as described herein in combination with at least one active agent, or as a separate formulation.
  • non-limiting examples of a second agent and/or treatment for treatment of cancer can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacin
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxy doxorubicin
  • irinotecan Camptosar, CPT-11 (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC -alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the methods of PKC -alpha, Raf, H-Ras, EGFR e.g., erlotinib (Tarceva®)
  • VEGF-A that reduce cell proliferation and pharmaceutically
  • the active agent can be an agent effective for the treatment of macular degeneration, e.g., a photosensitizer (for example verteporfm).
  • a photosensitizer for example verteporfm.
  • described herein is a method of treating macular degeneration, the method comprising administering a composition described herein, comprising at least one IF and at least one active agent, to the subject.
  • the at least one active agent is an agent effective for the treatment of macular degeneration.
  • the composition is administered intraocularly.
  • an effective dose of a composition described herein can be administered to a patient once.
  • an effective dose a composition described herein e.g., a composition comprising at least one IF as described herein in combination with at least one active agent
  • subjects can be administered a therapeutic amount of a composition described herein, e.g., a composition comprising at least one IF as described herein in combination with at least one active agent, such as, e.g.
  • the at least one active agent is present in the combination at a dose of from about 1.0-40.0 mg/kg. In some embodiments of any of the aspects, the at least one active agent is present in the combination at a dose of from 1.0-40.0 mg/kg. In some embodiments of any of the aspects, the at least one active agent is present in the combination at a dose of from about 1.0- 20.0 mg/kg. In some embodiments of any of the aspects, the at least one active agent is present in the combination at a dose of from 1.0-20.0 mg/kg.
  • the treatments after an initial treatment regimen, can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
  • Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active agent.
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
  • dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
  • a composition described herein, e.g., a composition comprising at least one IL in combination with at least one active agent can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the dosage ranges for the administration of the compositions described herein, according to the methods described herein depend upon, for example, the form of the active agent, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for symptoms or markers.
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • compositions described in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician.
  • a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate.
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of diabetes or cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.
  • compositions described herein e.g., a composition comprising at least one IL in combination with at least one active agent.
  • the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising).
  • other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.
  • compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.
  • the terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments of any of the aspects, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g.
  • the absence of a given treatment or agent can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4- fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of conditions described herein.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • the terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, of the full length polypeptide.
  • Conservative substitution variants that maintain the activity of wildtype protein will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • the nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • composition or active agent described herein is exogenous. In some embodiments of any of the aspects, the composition or active agent described herein is ectopic. In some embodiments of any of the aspects, the composition or active agent described herein is not endogenous.
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • exogenous can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • endogenous refers to a substance that is native to the biological system or cell.
  • ectopic refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.
  • a nucleic acid encoding a polypeptide as described herein is comprised by a vector.
  • a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof is operably linked to a vector.
  • the term "vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • a vector can be viral or non- viral.
  • the term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells.
  • a vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
  • the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).
  • non-native e.g., heterologous
  • the vector or nucleic acid described herein is codon-optomized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system.
  • the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism).
  • the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector.
  • sequences expressed will often, but not necessarily, be heterologous to the cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes.
  • the vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies.
  • the vector is episomal.
  • the use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer or macular degeneration.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with, e.g, cancer or macular degeneration.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • prophylactic refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom.
  • Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to metastasis or vision worsening. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
  • the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • nanoparticle refers to particles that are on the order of about 1 to 1,000 nanometers in diameter or width.
  • nanoparticle includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork.
  • nanoparticles also encompasses liposomes and lipid particles having the size of a nanoparticle. Exemplary nanoparticles include lipid nanoparticles or ferritin nanoparticles.
  • Lipid nanoparticles can comprise multiple componenents, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.
  • ionizable lipids such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102
  • pegylated lipids such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159
  • phospholipids such as DSPC
  • Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.
  • mPEG-DSPE methoxypolyethylene glycol
  • PC phosphatidyl choline
  • PG phosphatidyl glycerol
  • distearoylphosphatidylcholine and combinations thereof.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • statically significant or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • the present technology may be defined in any of the following numbered paragraphs:
  • a composition comprising: a) an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion; and b) an active agent.
  • composition of paragraph 1, wherein the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da.
  • composition of any one of the preceding paragraphs, wherein the at least one drug is selected from a photosensitizer, an anthracylcine, ataxane, or camptothecin and camptothecin analogs.
  • composition of any one of the preceding paragraphs, wherein the at least one drug is selected from verteporfm, gemcitabine, doxorubicin, paclitaxel, and camptothecin.
  • composition of any one of the preceding paragraphs, wherein the at least one drug is selected from verteporfm, doxorubicin, paclitaxel, and camptothecin.
  • composition of any one of the preceding paragraphs, wherein the at least one drug is selected from verteporfm, paclitaxel, and camptothecin.
  • the composition of any one of the preceding paragraphs, wherein the at least one drug comprises verteporfm.
  • the composition of any one of the preceding paragraphs, wherein the active agent is present at a concentration of at least 1 mg/mL.
  • the composition of any one of the preceding paragraphs, wherein the active agent is present at a concentration of at least 2 mg/mL.
  • the ionic liquid is at a concentration of at least 20 mg/mL in water.
  • composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration of 20-40 mg/mL in water.
  • the composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 1-lOOx the concentration of the agent.
  • the composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 5 -5 Ox the concentration of the agent.
  • the composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 10-3 Ox the concentration of the agent.
  • concentration of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 15 -2 Ox the concentration of the agent.
  • composition of any one of the preceding paragraphs, wherein the cation is choline.
  • the composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 2:1 to 1:4, inclusive.
  • the composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1.5:1 to 1:4, inclusive.
  • the composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1:1 to 1:4, inclusive.
  • the composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 2:1 to 1:1, inclusive.
  • composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1.7: 1 to 2: 1, inclusive.
  • the composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1.87:1.
  • the composition of any one of the preceding paragraphs, wherein the ionic liquid and active agent are present together in nanocomplexes.
  • a method of drug delivery comprising administering a composition of any of the preceding paragraphs to a subject in need of the active agent.
  • composition of paragraph 43, wherein the administration is intravenous, parenteral, or intraocular.
  • EXAMPLE 1 A Deep Eutectic Solvent-based approach to intravenous formulation
  • Clinically viable formulations of hydrophobic drugs, for example, chemotherapeutics require strategies to promote sufficient drug solubilization. However, such strategies often involve the use of organic solvents that pose a significant risk in generating toxic, unstable products.
  • verteporfm as a drug
  • a deep eutectic solvent-based approach to solvate drugs in a simple one-step process is reported. Lipoidal deep eutectic solvent composed of choline and oleate is used to successfully solvate verteporfm, resulting in stable sub- 100 nm nanocomplexes.
  • the nanocomplexes successfully demonstrate efficient cellular uptake as well as retention, tumor spheroid penetration, and tumor accumulation in vivo.
  • Systemic administration of the formulation significantly inhibits the primary tumor growth and its lung metastasis in the orthotopic 4T1 murine breast tumor model.
  • biocompatible deep eutectic solvent shows great potential as a novel material for intravenous formulation of chemotherapeutics.
  • Deep eutectic solvent is introduced as a novel, biocompatible agent for dissolving water- insoluble drugs.
  • Deep eutectic solvent comprised of choline and oleate demonstrated high aqueous solubility of a model chemotherapeutic drug verteporfm via nanocomplexation, and exhibited the ability to overcome multiple biological barriers to achieve successful cancer therapy in vivo against metastatic breast tumor in mice.
  • co-solvents and solubilization agents such as dimethyl sulfoxide (DMSO), ethanol, glycerol, n-methyl pyrrolidone, polyvinylpyrrolidone, polyethylene glycol, Cremophor EL, and polysorbate have a long-standing history of use for this purpose, 131 but many of these agents are associated with significant toxicity, hypersensitivity reactions, and instability at the concentrations required to achieve therapeutic doses.
  • DMSO dimethyl sulfoxide
  • glycerol glycerol
  • n-methyl pyrrolidone polyvinylpyrrolidone
  • polyethylene glycol polyethylene glycol
  • Cremophor EL Cremophor EL
  • polysorbate have a long-standing history of use for this purpose, 131 but many of these agents are associated with significant toxicity, hypersensitivity reactions, and instability at the concentrations required to achieve therapeutic doses.
  • 141 Chemical drug modification is another strategy explored for drug modification. [2a ’ 51
  • DESs deep eutectic solvents
  • ILs ionic liquids
  • DESs are eutectic mixture of cationic and anionic species with a melting point below either of the individual component.
  • ILs are simple salts in a viscous liquid state at temperature below 100°C, respectively.
  • DESs and ILs have been explored as biocompatible liquids with high solvation properties for many hydrophobic molecules. 171 In particular, small molecules and biological macromolecules have been dissolved in selective neat or dilute choline-based DESs and ILs to facilitate transdermal, oral and intratumoral drug delivery. 181 However, the use of ILs or DESs for solvation and intravenous administration of hydrophobic drugs has not been demonstrated.
  • CODES Choline and Oleic acid DES
  • DOPC synthetic phospholipid dioleylphosphatidylcholine
  • Verteporfm is a highly water insoluble drug (water solubility of 20 mM) that is used in light-dependent generation of reactive oxygen species as well as light-independent inhibition of cancer cell proliferation.
  • a current liposomal product, Visudyne uses a 2 mg mL 1 verteporfm formulation for the photodynamic treatment of age-related macular degeneration.
  • Previous studies on Visudyne have reported its instability in plasma and lack of tumor targeting in the absence of light, 191 thereby limiting intravenous administration of the liposomal formulation in non-photodynamic cancer therapy.
  • small amounts of CODES can successfully solvate verteporfm in water at concentrations greater than 2 mg mL 1 and form nanocomplexes as a novel intravenous formulation alternative to verteporfm solubilized in DMSO (VPDMSO), a co-solvent used to solubilize verteporfm in many previous studies.
  • VPDMSO DMSO
  • COVERT CODES-Verteporfm
  • the resulting product was a yellow paste at room temperature and 37 °C, but it transitioned into a viscous liquid when heated to 100 °C (Figs. 1C, 7B).
  • Use of other molar ratios of choline:oleic acid was attempted as well.
  • reaction of 1 : 1 molar ratio of choline bicarbonate to oleic acid yielded a gel-like paste that remained in the gel phase even at 100 °C, and thus was not explored further for formulating verteporfm.
  • Verteporfm was solubilized at 2.5 mg mL 1 using aqueous dilution of CODES.
  • Three different concentrations of CODES were tested to prepare COVERT (20, 40 and 80 mg mL 1 in water) labeled COVERT20, COVERT40 and COVERTso, respectively, to limit the concentration of CODES to only what is needed to solubilize verteporfm.
  • COVERT20, 40 and 80 mg mL 1 in water labeled COVERT20, COVERT40 and COVERTso, respectively.
  • Freshly prepared COVERT40 exhibited a size of 85 nm (PDI: 0.226 ⁇ 0.005, mean ⁇ sd) and zeta potential of -80 mV (Fig. 2A).
  • COVERT20 and COVERTso exhibited hydrodynamic diameters of 114 and 30 nm, respectively (Fig. 8A).
  • the process of making these nanocomplexes with CODES is much simpler than that to produce liposomal formulation of similar size, for which nanoprecipitation, extrusion, or drying-hydration steps are necessary.
  • the size and zeta potential were determined for both formulations following a freeze-thaw cycle and additional incubation for 48 hr at room temperature.
  • COVERT40 exhibited greater stability than COVERT20 and COVERTso, retaining its original size and surface charge. Moreover, COVERT20 resulted in aggregation of nanocomplexes following a 24-hr incubation in the presence of serum, while COVERT40 and COVERTso remained stable (Fig. 8B). Representative transmission electron microscopy image of COVERT40 also confirmed a nanocomplex of approximately 100 nm (Fig. 2B).
  • COVERT formulations exhibited similar IC50 values compared to VPDMSO- However, COVERT exhibited a significantly greater Hill coefficients (HC) than VPDMSO indicated by the steeper curve, indicating that COVERT formulations lead to increased drug sensitivity.
  • HC Hill coefficients
  • Intracellular uptake and retention [00187] The cellular uptake and retention of the drug in 4T1 cells were measured by flow cytometry after 2 hr incubation with COVERT or VPDMSO ⁇ In all three drug concentrations tested, nearly 100% of the cells showed intracellular presence of verteporfm for all groups. However, there was a significant increase in the cellular uptake of verteporfm per cell in the COVERT group (Figs. 4A, 10A). Interestingly, the increase in COVERT uptake was greater at higher verteporfm concentration of 2.5 x 10 6 M, indicating that greater absolute amount of CODES may induce energy- dependent endocytosis when presented to the cell membrane.
  • 4C, 10D shows significant enhancement of the verteporfin’s intracellular retention in 4T1 cells as nanocomplexes over VPDMSO-
  • the cells were allowed to exocytose intracellular drug for 60 mins in fresh media following 2 hr incubation of 2.5 x 10 6 M verteporfm, approximately 45% of verteporfm remained in the cells when formulated into COVERT, in comparison to 29% for VPDMSO-
  • COVERT entered 4T1 cells more effectively than DMSO-solubilized verteporfm at around their IC50 values via active cellular uptake mechanism and showed prolonged intracellular retention, resulting in increased drug sensitivity.
  • COVERT40 was used in the following in vitro tumor spheroid and in vivo experiments.
  • Penetration in three-dimensional multicellular tumor spheroid [00188]
  • One of the major barriers to effective tumor treatment is the drug’s limited penetration into densely packed tumor tissue to target the cancer cells in the core.
  • COVERT 40 and VPDMSO were incubated with three-dimensional tumor spheroid consisting of 4T1 cancer cells and 3T3 fibroblasts to determine the drug diffusion into the core and overall cell killing efficacy.
  • Nuclei-staining Hoescht dye was used to distinguish cells in two layers of the spheroids based on the fluorescence intensity (Fig. 11A), and 4T1 and 3T3 cells were identified based on distinct populations in the forward vs. side scatter plot. As shown in Figs.
  • COVERT 40 and VPDMSO resulted in a similar level of verteporfm uptake by both cell types collectively in the outer layer, but COVERT 40 demonstrated significantly greater penetration into the inner layer than VPDMSO- More importantly, COVERT 40 showed 27% increase in the amount of verteporfm delivered to 4T1 cells in the inner layer of the spheroid in comparison to VPDMSO- Higher verteporfm uptake in the form of COVERT 40 was also shown in the outer layer, as expected based on the result from the two-dimensional cellular uptake. When incubated with the equivalent concentration of 1 x 10 5 M verteporfm, significantly higher cell death from tumor spheroid was measured with COVERT 40 than VPDMSO (Fig. 5B).
  • COVERT 40 nanocomplex was investigated in a murine orthotopic breast tumor model.
  • VPDMSO was used as the free drug control.
  • Formulations were administered intravenously in B ALB/c female mice bearing 4T1 tumor in the mammary pad, and major organs including the tumors were harvested 3 hr post injection to determine the biodistribution of verteporfin (Fig. 6A).
  • COVERT 40 followed similar clearance pathway as lipid nanoparticles via the reticuloendothelial system and the kidneys.
  • COVERT 40 Similar level of VPDMSO was detected in the lungs as COVERT 40 , indicating lung entrapment of amorphous verteporfin aggregates in VPDMSO formulation. Importantly, COVERT 40 showed a significant increase of 66% in accumulation of verteporfin in tumor relative to VPDMSO control (Fig. 6B). Verteporfin continued to be detected in the tumor and all organs except the heart at 24 hr post injection (Fig. 12A). Interestingly, COVERT 40 showed significantly greater amount of verteporfin in the lungs at 24 hr than VPDMSO, indicating better retention of the drug in the tissue.
  • Tumor-bearing mice were administered with four doses of COVERT 40 or VPDMSO every 3 days and the primary tumor growth was monitored. Tumor growth was significantly inhibited by more than 70% in mice injected with COVERT 40 (Fig. 6C) compared to the saline control, CODES control, and VPDMSO, correlating with the greater Hill coefficient, efficient cellular uptake and retention, enhanced tumor spheroid penetration, and increased tumor accumulation of COVERT 40 over VPDMSO- Excised tumors also showed significant decrease in size and mass in COVERT 40 group compared to the saline control, CODES control, and VPDMSO (Fig. 12B and Fig. 12C). All mice maintained healthy body weight throughout the duration of the experiment (Fig. 6D).
  • 6E, 12E show that verteporfin in both DMSO- and CODES-based formulations significantly inhibited the progression of lung metastasis in the 4T1 tumor model compared to the saline control, while CODES control showed no statistical difference.
  • This observation is in agreement with the similar level of COVERT 40 and VPDMSO accumulation in the lungs at early timepoints, potentially leading to verteporfm-mediated suppression of metastatic nodule formation in the tissue.
  • lower VPDMSO accumulation at the primary tumor site may be sufficient to localize to the periphery of the tumor tissue and successfully inhibit the generation and shedding of circulating tumor cells.
  • Verteporfin is a hydrophobic small molecule drug that is clinically approved for use in photodynamic therapy (PDT) of neovascular age-related macular degeneration but is also being explored further in cancer chemotherapy.
  • PDT photodynamic therapy
  • anon-PDT mechanism was also elucidated for verteporfm, involving disruption of interaction between yes-associated protein (Y AP) and TEA domain transcription factor (TEAD) in the cancer cell-specific Hippo pathway, which leads to decreased cancer cell proliferation in vitro and tumor growth in vivo without any photoactivation in various cancer models.
  • Y AP yes-associated protein
  • TEAD TEA domain transcription factor
  • COVERT formulations were explored against two major biological bottlenecks in vitro. Efficient cellular uptake is critical at the cellular level for drugs with intracellular targets. While free verteporfm also showed high level of cellular uptake, COVERT formulations entered the cells more efficiently via energy-dependent pathways. By slowing exocytosis rate upon cellular uptake, which parallels previous reporting with another DES composed of choline and geranate, [15] COVERT40 nanocomplex allows for greater cytosolic concentration of verteporfm. This may explain over 2-fold increase in Hill coefficient with COVERT40 in comparison to the free drug, suggesting greater drug sensitivity and leading to more efficient cell killing.
  • COVERT can locally present multiple verteporfm molecules and mediate their cooperative binding to multiple YAP residues (Gln82, Val84, Met86 and Arg89) involved in interaction with TEAD.
  • Penetration into the tumor tissue is another hurdle for cancer nanomedicines. Limited diffusion of free drug or its formulations to the core of the tumor tissue results not only in partial efficacy at the regions proximate to the blood vessels but also development of drug resistance. 1181 Greater amount of verteporfm was observed in the cells located at the inner layer of tumor spheroids when delivered with 100-nm COVERT40 nanocomplexes than the free drug. This penetration is achieved with DES-based formulation in the absence of any surface modification that is known to facilitate diffusion of other nanovesicles.
  • Lipoidal deep eutectic solvent CODES was synthesized following previously described method. [8aI Briefly, choline bicarbonate (80% solution in water) and oleic acid were separately weighed and mixed in a round-bottom flask at a predetermined molar ratio. The mixture was allowed to react while stirring at 40 °C for 12 hr. Then, the product was purified by removing water using a rotary evaporator at 60 °C for 4 hr, followed by additional drying in a vacuum oven at 60 °C for 48 hr.
  • CODES was characterized by both 'H nuclear magnetic resonance (NMR) spectroscopy with CDCE using Agilent DD2600 MHz spectrometer and Fourier-transform infrared (FT-IR) spectra using Bruker ALPHA ATR-FTIR (64 scans).
  • NMR nuclear magnetic resonance
  • FT-IR Fourier-transform infrared
  • COVERT80, COVERT40, and COVERT20 were formulated by simple dissolution of verteporfm. Verteporfm was dissolved at 2.5 mg mL 1 using CODES diluted in water at 80, 40 or 20 mg mL 1 , respectively, by vortexing for 1 min vortexing followed by sonicating for 10 min. The solution was filtered through PD- 10 desalting column (Cytiva) to remove free drug not complexed with CODES, and the product was stored at -20 °C, and thawed and sonicated for 10 min before use.
  • COVERT nanocomplexes were diluted 100-fold with 9: 1 v/v acetonitrile: water, sonicated, and injected into reverse phase high performance liquid chromatography system with a column (Agilent, Santa Clara, CA).
  • Verteporfm was detected at 450 nm with DAD at 1 mL min 1 flow rate using mobile phases consisting of (A) 5 x 10 5 M monobasic sodium phosphate (pH 3.5) and (B) methanol, with gradient applied as follows: 0-4 min from 50 to 65% eluent B; 4-8 min from 65 to 85% eluent B; 8-22 min from 85 to 99% eluent B; 22-27 min from 99 to 10% eluent B; 27-30 min from 10 to 50% eluent B; 30-40 min 50% eluent B. Linear calibration curve was established from 0 to 50 mg/mL for quantification.
  • CODES was detected using Corona Veo Charged Aerosol Detector (CAD) (ThermoFisher Scientific, Waltham, MA).
  • CAD Corona Veo Charged Aerosol Detector
  • Mobile phases consisted of (A) 9: 1 v/v acetonitrile: water with 0.1% trifluoroacetic acid (TFA) and (B) methanol with 0.1% TFA, with gradient increasing from 0-15 min from 40 to 100% eluent B and 15-20 min from 100 to 40% eluent B. Finear calibration curve was established from 0 to 800 pg mF 1 for quantification.
  • Encapsulation efficiency (EE) and loading capacity (EC) are calculated according to the following equations:
  • COVERT40 X-ray diffraction of verteporfm power, neat CODES, and COVERT40 were detected with Bruker D2 Phaser Benchtop XRD System (Bruker USA). Turbidity of COVERT40 and VPDMSO was measured by absorbance at the wavelength (520 nm) verteporfm minimally absorbs to detect light scattering from aggregation in the formulations.
  • 1% penicillin/streptomycin at 37 °C and 5% CO2 and 3T3 fibroblasts were grown in high-glucose Dulbecco’s modified Eagle medium (DMEM) with 10% FBS at 37 °C and 5% CO2.
  • DMEM high-glucose Dulbecco’s modified Eagle medium
  • 4T1 cells were seeded in 96-well plates at 15,000 cells per well and incubated for 24 hr to allow adherence. Media was replaced with 100 pL fresh media containing dilutions of 1) CODES from 0 to 2.5 mg mL 1 , 2) stock verteporfm prepared at 3.5 mM in DMSO from 0.625 to 40 x 10 6 M, and 3) COVERT formulations to equivalent verteporfm concentrations from 0.625 to 40 x 10 6 M.
  • Three-dimensional tumor spheroids consisting of 4T1 cells and fibroblasts were constructed using the previously reported method [21] . Briefly, single-cell suspension was prepared with 1:5 ratio of 4T1 cells and 3T3 fibroblasts at a total cell concentration of 1 c 10 5 cells mF 1 , and added to U-bottomed 96-well spheroid microplates (Coming life sciences) at 20,000 cells per well. Cells were then incubated at 37 °C with 5% CO2 with medium replaced every other day. Spontaneously formed tumor spheroids of 500-600 pm diameter were visible in each well at day 6.
  • spheroid in each well was treated for 2 hr with 100 pF media containing COVERT40 or free verteporfm at equivalent verteporfm concentration of 2.5 x 10 6 M. During the last 30 min of treatment, 1 pF of Hoescht dye was added to the wells to a final concentration of 1 x 10 6 M. Spheroids were then washed once with IX PBS, trypsinized to achieve single cell suspension, and resuspended in IX PBS containing 2% v/v FBS for flow cytometry.
  • DAPI channel (laser 405 nm with bandpass filter 515/20) was used to separate two layers of the spheroids based on Hoescht fluorescence intensity, FSC-H vs. SSC-H was used to separate 4T1 cells and 3T3 fibroblasts, and PerCP Cy5.5 channel was used to quantify verteporfm uptake.
  • spheroid viability assay For spheroid viability assay, spheroid in each well was treated for 3 hr with 100 pF media containing COVERTso, COVERT40, or free verteporfm at equivalent verteporfm concentration of 2.5 x 10 6 M. Spheroids were then washed with IX PBS and further incubated in fresh media for additional 48 hr before viability quantification using CellTiter-Glo® 3D Cell Viability assay.
  • mice were purchased from Charles River Uaboratories.
  • Orthotopic breast tumor was developed by injection of 10 5 4T1 cells in IX PBS above the fourth mammary fat pad.
  • mice were intravenously injected with 5 mg kg 1 of verteporfm as either COVERT40 formulation or free verteporfm.
  • Verteporfm was recovered from the lysates by extraction in methanol, and quantified by fluorescence (430/20 nm excitation and 690/20 nm emission) on BioTek Neo2 plate reader. Uinear calibration curve was established from 1 x 10 9 to 3 x 10 6 M verteporfm in each of the organ lysates for quantification.
  • Orthotopic breast tumor was developed in female BAUB/c mice (aged 7-8 weeks) by injection of 10 5 4T1 cells in IX PBS above the fourth mammary fat pad.
  • mice When tumor size reached approximately 50 mm 3 on day 6, mice were treated with four intravenous injections three days apart of 5 mg kg 1 verteporfm as either COVERT40 formulation or free verteporfm, in addition to the saline and CODES controls. Tumor volume and body weight were monitored every other day. The study was terminated when the tumors in control group reached the endpoint criteria of 1000 mm 3 , at which point lungs were harvested to count surface-visible metastatic nodules and to visualize them in histological sections.
  • mice Healthy female BALB/c mice (aged 7-8 weeks) were treated with four intravenous injections three days apart of 5 mg kg 1 verteporfm as either COVERT40 formulation or free verteporfm, in addition to the saline and CODES controls. 24 hours post the fourth injection, mice were euthanized and major organs, including the spleen, kidneys, liver, lungs, and heart, were excised and fixed in paraformaldehyde. H&E-stained histological sections were obtained and imaged using AxioscanTM.

Abstract

The technology described herein is directed to ionic liquids and methods of drug delivery.

Description

IMPROVED METHODS AND COMPOSITIONS FOR DRUG DEUIVERY REUATING TO
IONIC UIQUIDS
CROSS-REFERENCE TO REUATED APPUICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/211, 261 filed June 16, 2021, the contents of which are incorporated herein by reference in their entirety.
TECHNICAU FIEUD
[0002] The technology described herein relates to ionic liquids for stabilization and delivery of active agents.
BACKGROUND
[0003] Effective administration of drugs often requires that the drug is adequately soluble, e.g., for intravenous administration. However, 60-90% of drug candidates exhibit limited solubility in water. Thus, intravenous delivery of small hydrophobic drugs for cancer presents a significant technical challenge due to their poor water solubility.
[0004] Current approaches to address solubility suffer from significant problems. For example, one approach to promote sufficient drug solubilization involves solvents. But the use of such solvents poses a significant risk because such solvents tend to generate toxic, unstable products. Another approach is the use of liposomes and polymeric nanoparticles. But, preparation of these systems also involves the use of solubilizing agents in the process and often results in low encapsulation efficiency or off-target effects. Hence, there is an outstanding need for safe materials that readily dissolve water- insoluble drugs, maintain stable formulation, and demonstrate effective pharmacokinetic and pharmacodynamic profiles.
SUMMARY
[0005] The inventors have found that ionic liquids, e.g., those with an oleic acid anion, can successfully solvate small hydrophic drugs. These compositions permit preparation of higher concentrations of drugs than older approaches such as DMSO. Such compositions, when comprising a chemotherapeutic drug, successfully demonstrate efficient cellular uptake as well as retention, tumor spheroid penetration, and tumor accumulation in vivo. Systemic administration of the formulation significantly inhibits primary tumor growth and metastasis in vivo. Thus, these compositions display a surprising ability to overcome multiple biological barriers to achieve successful cancer therapy in vivo. Accordingly, these ionic liquids are described herein as biocompatible agents for dissolving poorly water-soluble drugs.
[0006] In one aspect of any of the embodiments, described herein is a composition comprising: a) an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion; and b) an active agent. In some embodiments of any of the aspects, the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the aspects, the active agent comprises at least one drug with a water solubility of less than 10 mg/mL. In some embodiments of any of the aspects, the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the aspects, the drug has a water solubility of less than 1 mg/mL. In some embodiments of any of the aspects, the at least one drug has a water solubility of less than 1 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the aspects, the at least one drug is selected from a photosensitizer, an anthracylcine, ataxane, or camptothecin and camptothecin analogs. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, gemcitabine, doxorubicin, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, doxorubicin, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, doxorubicin, and paclitaxel. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug comprises verteporfm.
[0007] In some embodiments of any of the aspects, the active agent is present at a concentration of at least 1 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 2 mg/mL. In some embodiments of any of the aspects, the ionic liquid is at a concentration of at least 20 mg/mL in water. In some embodiments of any of the aspects, the ionic liquid is at a concentration of 20-40 mg/mL in water. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 1-lOOx the concentration of the agent. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 5 -5 Ox the concentration of the agent. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 10- 3 Ox the concentration of the agent. In some embodiments of any of the aspects, the ionic liquid is at a concentration which is 15-20x the concentration of the agent.
[0008] In some embodiments of any of the aspects, the cation is choline. In some embodiments of any of the aspects, the molar ratio of cation to anion is 2: 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.5: 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1 : 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 2: 1 to 1:1, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.7: 1 to 2: 1, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.87: 1. In some embodiments of any of the aspects, the ionic liquid and active agent are present together in nanocomplexes. [0009] In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising administering a composition as described herein to the subject. In one aspect of any of the embodiments, described herein is a composition as described herein, for use in a method of treating cancer in a subject in need thereof. In some embodiments of any of the aspects, the cancer is epithelial cancer, e.g., breast cancer. In some embodiments of any of the aspects, the composition is administered intravenously.
[0010] In one aspect of any of the embodiments, described herein is a method of treating macular degeneration in a subject in need thereof, the method comprising administering a composition as described herein to the subject. In one aspect of any of the embodiments, described herein is a composition as described herein, for use in a method of treating macular degeneration in a subject in need thereof. In some embodiments of any of the aspects, the at least one drug comprises verteporfm. In some embodiments of any of the aspects, the composition is administered intraocularly.
[0011] In one aspect of any of the embodiments, described herein is a method of drug delivery, the method comprising administering a composition as described herein to a subject in need of the active agent. In one aspect of any of the embodiments, described herein is a composition as described herein, for use in a method of drug delivery. In some embodiments of any of the aspects, the administration and/or drug delivery is intravenous, parenteral, or intraocular.
BRIEF DESCRIPTION OF THE DRAWINGS [0012] Figs. 1A-1C depict Choline and Oleic acid Deep Eutectic Solvent (CODES). Fig. 1A) Synthesis of CODES via metathesis reaction between choline bicarbonate and oleic acid, Fig. IB) 'H NMR spectrum of CODES indicating 1.87: 1 ratio of choline to oleate, and Fig. 1C) photographs of CODES at room temperature (top) and 100 °C (bottom).
[0013] Figs. 2A-2E depict the characterization of COVERT40. Fig. 2A) Hydrodynamic diameter and zeta potential of COVERT40 before and after freeze/thaw cycle, measured by dynamic light scattering (n=3), Fig. 2B) transmission electron microscopy image of COVERT40 and VPDMSO (scale bar = 100 nm for COVERT40 and 500 nm for VPDMSO), Fig. 2C) loading of verteporfm with CODES determined by HPLC (black bar: encapsulation efficiency, white bar: loading capacity, n=3), Fig. 2D) x-ray diffraction of verteporfm powder, COVERT40, and VPDMSO, and Fig. 2E) turbidity of COVERT40 and VPDMSO formulations based on absorbance at 520 nm with background absorbance from CODES and DMSO subtracted (n=3, unpaired/two-tailed student’s t-test). All data are presented as mean ± sd.
[0014] Figs. 3A-3C depict the in vitro cell killing efficacy in 4T1 murine breast cancer cells. Fig. 3A) Cytotoxicity of COVERT and VPDMSO formulations with 3 hr treatment followed by 24 hr incubation in fresh media, Fig. 3B) IC50 values, and Fig. 3C) Hill coefficients (HC) (n=6). All data are presented as mean ± sd and analyzed by one-way ANOVA with Tukey multiple comparison test. [0015] Figs. 4A-4C depict in vitro cellular uptake and retention in 4T1 murine breast cancer cells. Fluorescence intensity of verteporfm per cell was detected by flow cytometry. Fig. 4A) Cellular uptake of COVERT40 and VPDMSO after 2 hr treatment at 37 °C (n=4, one-way ANOVA with Sidak multiple comparison test) and Fig. 4B) 4 °C (n=4, unpaired/two-tailed student’s t-test), and Fig. 4C) their intracellular retention at various timepoints following 2 hr cellular uptake at 37 °C (n=4, one way ANOVA with Sidak multiple comparison test). All data are presented as mean ± sd.
[0016] Figs. 5A-5B depict three-dimensional multicellular tumor spheroid penetration. Fig. 5A) Amount of COVERT40 and VPDMSO penetration into respective layers and cell types forming the spheroid (n=3, one-way ANOVA with Sidak multiple comparison test), and Fig. 5B) cell killing efficacy of COVERT40 and VPDMSO in tumor spheroid (n=4, unpaired/two-tailed student’s t-test between COVERT40 and VPDMSO, CODES40: equivalent amount of CODES as in COVERT40, DMSO: equivalent amount of DMSO as in VPDMSO). All data are presented as mean ± sd.
[0017] Figs. 6A-6E depict the in vivo biodistribution and efficacy on orthotopic 4T1 breast tumor. Fig. 6A) Biodistribution of COVERT40 and VPDMSO in major organs and tumor at 3 hr post intravenous injection of 5 mg/kg drug dose and Fig. 6B) tumor alone (n=6, one-way ANOVA with Sidak multiple comparison test), Fig. 6C) primary tumor growth of 4T1 inoculated in mammary fat pad with four tail-vein injections of therapeutics 3 days apart (n>5, one-way ANOVA with Sidak multiple comparison test), Fig. 6D) body weight (n>5), and Fig. 6E) number of metastatic nodules from lungs harvested at the termination of tumor growth inhibition study on day 26 (n>5, one-way ANOVA with Tukey multiple comparison test). All data are presented as mean ± sd.
[0018] Figs. 7A-7B depict the characterization of CODES. Fig. 7A) FT-IR spectrum of CODES showing characteristic peaks at 1400 cm 1 for v(O-H) and 1650 cm 1 for v(C=C) in oleate and at 2900 cm 1, for v(O-H) and 1075 cm 1 for v(C-N) in choline, and Fig. 7B) photograph of paste-like CODES at 37 °C.
[0019] Figs. 8A-8E depict the characterization of COVERT systems. Hydrodynamic diameter of COVERT20 and COVERTso Fig. 8A) before and after freeze/thaw cycle and Fig. 8B) 3 hr and 24 hr post incubation in 10% serum by volume in IX PBS, measured by dynamic light scattering (n=3),
Fig. 8C) loading of verteporfm with CODES determined by HPLC (black bar: encapsulation efficiency, white bar: loading capacity, n=3), Fig. 8D) turbidity of COVERTso and VPDMSO formulations based on absorbance at 520 nm with background absorbance from CODES and DMSO subtracted (n=3, unpaired/two-tailed student’s t-test with * as p<0.05), and Fig. 8E) in vitro release of verteporfm from COVERT40 in water over 24 hr (n=3). All data are presented as mean ± sd.
[0020] Fig. 9 depicts the in vitro cytotoxicity of CODES. 4T1 cell viability after 3 hr incubation with serial dilutions of CODES, followed by 24 hr incubation in fresh media (n=6). Data are presented as mean ± sd. [0021] Figs. 10A-10D depict the in vitro cellular uptake and retention of COVERTso in 4T1 murine breast cancer cells. Fluorescence intensity of verteporfm per cell was detected by flow cytometry. Fig. 10A) Cellular uptake of COVERTso and VPDMSO after 2 hr treatment at 37 °C (n=4, one-way ANOVA with Sidak multiple comparison test) and Fig. 10B) 4 °C (n=4, unpaired/two-tailed student’s t-test), Fig. IOC) comparison of the cellular uptake at 37 °C vs. 4 °C for each formulation (n=4, one-way ANOVA with Sidak multiple comparison test), and Fig. 10D) their intracellular retention at various timepoints following 2 hr cellular uptake at 37°C (n=4, one-way ANOVA with Sidak multiple comparison test). All data are presented as mean ± sd.
[0022] Figs. 1 lA-1 IB depict three-dimensional multicellular tumor spheroid penetration. Fig.
11A) Histogram plot of Hoescht staining of tumor spheroid by flow cytometry used to separate the spheroid into inner core and outer perimeter layers, Fig. 1 IB) amount of COVERT40 and free verteporfm penetration into respective layers of all cells in the spheroid (n=3, one-way ANOVA with Sidak multiple comparison test). Data are presented as mean ± sd.
[0023] Figs. 12A-12E depict the in vivo biodistribution, efficacy, and safety on orthotopic 4T1 breast tumor. Fig. 12A) Biodistribution of COVERT40 and VPDMSO in major organs and tumor at 24 hr post intravenous injection of 5 mg/kg drug dose (n=5, one-way ANOVA with Sidak multiple comparison test), Fig. 12B) mass (n=5, one-way ANOVA with Sidak multiple comparison test) and Fig. 12C) photographs of primary 4T1 tumor tissue excised from the mammary fat pad at the termination of the tumor growth inhibition study on day 26, Fig. 12D) representative H&E-stained sections of the major organs harvested following four intravenous injections of the formulations, and Fig. 12E) metastatic nodules visualized from histological sections of the lungs harvested at the termination of tumor growth inhibition study on day 26 (black arrows indicate metastatic nodules).
All data are presented as mean ± sd.
DETAILED DESCRIPTION
[0024] It is demsontrated herein that compositions comprising certain ionic liquids, e.g., those with an oleic acid anion, and an active agent, e.g., a small hydrophic drug, can provide solubilized drugs at significantly higher concentrations than prior art approaches. Additionally, these compositions display a surprising ability to overcome multiple biological barriers to achieve successful therapy in vivo, providing effective and superior parenteral (e.g., intravenous) delivery. Accordingly, in one aspect of any of the embodiments, described herein is a composition comprising: a) an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion (CODES); and b) an active agent.
In one aspect of any of the embodiments, described herein is a composition comprising: a) an ionic liquid comprising i) a choline cation and ii) an oleate and/or oleic acid anion (CODES); and b) an active agent.
[0025] The term "ionic liquids (ILs)" as used herein refers to organic salts or mixtures of organic salts which are in liquid state at room temperature. This class of solvents has 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 at least one anionic and at least one cationic component may be present in any molar ratio. Exemplary molar ratios (cation: anion) include but are not limited to 1 : 1, 1:2, 2: 1, 1 :3, 3: 1, 2:3, 3:2, and ranges between these ratios. For further discussion of ionic liquids, see, e.g., Hough, et ah , "The third evolution of ionic liquids: active pharmaceutical ingredients", New Journal of Chemistry, 31 : 1429 (2007) and Xu, et al., "Ionic Liquids: Ion Mobilities, Glass Temperatures, and Fragilities", Journal of Physical Chemistry B, 107(25): 6170-6178 (2003); each of which is incorporated by reference herein in its entirety. In some embodiments of any of the aspects, the ionic liquid or solvent exists as a liquid below 100 °C. In some embodiments of any of the aspects, the ionic liquid or solvent exists as a liquid at room temperature.
[0026] The cation of an IL described herein can be a cation comprising a quaternary ammonium. A quaternary ammonium is a positively charged polyatomic ion of the structure NR_f. each R independently being an alkyl group or an aryl group. The general term “quaternary ammonium” relates to any compound that can be regarded as derived from ammonium hydroxide or an ammonium salt by replacement of all four hydrogen atoms of the NFLt + ion by organic groups. For example, the quaternary ammonium has the structure of NRi+, where each R is independently selected from hydroxyl, optionally substituted Ci-Cioalkyl, optionally substituted C2-Cioalkenyl, optionally substituted C2-Cioalkynyl, optionally substituted aryl, or optionally substituted heteroaryl.
[0027] In some embodiments of any of the aspects, the cation has a molar mass equal to or greater than choline, e.g., a molar mass equal to or greater than 104.1708 g/mol. In some embodiments of any of the aspects, the cation has a molar mass greater than choline, e.g., a molar mass equal greater than 104.1708 g/mol.
[0028] In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl, alkane, alkene, or aryl. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl, alkane, or alkene. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkane or alkene. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 10 carbon atoms in length, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms in length. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 12 carbon atoms in length. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 15 carbon atoms in length. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 20 carbon atoms in length.
[0029] In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 10 carbon atoms, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 12 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 15 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises a carbon chain of no more than 20 carbon atoms.
[0030] In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl group of no more than 10 carbon atoms, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl group of no more than 12 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl group of no more than 15 carbon atoms. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkyl group of no more than 20 carbon atoms.
[0031] In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an alkane, alkene, aryl, heteroaryl, alkyl, or heteroalkyl. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an unsubstituted alkane, unsubstituted alkene, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkyl, or unsubstituted heteroalkyl. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an unsubstituted alkane. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises an unsubstituted alkene. In some embodiments of any of the aspects, each R group of the quaternary ammonium independently comprises one or more substituent groups.
[0032] In some embodiments of any of the aspects, at least one R group of the quaternary ammonium comprises a hydroxy group. In some embodiments of any of the aspects, one R group of the quaternary ammonium comprises a hydroxy group. In some embodiments of any of the aspects, only one R group of the quaternary ammonium comprises a hydroxy group. [0033] Exemplary, non-limiting cations can include choline, and any of the cations designated C1-C7 which are defined by structure below.
Figure imgf000010_0001
[0034] Further non-limiting examples of cations include the following: 1 -(hydroxymethyl)- 1 -methylpyrrolidin- 1 -ium 1 -(2-hydroxy ethyl) - 1 -methylpyrrolidin- 1 -ium 1 -ethyl- 1 -(3 -hydroxypropyl)pyrrolidin- 1 -ium 1 -(3-hydroxypropyl)- 1 -methylpyrrolidin- 1 -ium 1 -(4-hydroxybutyl)- 1 -methylpyrrolidin- 1 -ium 1 -ethyl- 1 -(4-hydroxybutyl)pyrrolidin- 1 -ium 1 -(4-hydroxybutyl)- 1 -propylpyrrolidin- 1 -ium 1 -(5-hydroxypentyl)- 1 -propylpyrrolidin- 1 -ium 1 -ethyl- 1 -(5-hydroxypentyl)pyrrolidin- 1 -ium 1 -(5 -hydroxypentyl)- 1 -methylpyrrolidin- 1 -ium 1 -(hydroxymethyl)- 1 -methylpiperidin- 1 -ium 1 -(2-hydroxy ethyl) - 1 -methylpiperidin- 1 -ium 1 -ethyl- 1 -(2-hydroxy ethyl)piperidin- 1 -ium -ethyl- 1 -(3 -hydroxypropyl)piperidin- 1 -ium -(3-hydroxypropyl)- 1 -propylpiperidin- 1 -ium -(3-hydroxypropyl)- 1 -methylpiperidin- 1 -ium -(4-hydroxybutyl)- 1 -methylpiperidin- 1 -ium -ethyl- 1 -(4-hydroxybutyl)piperidin- 1 -ium -(4-hydroxybutyl)- 1 -propylpiperidin- 1 -ium -butyl- 1 -(5 -hydroxypentyl)piperidin- 1 -ium -(5-hydroxypentyl)- 1 -propylpiperidin- 1 -ium -ethyl- 1 -(5 -hydroxypentyl)piperidin- 1 -ium -(5-hydroxypentyl)- 1 -methylpiperidin- 1 -ium -ethyl- 1 -methyl- lH-imidazol-3 -ium -methyl-3 -propyl- lH-imidazol-3 -ium -butyl- 1 -methyl- lH-imidazol-3 -ium -methyl-3 -pentyl- lH-imidazol-3 -ium .2-dimethyl-3-pentyl-lH-imidazol-3-ium -butyl- 1 ,2-dimethyl- lH-imidazol-3 -ium .2-dimethyl-3 -propyl- lH-imidazol-3 -ium -(hydroxymethyl)- 1 ,2-dimethyl- lH-imidazol-3 -ium -(2-hydroxyethyl)-l,2-dimethyl-lH-imidazol-3-ium -(3-hydroxypropyl)-l,2-dimethyl-lH-imidazol-3-ium -(4-hydroxybutyl)-l,2-dimethyl-lH-imidazol-3-ium -(5-hydroxypentyl)-l,2-dimethyl-lH-imidazol-3-ium -(5-hydroxypentyl)- 1 -methyl- lH-imidazol-3 -ium -(4-hydroxybutyl)- 1 -methyl- lH-imidazol-3 -ium -(3 -hydroxypropyl)- 1 -methyl- lH-imidazol-3 -ium -(2-hydroxy ethyl) - 1 -methyl- 1 H-imidazol-3 -ium -(hydroxymethyl)- 1 ,2,4,5 -tetramethyl- lH-imidazol-3 -ium-(2-hydroxyethyl)-l,2,4,5-tetramethyl-lH-imidazol-3-ium-(3-hydroxypropyl)-l,2,4,5-tetramethyl-lH-imidazol-3-ium -(4-hydroxybutyl)- 1 ,2,4,5 -tetramethyl- lH-imidazol-3 -ium-(5-hydroxypentyl)-l,2,4,5-tetramethyl-lH-imidazol-3-ium -(5-hydroxypentyl)pyridin- 1 -ium -(4-hydroxybutyl)pyridin- 1 -ium -(3-hydroxypropyl)pyridin- 1 -ium -(2-hydroxyethyl)pyridin- 1 -ium -(hydroxymethyl)pyridin- 1 -ium 1 -hydroxypyridin- 1 -ium
(hydroxymethyl)trimethylphosphonium triethyl(hydroxymethyl)phosphonium triethyl(2-hydroxyethyl)phosphonium
(2-hydroxyethyl)tripropylphosphonium
(3-hydroxypropyl)tripropylphosphonium tributyl(3-hydroxypropyl)phosphonium
(3-hydroxypropyl)tripentylphosphonium
(4-hydroxybutyl)tripentylphosphonium
(5-hydroxypentyl)tripentylphosphonium
[0035] In some embodiments of any of the aspects, the cation is choline, Cl, C6, and/or C7. In some embodiments of any of the aspects, the cation is Cl, C6, and/or C7. In some embodiments of any of the aspects, the cation is choline.
[0036] The anion of an ionic liquid described herein can comprise oleate and/or oleic acid. The structure of oleic acid is:
Figure imgf000012_0001
Salts and esters of oleic acids are known as oleates.
[0037] In some embodiments of any of the aspects wherein the composition comprises two or more ionic liquids, a first and second ionic liquid have the same cation, e.g., choline. In some embodiments of any of the aspects wherein the composition comprises two or more ionic liquids, a first and second ionic liquid have different anions.
[0038] In some embodiments of any of the aspects, the IL is at a concentration of at least 0.01% w/v. In some embodiments of any of the aspects, the IL is at a concentration of at least 0.05% w/v.
In some embodiments of any of the aspects, the IL is at a concentration of at least 0.1% w/v. In some embodiments of any of the aspects, the IL is at a concentration of at least 0.2% w/v, at least 0.3% w/v, at least 0.4% w/v, at least 0.5% w/v, at least 1% w/v or greater. In some embodiments of any of the aspects, the IL is at a concentration of from about 0.01% w/v to about 1% w/v. In some embodiments of any of the aspects, the IL is at a concentration of from 0.01% w/v to 1% w/v. In some embodiments of any of the aspects, the IL is at a concentration of from about 0.05% w/v to about 0.5% w/v. In some embodiments of any of the aspects, the IL is at a concentration of from 0.05% w/v to 0.5% w/v. [0039] In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in water. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in saline or a physiologically compatible buffer.
[0040] In some embodiments of any of the aspects, the IL is at a concentration of from about 5% w/w to about 75% w/w. In some embodiments of any of the aspects, the IL is at a concentration of from 5% w/w to 75% w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 5% w/w to about 75% w/w in water, saline or a physiologically compatible buffer. In some embodiments of any of the aspects, the IL is at a concentration of from 5% w/w to 75% w/w in water, saline or a physiologically compatible buffer.
[0041] In some embodiments of any of the aspects, the IL is at a concentration of at least about 0.1 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of at least 0.1 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 10 % w/w to about 70 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from 10 % w/w to 70 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 30 % w/w to about 50 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from 30 % w/w to 40 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from about 30 % w/w to about 50 % w/w. In some embodiments of any of the aspects, the IL is at a concentration of from 30 % w/w to 40 % w/w.
[0042] In some embodiments of any of the aspects, the % w/w concentration of the IL is % w/w concentration in water, saline, or a physiologically compatible buffer.
[0043] In some embodiments of any of the aspects, the IL is 100% by w/w or w/v.
[0044] In some embodiments, the IL is an anhydrous salt, e.g., an ionic liquid not diluted or dissolved in water. In some embodiments, the IL is provided as an aqueous solution.
[0045] In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w and has a ratio of catiomanion of at least 1:3. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in water and has a ratio of catiomanion of at least 1:3. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w and has a ratio of catiomanion of 1:3 or 1:4. In some embodiments of any of the aspects, the IL is at a concentration of at least 25% w/w in water and has a ratio of cation: anion of 1:3 or 1:4. In some embodiments of any of the aspects, the IL is a gel, or a shear-thining Newtonian gel.
[0046] In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from about 10: 1 to about 1: 10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 10: 1 to 1 : 10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 5: 1 to about 1:5. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 5: 1 to 1:5. In some embodiments of any of the aspects, the IL has a ratio of cation:anion of from about 2: 1 to about 1:4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 2: 1 to 1 :4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 2: 1 to about 1:10. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from about 2: 1 to about 1 : 1. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 2: 1 to 1:10. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from 2: 1 to 1 : 1. In some embodiments of any of the aspects, the IL has a ratio of catiomanion such that there is a greater amount of anion, e.g., a ratio of less than 1:1. In some embodiments of any of the aspects, the IL has a ratio of catiomanion such that there is an excess of anion. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 1: 1 to about 1:10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 1 : 1 to 1 : 10. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from about 1: 1 to about 1:4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 1:1 to 1:4. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from about 1: 1 to about 1:3. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of from 1 : 1 to 1:3. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from about 1 : 1 to about 1 :2. In some embodiments of any of the aspects, the IL has a ratio of cation: anion of from 1:1 to 1:2. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of about 1:1, 1:2, 1:3, or 1:4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion of 1 : 1, 1:2, 1 :3, or 1:4. In some embodiments of any of the aspects, the IL has a ratio of catiomanion less than about of 1 : 1. In some embodiments of any of the aspects, the IL has a ratio of catiomanion less than 1:1. Without wishing to be constrained by theory, compositions with higher amounts of anion relative to cation display greater hydrophobicity. [0047] In some embodiments of any of the aspects, the IL has a catiomanion ratio with an excess of cation.
[0048] In some embodiments of any of the aspects, the molar ratio of cation to anion is 2: 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.5:1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1 : 1 to 1:4, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 2: 1 to 1:1, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is
1.7:1 to 2: 1, inclusive. In some embodiments of any of the aspects, the molar ratio of cation to anion is 1.87:1.
[0049] In some embodiments of any of the aspects, the IL is at a concentration of at least 20 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least about 20 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least 25 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least about 25 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least 50 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least about 50 mM. In some embodiments of any of the aspects, the IL is at a concentration of at least 100 mM, 500 mM, 1 M, 2 M, 3 M or greater. In some embodiments of any of the aspects, the IL is at a concentration of at least about 100 mM, 500 mM, 1 M, 2 M, 3 M or greater.
[0050] In some embodiments of any of the aspects, the IL is at a concentration of from about 50 mM to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 50 mM to 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from about 500 mM to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 500 mM to 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from about 1 M to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 1 M to 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from about 2 M to about 4 M. In some embodiments of any of the aspects, the IL is at a concentration of from 2 M to 4 M.
[0051] In some embodiments of any of the aspects, the IL concentration in the composition or formulation is about 0.1 mM to 20 mM. In some embodiments of any of the aspects, the IL concentration in the composition or formulation is about 0.5 mM to 20 mM, 0.5 mM to 18 mM, 0.5 mM to 16 mM, 0.5 mM to 14 mM, 0.5 mM to 12 mM, 0.5 mM to 10 mM, 0.5 mM to 8 mM, 1 mM to 20 mM, 1 mM to 18 mM, 1 mM to 16 mM, 1 mM to 14 mM, ImM to 12 mM, 1 mM to 10 mM, 1 mM to 8 mM, 2 mM to 20 mM, 2 mM to 18 mM, 2 mM to 16 mM, 2 mM to 14 mM, 2 mM to 12 mM, 2 mM to 10 mM, 2 mM to 8 mM, 4 mM to 20 mM, 4 mM to 18 mM, 4 mM to 16 mM, 4 mM to 14 mM, 4 mM to 12 mM, 4 mM to 10 mM, 4 mM to 8 mM, 6 mM to 20 mM, 6 mM to 18 mM, 6 mM to 14 mM, 6 mM to 12 mM, 6 mM to 10 mM, 6 mM to 8 mM, 8 mM to 20 mM, 8 mM to 18 mM, 8 mM to 16 mM, 8 mM to 14 mM, 8 mM to 12 mM, 8 mM to 10 mM, 10 mM to 20 mM, 10 mM to 18 mM, 10 mM to 16 mM, 10 mM to 14 mM, 10 mM to 12 mM, 12 mM to 20 mM, 12 mM to 18 mM, 12 mM to 16 mM, 12 mM to 14 mM, 14 mM to 20 mM, 14 mM to 18 mM, 14 mM to 16 mM, 16 mM to 20 mM, 16 mM to 18 mM, or 18 mM to 20 mM. In some embodiments of any of the aspects, the IL concentration in the composition or formulation is about ImM, about 2 mM, about 3mM, about 4mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM or about 20 mM.
[0052] It is specifically contemplated that a composition or combination described herein can comprise one, two, three, or more of any of the types of components described herein. For example, a composition can comprise a mixture, solution, combination, or emulsion of two or more different ionic liquids (e.g., different ionic liquids described herein), and/or a mixture, solution, combination, or emulsion of two or more different active agents. [0053] In some embodiments of any of the aspects, the one or more ILs can be in combination with at least one active agent. As used herein, “in combination with” refers to two or more substances being present in the same formulation in any molecular or physical arrangement, e.g., in an admixture, in a solution, in a mixture, in a suspension, in a colloid, in an emulsion. The formulation can be a homogeneous or heterogenous mixture. In some embodiments of any of the aspects, the active agent(s) can be comprised by a superstructure, e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like, said superstructure is which in solution, mixture, admixture, suspension, etc., with the IL. [0054] As used herein, an “active compound” or “active agent” is any agent which will exert an effect on a target cell or organism. The terms “compound” and “agent” refer to any entity which is normally not present or not present at the levels being administered and/or provided to a cell, tissue or subject. An agent can be selected from a group comprising: chemicals; small organic or inorganic molecules; signaling molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; enzymes; aptamers; peptidomimetic, peptide derivative, peptide analogs, antibodies; intrabodies; biological macromolecules, extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions or functional fragments thereof. In some embodiments of any of the aspects, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds. Non-limiting examples of active agents contemplated for use in the methods described herein include small molecules, drugs, polypeptides, nucleic acids, chemotherapies/chemotherapeutic compounds, antibodies, and antibody reagents.
[0055] In some embodiments of any of the aspects, the active agent can be a therapeutic compound or drug, e.g., an agent or compound which is therapeutically effective for the treatment of at least one condition in a subject. Therapeutic compounds are known in the art for a variety of conditions, see, e.g., the database available on the world wide web at drugs.com or the catalog of FDA-approved compounds available on the world wide web at catalog.data.gov/dataset/drugsfda- database; each of which is incorporated by reference herein in its entirety.
[0056] In some embodiments of any of the aspects, the active agent comprises, consists of, consists essentially of, or is at least one drug. In some embodiments of any of the aspects, the active agent comprises, consists of, consists essentially of, or is one drug. In some embodiments of any of the aspects, the active agent comprises, consists of, consists essentially of, or is a combination of two or more drugs.
[0057] In some embodiments of any of the aspects, the drug is a photosensitizer. Non-limiting examples of photosensitizers include 5-Aminolaevulinic acid, verteporfm, purlytin, foscan, lutex, ATMPn, zinc phthalocyanine, naphthalocyanines, expanded metallo-porphyrins, metallochlorins/bacteriochlorins, metallo-phthalocyanines, metallo-naphthocyaninesulfobenzo- porphyrazines (M-NSBP), and metallo-naphthalocyanines.
[0058] Drugs include chemotherapeutic agents. As used herein the term “chemotherapeutic agent" refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation. In some embodiments of any of the aspects, a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. [0059] One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).
[0060] Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells. In some embodiments of any of the aspects, a chemotherapeutic agent is a radioactive molecule.
[0061] In some embodiments of any of the aspects, the chemotherapeutic agent is a taxane (e.g., paclitaxel, docetaxel, cabazitaxel, and taxine).
[0062] In some embodiments of any of the aspects, the chemotherapeutic is a topoisomerase inhibitor. In some embodiments of any of the aspects, the topoisomerase inhibitor is camptothecin or a camptothecin analog (e.g., topotecan, irinotecan, belotecan, and tastuzumab deruxtecan).
[0063] In some embodiments of any of the aspects the chemotherapeutic is an anthracycline (e.g., doxorubicin, daunorubicin, epirubicin, and idarubicin).
[0064] In some embodiments of any of the aspects, the active agent, e.g., the drug, is a small molecule. As used herein, the term “small molecule” refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. [0065] In some embodiments of any of the aspects, the active agent has a molecular weight of less than 1000 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 900 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 800 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 700 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 600 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 500 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 400 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 300 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 200 Da. In some embodiments of any of the aspects, the active agent has a molecular weight of less than 100 Da.
[0066] In some embodiments of any of the aspects, the active agent, e.g., the drug, is poorly water soluble. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 10 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 9 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 8 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 7 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 6 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 5 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 4 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 3 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 2 mg/mL. In some embodiments of any of the aspects, the active agent, e.g., the drug, has a water solubility of less than 1 mg/mL.
[0067] The water solubilities for active agents, e.g., drugs, are known in the art and/or can be calculated by one of skill in the art. For example, PubChem, SpiderChem, and the Human Metabolome Database (available on the world wide web at hmdb.ca) provide these values for various agents, and chemical manufacturers typically provide them as part of the product information or catalog listings for their products. In some embodiments of any of the aspects, the water solubility is the water solubility at a physiological pH. In some embodiments of any of the aspects, the water solubility is the water solubility over the physiological pH range (e.g., 7.35-7.45). In some embodiments of any of the aspects, the water solubility is the water solubility at pH 7.4.
[0068] By way of non-limiting example, the water solubility for several small molecule drugs are:
Figure imgf000018_0001
Figure imgf000019_0001
[0069] Further water insoluble or poorl soluble drugs are well known in the art and are described at, e.g., Liu “Water-Insoluble Drug Formulation” CRC Press 2022 ISBN 9781032339214; and Savjani et al. ISRN Pharm 2012: 195727 doi: 10.5402/2012/195727; the contents of each of which are incorporated by reference herein in their entirties.
[0070] In some embodiments of any of the asepcts, a drug described herein has a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the asepcts, a drug described herein has a water solubility of less than 5 mg/mL and a molecular weight of less than 1000 Da. In some embodiments of any of the asepcts, a drug described herein has a water solubility of less than 1 mg/mL and a molecular weight of less than 1000 Da.
[0071] Non-limiting examples of drugs include verteporfm, gemcitabine, doxorubicin, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug is selected from verteporfm, doxorubicin, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug is selected from the at least one drug is selected from verteporfm, doxorubicin, and paclitaxel. In some embodiments of any of the aspects, the at least one drug is selected from the at least one drug is selected from verteporfm, paclitaxel, and camptothecin. In some embodiments of any of the aspects, the at least one drug comprises, consists of, or consists essentially of paclitaxel. In some embodiments of any of the aspects, the at least one drug comprises, consists of, or consists essentially of camptothecin. In some embodiments of any of the aspects, the at least one drug comprises, consists of, or consists essentially of verteporfm.
[0072] In some embodiments of any of the aspects, the active agent is present at a concentration of at least 1 mg/mL, e.g, at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 20 mg/mL, at least 30 mg/mL, at least 40 mg/mL, or more. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 2 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 5 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 10 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 20 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 30 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 40 mg/mL.
[0073] In some embodiments of any of the aspects, the active agent is present at a concentration of 1-100 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-50 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-40 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-100 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-50 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-40 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-100 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-50 mg/mL. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-40 mg/mL.
[0074] In some embodiments of any of the aspects, the active agent is present at a concentration of at least 1 mg/mL, e.g, at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 20 mg/mL, at least 30 mg/mL, at least 40 mg/mL, or more, in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 2 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 5 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 10 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 20 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 30 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of at least 40 mg/mL in water.
[0075] In some embodiments of any of the aspects, the active agent is present at a concentration of 1-100 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-50 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 1-40 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-100 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-50 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 10-40 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-100 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-50 mg/mL in water. In some embodiments of any of the aspects, the active agent is present at a concentration of 20-40 mg/mL in water.
[0076] In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 1-lOOx the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 5-50x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 10-3 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 15 -2 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL.
[0077] In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least 5x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least lOx the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least 15x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than lOOx the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 5 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 3 Ox the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 200x the concentration of the active agent, e.g., where both concentrations are expressed as mg/mL.
[0078] In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 1-lOOx the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 5 -5 Ox the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 10-30x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is 15 -2 Ox the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. [0079] In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least 5x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least lOx the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is at least 15x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than lOOx the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 5 Ox the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 30x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL. In some embodiments of any of the aspects, the the ionic liquid is at a concentration which is no greater than 200x the concentration of the active agent in water, e.g., where both concentrations are expressed as mg/mL.
[0080] As used herein, “composition” refers to any IL, combination of ILs, or combination of one or more ILs and one or more active agents described herein, unless further specified.
[0081] In some embodiments of any of the aspects, a composition or combination as described herein, comprising at least one IL and optionally an active agent can be formulated as an oral, subcutaneous, transdermal, intratumoral, intravenous, intradermal, or parenteral formulation. In some embodiments of any of the aspects, the composition or combination as described herein can be formulated for delivery to a mucus membrane, e.g., to a nasal, oral, or vaginal membrane. In some embodiments of any of the aspects, an oral formulation can be a degradable capsule comprising the composition comprising the at least one IL and optionally, an active agent. In some embodiments of any of the aspects, the composition or combination as described herein can be formulated for intravenous or intraocular delivery. In some embodiments of any of the aspects, the composition or combination as described herein can be formulated for intravenous delivery. In some embodiments of any of the aspects, the composition or combination as described herein can be formulated for in intraocular delivery.
[0082] In some embodiments of any of the aspects, described herein is a composition comprising at least one IL as described herein and at least one active agent. In some embodiments of any of the aspects, described herein is a composition consisting essentially of at least one IL as described herein and at least one active agent. In some embodiments of any of the aspects, described herein is a composition consisting of at least one IL as described herein and at least one active agent. In some embodiments of any of the aspects, the composition comprising at least one IL as described herein and at least one active agent is administered as a monotherapy, e.g., another treatment for the condition is not administered to the subject.
[0083] In one aspect of any of the embodiments, described herein is a pharmaceutical composition comprising at least one active agent in combination with at least one IL as described herein. In some embodiments of any of the aspects, the pharmaceutical composition comprises the at least one IL and the one or more active agents as described herein. In some embodiments of any of the aspects, the pharmaceutical composition consists essentially of the at least one IL and the one or more active agents as described herein. In some embodiments of any of the aspects, the pharmaceutical composition consists of the at least one IL and the one or more active agents as described herein. In some embodiments of any of the aspects, the pharmaceutical composition consists essentially of an aqueous solution of the at least one IL and the one or more active agents as described herein. In some embodiments of any of the aspects, the pharmaceutical composition consists of an aqueous solution of the at least one IL and the one or more active agents as described herein.
[0084] The compositions, formulations, and combinations described herein can comprise at least one IL as described herein, e.g., one IL, two ILs, three ILs, or more. In some embodiments of any of the aspects, a composition, formulation, or combination as described herein can comprise at least one IL as described herein and CAGE (Choline And GEranate).
[0085] In some embodiments of any of the aspects, the at least one active agent and the at least one ionic liquid are further in combination with at least one non-ionic surfactant. As used herein, “non-ionic surfactant” refers to a surfactant that lacks a net ionic charge and does not dissociate to an appreciable extent in aqueous media. The properties of non-ionic surfactants are largely dependent upon the proportions of the hydrophilic and hydrophobic groups in the molecule. Hydrophilic groups include the oxyethylene group (— OCH2 CH2 — ) and the hydroxy group. By varying the number of these groups in a hydrophobic molecule, such as a fatty acid, substances are obtained which range from strongly hydrophobic and water insoluble compounds, such as glyceryl monostearate, to strongly hydrophilic and water-soluble compounds, such as the macrogols. Between these two extremes types include those in which the proportions of the hydrophilic and hydrophobic groups are more evenly balanced, such as the macrogol esters and ethers and sorbitan derivatives. Suitable non ionic surfactants may be found in Martindale, The Extra Pharmacopoeia, 28th Edition, 1982, The Pharmaceutical Press, London, Great Britain, pp. 370 to 379. Non-limiting examples of non-ionic surfactants include polysorbates, a Tween™, block copolymers of ethylene oxide and propylene oxide, glycol and glyceryl esters of fatty acids and their derivatives, polyoxyethylene esters of fatty acids (macrogol esters), polyoxyethylene ethers of fatty acids and their derivatives (macrogol ethers), polyvinyl alcohols, and sorbitan esters, sorbitan monoesters, ethers formed from fatty alcohols and polyethylene glycol, polyoxyethylene-polypropylene glycol, alkyl polyglycoside, Cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, decyl polyglucose, glycerol monostearate, IGEPAL CA-630, isoceteth-20, lauryl glucoside, maltosides, monolaurin, mycosubtilin, Nonidet P-40, nonoxynol-9, nonoxynols, NP-40, octaethylene glycol monododecyl ether, N-Octyl beta-D-thioglucopyranoside, octyl glucoside, oleyl alcohol, PEG- 10 sunflower glycerides, pentaethylene glycol monododecyl ether, polidocanol, poloxamer, poloxamer 407, polyethoxylated tallow amine, polyglycerol polyricinoleate, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, Triton X-100, and the like. In some embodiments of any of the aspects, the at least one non-ionic surfactant has a neutral hydrophilic head group.
[0086] As used herein, “polysorbate” refers to a surfactant derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Common brand names for polysorbates include Scattics™, Alkest™, Canarcel™, and Tween™. Exemplary polysorbates include polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
[0087] In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 0.1% to about 50% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of 0.1% to 50% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 1% to about 5% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of 1% to 5% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 3% to about 10% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of 3% to 10% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of less than about 5% w/v. In some embodiments of any of the aspects, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of less than 5% w/v.
[0088] In some embodiments of any of the aspects, the combination of the at least one active agent and at least one IL as described herein is provided in one or more nanoparticles. In some embodiments of any of the aspects, the combination of the at least one active agent and at least one IL as described herein comprises nanoparticles comprising the active agent, the nanoparticles in solution or suspension in a composition comprising at least one IL as described herein.
[0089] In some embodiments of any of the aspects, the combination of the at least one active agent and at least one IL as described herein is provided in one or more nanocomplexes. [0090] In some embodiments of any of the aspects, a composition as described herein, e.g., a composition comprising at least one IL and an active agent, can further comprise a pharmaceutically acceptable carrier. As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition of the present disclosure can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field of art. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
[0091] The term "carrier" in the context of a pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations, and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). The formulation should suit the mode of administration.
[0092] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol;
(11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments of any of the aspects, the carrier inhibits the degradation of the active agent. The term "pharmaceutically acceptable carrier" excludes tissue culture medium.
[0093] In some embodiments of any of the aspects, a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent, can be formulated as an oral, intraocular, subcutaneous, intravenous, intradermal, or parenteral formulation. In some embodiments of any of the aspects, a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent, can be formulated as an intraocular, subcutaneous, intravenous, intradermal, or parenteral formulation. In some embodiments of any of the aspects, a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent, can be formulated as a subcutaneous, intravenous, intradermal, or parenteral formulation. In some embodiments of any of the aspects, a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent, can be formulated as a intravenous or parenteral formulation. In some embodiments of any of the aspects, a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent, can be formulated as an intravenous formulation. In some embodiments of any of the aspects, a composition as described herein, e.g., a composition comprising at least one IL as described herein and an active agent, can be formulated as an intraocular formulation. In some embodiments of any of the aspects, an oral formulation can be a degradable capsule comprising the composition described herein, e.g., a composition comprising at least one IL as described herein and an active agent. In some embodiments of any of the aspects, an oral formulation can be a capsule comprising a degradable coating or enteric coating, and further comprising the composition described herein, e.g., a composition comprising at least one IL as described herein and an active agent.
[0094] In some embodiments of any of the aspects described herein, the biological activity of the active agent is improved or stabilized as compared to the activity in the absence of the at least one IL. In some embodiments of any of the aspects described herein, the IL greatly enhances permeation of the active agent across the skin compared to a control where the at least one IL is absent.
[0095] In one aspect of any of the embodiments, the composition or combination described herein is for a method of administering or delivering at least one active agent, e.g., for the treatment of a disease. In one aspect of any of the embodiments, described herein is a method of administering at least one active agent, the method comprising administering the active agent in combination with at least one IL as described herein. In one aspect of any of the embodiments, described herein is a method of treating a disease by administering at least one active agent, the method comprising administering the active agent in combination with at least one IL as described herein. [0096] The disease treated by the methods described herein can be, e.g., cancer (breast cancer, leukemia, lymphoma, B-cell chronic lymphocytic leukemia, glioblastoma, carcinoma, urothelial carcinoma, lung cancer, colorectal cancer, lymphoblastic leukemia, lymphocytic leukemia, sarcoma, melanoma, prostate cancer, myeloma, multiple myeloma, Non-Hodgkin’s lymphoma), neuroblastoma, macular degeneration, and the like.
[0097] In some embodiments of any of the aspects, the methods described herein relate to treating a subject having or diagnosed as having a condition with a composition as described herein, e.g., a comprising at least one IL and an active agent. Subjects having a condition, e.g., cancer, can be identified by a physician using current methods of diagnosing cancer. Symptoms and/or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, for example, a lump/mass/tumor, swelling, or pain. Tests that may aid in a diagnosis of, e.g. cancer include, but are not limited to, x-rays, MRI, ultrasound, a biopsy, or tests for the function/activity of affected organs or systems. A family history of cancer or exposure to risk factors for cancer (e.g. smoke, radiation, pollutants, mutation, etc.) can increase the risk of a subject having cancer.
[0098] The compositions and methods described herein can be administered to a subject having or diagnosed as having a condition described herein. In some embodiments of any of the aspects, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a composition comprising at least one IL as described herein and an active agent, to a subject in order to alleviate a symptom of a condition described herein. As used herein, "alleviating a symptom" is ameliorating any marker or symptom associated with a condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration can be local or systemic.
[0099] In some embodiments of any of the aspects, the administration is intravenous. In some embodiments of any of the aspects, the administration is intraocular. In some embodiments of any of the aspects, the administration is transdermal. In some embodiments of any of the aspects, the administration is transdermal, to a mucus membrane (e.g., to a nasal, oral, or vaginal membrane), oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous.
[00100] Oral administration can comprise providing tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Oral formulations can comprise discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the IL and the at least one active agent, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
[00101] In one aspect of any of the embodiments, described herein is a method of delivery of at least one active agent by intraocular, subcutaneous, intradermal or intravenous administration, the method comprising administering the active agent in combination with at least one IL as described herein. In some embodiments of any of the aspects, intraocular, subcutaneous, intradermal or intravenous administration comprises administration via injection, catheter, port, or the like.
[00102] In one aspect of any of the embodiments, described herein is a method of parenteral delivery of at least one active agent, the method comprising parenterally administering the active agent in combination with at least one IL as described herein. In some embodiments of any of the aspects, the parenteral administration comprises delivery to a tumor, e.g., a cancer tumor. In some embodiments of any of the aspects, the composition or combination described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.
[00103] Suitable vehicles that can be used to provide parenteral dosage forms of a composition comprising at least one IL (e.g., CODES) in combination with at least one active agent as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of an ingredient in a composition as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms. [00104] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. While as noted above herein, the compositions comprising the at least one IL in combination with at least one active agent can obviate certain reasons for using a controlled-release formulation, it is contemplated herein that the methods and compositions can be utilized in controlled-release formulations in some embodiments. For example, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments of any of the aspects, the composition comprising the at least one IL in combination with at least one active agent can be administered in a sustained release formulation.
[00105] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
[00106] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
[00107] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
[00108] The term “effective amount" as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of a composition that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
[00109] In some embodiments of any of the aspects, a treatment for cancer is effective when the rate of growth of a tumor or cancer cell population is slowed. In some embodiments of any of the aspects, a treatment for cancer is effective when the growth of a tumor or cancer cell population is stopped. In some embodiments of any of the aspects, a treatment for cancer is effective when a tumor or cancer cell population is reduced in size. In some embodiments of any of the aspects, a treatment for cancer is effective when the rate of metastasis is slowed. In some embodiments of any of the aspects, a treatment for cancer is effective when the rate of metastasis is slowed. In some embodiments of any of the aspects, a treatment for cancer is effective when the rate of cell death in a tumor or cancer cell population is increased.
[00110] In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of growth of one or more blood vessels under the macular is slowed. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the growth of one or more blood vessels under the macular is stopped. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of blood or fluid leakage into the retina is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of blood or fluid leakage into the retina is slowed. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of retinal pigment epithelium thinning or depigrmentation is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when retinal pigment epithelium thinning or depigrmentation is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when retinal pigment epithelium thinning or depigrmentation is stopped. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when the rate of photoreceptor death is reduced. In some embodiments of any of the aspects, a treatment for macular degeneration is effective when photoreceptor death is stopped.
[00111] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e.. the concentration of the active agent, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor cell growth, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
[00112] In some embodiments of any of the aspects, the active agent can be a chemotherapeutic agent or agent effective for the treatment of cancer. In one aspect of any of the embodiments, described herein is a method of treating cancer, the method comprising administering a composition described herein, comprising at least one IL and at least one active agent, to the subject. In some embodiments of any of the aspects, the at least one active agent is a chemotherapeutic agent or agent effective for the treatment of cancer.
[00113] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
[00114] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e.. intrusion on and destruction of adjacent tissues), and metastasis (i.e. , spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
[00115] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre -malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
[00116] As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.
[00117] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastases. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
[00118] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome.
[00119] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
[00120] In some embodiments of any of the aspects, the cancer is an epithelial cancer. In some embodiments of any of the aspects, the cancer is breast cancer.
[00121] In some embodiments of any of the apsects, the composition as described herein, e.g., a composition comprising at least one IL as described herein in combination with at least one active agent, is administered as a monotherapy, e.g., another treatment for the condition is not administered to the subject.
[00122] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy, either in the composition described herein, e.g., a composition comprising at least one IL as described herein in combination with at least one active agent, or as a separate formulation. For example, non-limiting examples of a second agent and/or treatment for treatment of cancer can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizof iran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxabplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC -alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
[00123] In some embodiments of any of the aspects, the active agent can be an agent effective for the treatment of macular degeneration, e.g., a photosensitizer (for example verteporfm). In one aspect of any of the embodiments, described herein is a method of treating macular degeneration, the method comprising administering a composition described herein, comprising at least one IF and at least one active agent, to the subject. In some embodiments of any of the aspects, the at least one active agent is an agent effective for the treatment of macular degeneration. In some embodiments of any of the aspects, the composition is administered intraocularly.
[00124] In certain embodiments, an effective dose of a composition described herein, e.g., a composition comprising at least one IF as described herein in combination with at least one active agent, can be administered to a patient once. In certain embodiments, an effective dose a composition described herein, e.g., a composition comprising at least one IF as described herein in combination with at least one active agent, can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition described herein, e.g., a composition comprising at least one IF as described herein in combination with at least one active agent, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. In some embodiments of any of the aspects, the at least one active agent is present in the combination at a dose of from about 1.0-40.0 mg/kg. In some embodiments of any of the aspects, the at least one active agent is present in the combination at a dose of from 1.0-40.0 mg/kg. In some embodiments of any of the aspects, the at least one active agent is present in the combination at a dose of from about 1.0- 20.0 mg/kg. In some embodiments of any of the aspects, the at least one active agent is present in the combination at a dose of from 1.0-20.0 mg/kg.
[00125] In some embodiments of any of the aspects, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
[00126] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active agent. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments of any of the aspects, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition described herein, e.g., a composition comprising at least one IL in combination with at least one active agent, can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
[00127] The dosage ranges for the administration of the compositions described herein, according to the methods described herein depend upon, for example, the form of the active agent, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for symptoms or markers. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
[00128] The efficacy of a composition described in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of diabetes or cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.
[00129] In vitro and animal model assays are provided herein which allow the assessment of a given dose of a composition described herein, e.g., a composition comprising at least one IL in combination with at least one active agent.
[00130] The uptake of many active agents, e.g., pharmaceutically active agents, can be improved by delivering the compounds in solvents. However, such approaches are often unsuitable for in vivo use because most such solvents demonstrate toxic side effects and/or act as irritants to the point of delivery. Described herein are methods and compositions which can provide low toxicity with improved delivery kinetics.
[00131] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.
[00132] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
[00133] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments of any of the aspects, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[00134] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments of any of the aspects, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4- fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.
[00135] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments of any of the aspects, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein. [00136] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of conditions described herein. A subject can be male or female.
[00137] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
[00138] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
[00139] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, of the full length polypeptide. Conservative substitution variants that maintain the activity of wildtype protein will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species. Amino acids that are identical between homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and/or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of a condition as described herein. [00140] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.
[00141] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
[00142] In some embodiments of any of the aspects, the composition or active agent described herein is exogenous. In some embodiments of any of the aspects, the composition or active agent described herein is ectopic. In some embodiments of any of the aspects, the composition or active agent described herein is not endogenous.
[00143] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.
[00144] In some embodiments, a nucleic acid encoding a polypeptide as described herein is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non- viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
[00145] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).
[00146] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optomized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.
[00147] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
[00148] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
[00149] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
[00150] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer or macular degeneration. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with, e.g, cancer or macular degeneration. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
[00151] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to metastasis or vision worsening. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
[00152] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
[00153] As used herein, the term “nanoparticle” refers to particles that are on the order of about 1 to 1,000 nanometers in diameter or width. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. Exemplary nanoparticles include lipid nanoparticles or ferritin nanoparticles. Lipid nanoparticles can comprise multiple componenents, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.
[00154] Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.
[00155] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
[00156] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
[00157] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
[00158] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.
[00159] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
[00160] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[00161] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[00162] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00163] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00164] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [00165] Other terms are defined herein within the description of the various aspects of the invention.
[00166] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[00167] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[00168] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
[00169] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:
1. A composition comprising: a) an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion; and b) an active agent.
2. The composition of paragraph 1, wherein the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da.
3. The composition of any one of the preceding paragraphs, wherein the active agent comprises at least one drug with a water solubility of less than 10 mg/mL.
4. The composition of any one of the preceding paragraphs, wherein the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da.
5. The composition of any one of the preceding paragraphs, wherein the drug has a water solubility of less than 1 mg/mL.
6. The composition of any one of the preceding paragraphs, wherein the at least one drug has a water solubility of less than 1 mg/mL and a molecular weight of less than 1000 Da.
7. The composition of any one of the preceding paragraphs, wherein the at least one drug is selected from a photosensitizer, an anthracylcine, ataxane, or camptothecin and camptothecin analogs.
8. The composition of any one of the preceding paragraphs, wherein the at least one drug is selected from verteporfm, gemcitabine, doxorubicin, paclitaxel, and camptothecin.
9. The composition of any one of the preceding paragraphs, wherein the at least one drug is selected from verteporfm, doxorubicin, paclitaxel, and camptothecin.
10. The composition of any one of the preceding paragraphs, wherein the at least one drug is selected from verteporfm, doxorubicin, and paclitaxel.
11. The composition of any one of the preceding paragraphs, wherein the at least one drug is selected from verteporfm, paclitaxel, and camptothecin. The composition of any one of the preceding paragraphs, wherein the at least one drug comprises verteporfm. The composition of any one of the preceding paragraphs, wherein the active agent is present at a concentration of at least 1 mg/mL. The composition of any one of the preceding paragraphs, wherein the active agent is present at a concentration of at least 2 mg/mL. The composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration of at least 20 mg/mL in water. The composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration of 20-40 mg/mL in water. The composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 1-lOOx the concentration of the agent. The composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 5 -5 Ox the concentration of the agent. The composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 10-3 Ox the concentration of the agent. The composition of any one of the preceding paragraphs, wherein the ionic liquid is at a concentration which is 15 -2 Ox the concentration of the agent. The composition of any one of the preceding paragraphs, wherein the cation is choline. The composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 2:1 to 1:4, inclusive. The composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1.5:1 to 1:4, inclusive. The composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1:1 to 1:4, inclusive. The composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 2:1 to 1:1, inclusive. The composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1.7: 1 to 2: 1, inclusive. The composition of any one of the preceding paragraphs, wherein the molar ratio of cation to anion is 1.87:1. The composition of any one of the preceding paragraphs, wherein the ionic liquid and active agent are present together in nanocomplexes. A method of treating cancer in a subject in need thereof, the method comprising administering a composition of any of the preceding paragraphs to the subject. The method of paragraph 29, wherein the cancer is breast cancer. 31. The method of any one of paragraphs 29-30, wherein the composition is administered intravenously.
32. A method of treating macular degeneration in a subject in need thereof, the method comprising administering a composition of any of the preceding paragraphs to the subject.
33. The method of paragraph 32, wherein the at least one drug comprises verteporfm.
34. The method of any one of paragraphs 32-33, wherein the composition is administered intraocularly.
35. A method of drug delivery, the method comprising administering a composition of any of the preceding paragraphs to a subject in need of the active agent.
36. The method of paragraph 35, wherein the administration is intravenous, parenteral, or intraocular.
37. A composition of any one of paragraphs 1-28, for use in a method of treating cancer in a subject in need thereof.
38. The composition of paragraph 37, wherein the cancer is breast cancer.
39. The composition of any one of paragraphs 37-38, wherein the composition is administered intravenously.
40. A composition of any one of paragraphs 1-28, for use in a method of treating macular degeneration in a subject in need thereof.
41. The compsition of paragraph 40, wherein the at least one drug comprises verteporfm.
42. The composition of any one of paragraphs 40-41, wherein the composition is administered intraocularly.
43. A composition of any one of paragraphs 1-28, for use in a method of drug delivery.
44. The composition of paragraph 43, wherein the administration is intravenous, parenteral, or intraocular.
[00170] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
EXAMPLES
[00171] EXAMPLE 1: A Deep Eutectic Solvent-based approach to intravenous formulation [00172] Clinically viable formulations of hydrophobic drugs, for example, chemotherapeutics, require strategies to promote sufficient drug solubilization. However, such strategies often involve the use of organic solvents that pose a significant risk in generating toxic, unstable products. Using verteporfm as a drug, a deep eutectic solvent-based approach to solvate drugs in a simple one-step process is reported. Lipoidal deep eutectic solvent composed of choline and oleate is used to successfully solvate verteporfm, resulting in stable sub- 100 nm nanocomplexes. The nanocomplexes successfully demonstrate efficient cellular uptake as well as retention, tumor spheroid penetration, and tumor accumulation in vivo. Systemic administration of the formulation significantly inhibits the primary tumor growth and its lung metastasis in the orthotopic 4T1 murine breast tumor model. Collectively, biocompatible deep eutectic solvent shows great potential as a novel material for intravenous formulation of chemotherapeutics.
[00173] Deep eutectic solvent is introduced as a novel, biocompatible agent for dissolving water- insoluble drugs. Deep eutectic solvent comprised of choline and oleate demonstrated high aqueous solubility of a model chemotherapeutic drug verteporfm via nanocomplexation, and exhibited the ability to overcome multiple biological barriers to achieve successful cancer therapy in vivo against metastatic breast tumor in mice.
[00174] Introduction
[00175] Intravenous delivery of small hydrophobic drugs for cancer presents a significant technical challenge due to their poor water solubility. Currently 60-90% of drug candidates exhibit limited solubility.111 Current options to address this challenge include drug solubilization with water- miscible solvents, interfacial stabilization with surfactants, modifications with amphiphiles, and physical encapsulation in polymeric, lipid or inorganic nanovesicles.121 However, successful utilization of these strategies in the clinic is challenging especially for intravenous injections that cause systemic exposure of these solvating agents. In particular, co-solvents and solubilization agents, such as dimethyl sulfoxide (DMSO), ethanol, glycerol, n-methyl pyrrolidone, polyvinylpyrrolidone, polyethylene glycol, Cremophor EL, and polysorbate have a long-standing history of use for this purpose,131 but many of these agents are associated with significant toxicity, hypersensitivity reactions, and instability at the concentrations required to achieve therapeutic doses.141 Chemical drug modification is another strategy explored for drug modification. [2a51 However, this strategy poses a significant limitation by creating a new chemical entity, which increases the required efforts on establishment of safety. Engineered nanoparticle systems including liposomes and polymeric nanoparticles are also explored for drug encapsulation, however, preparation of these systems also involves the use of solubilizing agents in the process and often results in low encapsulation efficiency or off-target effects.161 Hence, there is an outstanding need for safe materials that readily dissolve water-insoluble drugs, maintain stable formulation, and demonstrate effective pharmacokinetic and pharmacodynamic profiles.
[00176] The inventors hypothesized that deep eutectic solvents (DESs) and ionic liquids (ILs) can provide an alternative to current options of solvents for solvation of hydrophobic drugs to enable their intravenous delivery. DESs are eutectic mixture of cationic and anionic species with a melting point below either of the individual component. ILs are simple salts in a viscous liquid state at temperature below 100°C, respectively. Recently, DESs and ILs have been explored as biocompatible liquids with high solvation properties for many hydrophobic molecules.171 In particular, small molecules and biological macromolecules have been dissolved in selective neat or dilute choline-based DESs and ILs to facilitate transdermal, oral and intratumoral drug delivery.181 However, the use of ILs or DESs for solvation and intravenous administration of hydrophobic drugs has not been demonstrated.
[00177] Described herein is the use of Choline and Oleic acid DES (CODES) for intravenous administration of a chemotherapeutic drug verteporfm. CODES is characterized by the conceptual resemblance to a synthetic phospholipid dioleylphosphatidylcholine (DOPC) that is typically used in liposomal formulation based on its choline and oleyl structures (Fig. 1A). Verteporfm is a highly water insoluble drug (water solubility of 20 mM) that is used in light-dependent generation of reactive oxygen species as well as light-independent inhibition of cancer cell proliferation. A current liposomal product, Visudyne, uses a 2 mg mL 1 verteporfm formulation for the photodynamic treatment of age-related macular degeneration. However, previous studies on Visudyne have reported its instability in plasma and lack of tumor targeting in the absence of light,191 thereby limiting intravenous administration of the liposomal formulation in non-photodynamic cancer therapy. In this study, it is demonstrated that small amounts of CODES can successfully solvate verteporfm in water at concentrations greater than 2 mg mL 1 and form nanocomplexes as a novel intravenous formulation alternative to verteporfm solubilized in DMSO (VPDMSO), a co-solvent used to solubilize verteporfm in many previous studies.1101 The resultant formulation, CODES-Verteporfm (COVERT), was stable and showed increased cellular uptake, tumor spheroid penetration, and tumor accumulation. Intravenously injected COVERT exhibited significant inhibition of primary tumor growth as well as its lung metastasis over VPDMSO in a murine orthotopic breast tumor model.
[00178] Results
[00179] Synthesis and characterization of CODES
[00180] Choline bicarbonate and oleic acid were reacted at 2: 1 molar ratio via metathesis to successfully synthesize lipid-like choline-oleate-bicarbonate deep eutectic solvent (Fig. 1A). 'H NMR analysis confirmed the stoichiometry of choline to oleic acid in CODES (choline :oleate ratio of 1.87:1 (Fig. IB). Fourier-transform infrared spectrum of CODES also exhibited specific peaks at 1650 cm 1 for v(C=C) in oleate and at 2900 cm 1, for v(O-H) in choline (Fig. 7A). The resulting product was a yellow paste at room temperature and 37 °C, but it transitioned into a viscous liquid when heated to 100 °C (Figs. 1C, 7B). Use of other molar ratios of choline:oleic acid was attempted as well. For example, reaction of 1 : 1 molar ratio of choline bicarbonate to oleic acid yielded a gel-like paste that remained in the gel phase even at 100 °C, and thus was not explored further for formulating verteporfm.
[00181] Preparation and characterization of COVERT
[00182] Verteporfm was solubilized at 2.5 mg mL 1 using aqueous dilution of CODES. Three different concentrations of CODES were tested to prepare COVERT (20, 40 and 80 mg mL 1 in water) labeled COVERT20, COVERT40 and COVERTso, respectively, to limit the concentration of CODES to only what is needed to solubilize verteporfm. Following 10 min sonication and fdtration through a desalting column, both formulations displayed a homogenous solution with no apparent precipitation of the drug. Freshly prepared COVERT40 exhibited a size of 85 nm (PDI: 0.226 ± 0.005, mean ± sd) and zeta potential of -80 mV (Fig. 2A). COVERT20 and COVERTso exhibited hydrodynamic diameters of 114 and 30 nm, respectively (Fig. 8A). The process of making these nanocomplexes with CODES is much simpler than that to produce liposomal formulation of similar size, for which nanoprecipitation, extrusion, or drying-hydration steps are necessary. To test the stability of COVERT upon storage at -20 °C, the size and zeta potential were determined for both formulations following a freeze-thaw cycle and additional incubation for 48 hr at room temperature. COVERT40 exhibited greater stability than COVERT20 and COVERTso, retaining its original size and surface charge. Moreover, COVERT20 resulted in aggregation of nanocomplexes following a 24-hr incubation in the presence of serum, while COVERT40 and COVERTso remained stable (Fig. 8B). Representative transmission electron microscopy image of COVERT40 also confirmed a nanocomplex of approximately 100 nm (Fig. 2B).
[00183] HPLC with diode-array and charged aerosol detectors was used to determine the verteporfm and CODES concentrations in the final formulations. Verteporfm concentration was determined to be 2.35 mg mL 1 for both COVERT formulations, yielding 94% loading efficiency of the drug in the nanocomplex (Figs. 2C, 8C). Final concentration of CODES in COVERT40 was determined to be 28.5 mg mL 1. Solvation of verteporfm in COVERT was assessed using X-ray diffraction (Fig. 2D). The crystalized structure, as observed in verteporfm powder, was not observed in COVERT40 nor VPDMSO at this concentration. However, the amorphous aggregation in VPDMSO as shown in the transmission electron microscopy image, significantly increased turbidity and thus scattering of light compared to COVERT (Figs. 2B, 2E, 8D). In vitro release of verteporfm from COVERT40 was characterized by a burst release within the first 2-4 hours, followed by an extended release (Fig. 8E).
[00184] In vitro toxicity and anti-cancer effect
[00185] Safety of CODES was assessed in vitro first to ensure verteporfm-specific cytotoxicity can be measured. Cytotoxicity of CODES on 4T1 murine breast cancer cells was measured after treating the cells with serial dilutions of CODES for 3 hr and incubating further in fresh media for 24 hr and the IC50 of CODES was determined to be 0.22 mg mL 1 (Fig. 9). Anti-cancer efficacy of COVERT and VPDMSO was assessed in serial dilutions. The dose-response curves for both COVERT formulations nearly overlapped with each other, indicating that CODES likely does not contribute significantly to COVERT’s cytotoxicity (Fig. 3). COVERT formulations exhibited similar IC50 values compared to VPDMSO- However, COVERT exhibited a significantly greater Hill coefficients (HC) than VPDMSO indicated by the steeper curve, indicating that COVERT formulations lead to increased drug sensitivity.
[00186] Intracellular uptake and retention [00187] The cellular uptake and retention of the drug in 4T1 cells were measured by flow cytometry after 2 hr incubation with COVERT or VPDMSO· In all three drug concentrations tested, nearly 100% of the cells showed intracellular presence of verteporfm for all groups. However, there was a significant increase in the cellular uptake of verteporfm per cell in the COVERT group (Figs. 4A, 10A). Interestingly, the increase in COVERT uptake was greater at higher verteporfm concentration of 2.5 x 106 M, indicating that greater absolute amount of CODES may induce energy- dependent endocytosis when presented to the cell membrane. This is further supported by COVERT showing similar level of energy-independent cellular uptake at 4 °C as VPDMSO (Figs. 4B, 10B), and therefore the increase in cellular uptake is likely due to energy-dependent endocytosis. Meanwhile, significant decrease in cellular uptake was observed at 4 °C compared to 37 °C for both COVERT40 and VPDMSO, indicating energy-dependent endocytosis as the major route of cellular entry for verteporfm (Fig. IOC). Moreover, Figs. 4C, 10D shows significant enhancement of the verteporfin’s intracellular retention in 4T1 cells as nanocomplexes over VPDMSO- When the cells were allowed to exocytose intracellular drug for 60 mins in fresh media following 2 hr incubation of 2.5 x 106 M verteporfm, approximately 45% of verteporfm remained in the cells when formulated into COVERT, in comparison to 29% for VPDMSO- Collectively, COVERT entered 4T1 cells more effectively than DMSO-solubilized verteporfm at around their IC50 values via active cellular uptake mechanism and showed prolonged intracellular retention, resulting in increased drug sensitivity. Based on these cellular responses to CODES-based nanocomplex formulations of verteporfm, COVERT40 was used in the following in vitro tumor spheroid and in vivo experiments.
[00188] Penetration in three-dimensional multicellular tumor spheroid [00189] One of the major barriers to effective tumor treatment is the drug’s limited penetration into densely packed tumor tissue to target the cancer cells in the core. COVERT40 and VPDMSO were incubated with three-dimensional tumor spheroid consisting of 4T1 cancer cells and 3T3 fibroblasts to determine the drug diffusion into the core and overall cell killing efficacy. Nuclei-staining Hoescht dye was used to distinguish cells in two layers of the spheroids based on the fluorescence intensity (Fig. 11A), and 4T1 and 3T3 cells were identified based on distinct populations in the forward vs. side scatter plot. As shown in Figs. 5A and 11B, COVERT40 and VPDMSO resulted in a similar level of verteporfm uptake by both cell types collectively in the outer layer, but COVERT40 demonstrated significantly greater penetration into the inner layer than VPDMSO- More importantly, COVERT40 showed 27% increase in the amount of verteporfm delivered to 4T1 cells in the inner layer of the spheroid in comparison to VPDMSO- Higher verteporfm uptake in the form of COVERT40 was also shown in the outer layer, as expected based on the result from the two-dimensional cellular uptake. When incubated with the equivalent concentration of 1 x 105 M verteporfm, significantly higher cell death from tumor spheroid was measured with COVERT40 than VPDMSO (Fig. 5B).
[00190] In vivo biodistribution and anti-tumor efficacy [00191] Finally, the efficacy of COVERT40 nanocomplex was investigated in a murine orthotopic breast tumor model. VPDMSO was used as the free drug control. Formulations were administered intravenously in B ALB/c female mice bearing 4T1 tumor in the mammary pad, and major organs including the tumors were harvested 3 hr post injection to determine the biodistribution of verteporfin (Fig. 6A). COVERT40 followed similar clearance pathway as lipid nanoparticles via the reticuloendothelial system and the kidneys.1111 Similar level of VPDMSO was detected in the lungs as COVERT40, indicating lung entrapment of amorphous verteporfin aggregates in VPDMSO formulation. Importantly, COVERT40 showed a significant increase of 66% in accumulation of verteporfin in tumor relative to VPDMSO control (Fig. 6B). Verteporfin continued to be detected in the tumor and all organs except the heart at 24 hr post injection (Fig. 12A). Interestingly, COVERT40 showed significantly greater amount of verteporfin in the lungs at 24 hr than VPDMSO, indicating better retention of the drug in the tissue.
[00192] Tumor-bearing mice were administered with four doses of COVERT40 or VPDMSO every 3 days and the primary tumor growth was monitored. Tumor growth was significantly inhibited by more than 70% in mice injected with COVERT40 (Fig. 6C) compared to the saline control, CODES control, and VPDMSO, correlating with the greater Hill coefficient, efficient cellular uptake and retention, enhanced tumor spheroid penetration, and increased tumor accumulation of COVERT40 over VPDMSO- Excised tumors also showed significant decrease in size and mass in COVERT40 group compared to the saline control, CODES control, and VPDMSO (Fig. 12B and Fig. 12C). All mice maintained healthy body weight throughout the duration of the experiment (Fig. 6D). Also, histopathological assessment of inflammation, fibrosis, ulceration, necrosis, and edema showed no visible toxicity in the major organs due to injections of VPDMSO, CODES, and COVERT40 (Fig. 12D). The study was terminated on day 26 when the tumor volume in the control group reached 1000 mm3, and lungs were harvested to quantify the number of metastatic nodules visible on the tissue. 4T1 breast cancer cells spontaneously metastasize to the draining lymph nodes, lungs and bone.[121 Figs. 6E, 12E show that verteporfin in both DMSO- and CODES-based formulations significantly inhibited the progression of lung metastasis in the 4T1 tumor model compared to the saline control, while CODES control showed no statistical difference. This observation is in agreement with the similar level of COVERT40 and VPDMSO accumulation in the lungs at early timepoints, potentially leading to verteporfm-mediated suppression of metastatic nodule formation in the tissue. Alternatively, lower VPDMSO accumulation at the primary tumor site may be sufficient to localize to the periphery of the tumor tissue and successfully inhibit the generation and shedding of circulating tumor cells.
[00193] Discussion
[00194] Verteporfin is a hydrophobic small molecule drug that is clinically approved for use in photodynamic therapy (PDT) of neovascular age-related macular degeneration but is also being explored further in cancer chemotherapy. [9bI More recently, anon-PDT mechanism was also elucidated for verteporfm, involving disruption of interaction between yes-associated protein (Y AP) and TEA domain transcription factor (TEAD) in the cancer cell-specific Hippo pathway, which leads to decreased cancer cell proliferation in vitro and tumor growth in vivo without any photoactivation in various cancer models. [10b131
[00195] Existing co-solvents, such as alcohol, polyethylene glycol, and polyvinylpyrrolidone, render high solubility of many hydrophobic compounds in aqueous mixture. However, drugs dissolved in such co-solvent system often require additional surfactants or pH modifiers to prevent precipitation.131 We demonstrated that CODES can both solvate a hydrophobic drug and maintain its stability upon further aqueous dilution through formation of nanocomplexes. Furthermore, the resulting nanocomplexes share many advantages of other nanoparticulate drug delivery systems to overcome biological barriers.
[00196] Drugs in systemic circulation are faced with various barriers leading to their site of action. The advantages of COVERT formulations were explored against two major biological bottlenecks in vitro. Efficient cellular uptake is critical at the cellular level for drugs with intracellular targets. While free verteporfm also showed high level of cellular uptake, COVERT formulations entered the cells more efficiently via energy-dependent pathways. By slowing exocytosis rate upon cellular uptake, which parallels previous reporting with another DES composed of choline and geranate,[15] COVERT40 nanocomplex allows for greater cytosolic concentration of verteporfm. This may explain over 2-fold increase in Hill coefficient with COVERT40 in comparison to the free drug, suggesting greater drug sensitivity and leading to more efficient cell killing. While IC50 is an extrinsic parameter affected by cell division rates, cell seeding density, and drug incubation time, Hill coefficient is intrinsic to the interaction between ligands to receptors and has been reported to have a stronger correlation to the in vivo efficacy than IC50 values.1161 Through nano-complexation and increased cytosolic concentration, COVERT can locally present multiple verteporfm molecules and mediate their cooperative binding to multiple YAP residues (Gln82, Val84, Met86 and Arg89) involved in interaction with TEAD.[171
[00197] Penetration into the tumor tissue is another hurdle for cancer nanomedicines. Limited diffusion of free drug or its formulations to the core of the tumor tissue results not only in partial efficacy at the regions proximate to the blood vessels but also development of drug resistance.1181 Greater amount of verteporfm was observed in the cells located at the inner layer of tumor spheroids when delivered with 100-nm COVERT40 nanocomplexes than the free drug. This penetration is achieved with DES-based formulation in the absence of any surface modification that is known to facilitate diffusion of other nanovesicles.
[00198] Reduced number of metastatic nodules in the lungs was observed upon both COVERT40 and VPDMSO treatments. It has previously been shown that non-photoactivated verteporfm suppresses interaction of yes-associated protein (Y AP) with transcriptional enhancer factor 1 in cancer cells, the complex of which is known to promote both tumor progression and metastasis in breast cancer and melanoma.1201 While VPDMSO significantly reduced lung metastasis, it failed to suppress the primary tumor growth. In contrast, COVERT40 successfully delivers verteporfm’s dual functions of slowing the primary tumor growth and metastasis to distant organs in highly metastatic orthotopic breast cancer. This deep eutectic solvent-based intravenous formulation thus maximizes the anti-cancer efficacy of verteporfm.
[00199] Experimental procedures [00200] Materials
[00201] Choline bicarbonate, verteporfm (Millipore Sigma), oleic acid (TCI America), and Hoescht 33342 (Thermo Fisher) were purchased and used as received. All organic solvents are of analytical grade. 4T1 murine breast cancer cell (ATCC CRL-2539) and 3T3 murine fibroblasts (ATCC CRL-1658) were purchased from ATCC (Manassas, VA). CyQUANT™ Direct Red Cell Proliferation assay (Invitrogen) and CellTiter-Glo® 3D Cell Viability assay (Promega) were used per manufacturer’s instructions.
[00202] Synthesis and characterization of CODES
[00203] Lipoidal deep eutectic solvent CODES was synthesized following previously described method. [8aI Briefly, choline bicarbonate (80% solution in water) and oleic acid were separately weighed and mixed in a round-bottom flask at a predetermined molar ratio. The mixture was allowed to react while stirring at 40 °C for 12 hr. Then, the product was purified by removing water using a rotary evaporator at 60 °C for 4 hr, followed by additional drying in a vacuum oven at 60 °C for 48 hr. CODES was characterized by both 'H nuclear magnetic resonance (NMR) spectroscopy with CDCE using Agilent DD2600 MHz spectrometer and Fourier-transform infrared (FT-IR) spectra using Bruker ALPHA ATR-FTIR (64 scans).
[00204] Formulation and characterization of COVERT
[00205] COVERT80, COVERT40, and COVERT20 were formulated by simple dissolution of verteporfm. Verteporfm was dissolved at 2.5 mg mL 1 using CODES diluted in water at 80, 40 or 20 mg mL 1, respectively, by vortexing for 1 min vortexing followed by sonicating for 10 min. The solution was filtered through PD- 10 desalting column (Cytiva) to remove free drug not complexed with CODES, and the product was stored at -20 °C, and thawed and sonicated for 10 min before use. [00206] To quantify verteporfm and CODES concentration in the final formulation, COVERT nanocomplexes were diluted 100-fold with 9: 1 v/v acetonitrile: water, sonicated, and injected into reverse phase high performance liquid chromatography system with a column (Agilent, Santa Clara, CA). Verteporfm was detected at 450 nm with DAD at 1 mL min 1 flow rate using mobile phases consisting of (A) 5 x 105 M monobasic sodium phosphate (pH 3.5) and (B) methanol, with gradient applied as follows: 0-4 min from 50 to 65% eluent B; 4-8 min from 65 to 85% eluent B; 8-22 min from 85 to 99% eluent B; 22-27 min from 99 to 10% eluent B; 27-30 min from 10 to 50% eluent B; 30-40 min 50% eluent B. Linear calibration curve was established from 0 to 50 mg/mL for quantification. CODES was detected using Corona Veo Charged Aerosol Detector (CAD) (ThermoFisher Scientific, Waltham, MA). Mobile phases consisted of (A) 9: 1 v/v acetonitrile: water with 0.1% trifluoroacetic acid (TFA) and (B) methanol with 0.1% TFA, with gradient increasing from 0-15 min from 40 to 100% eluent B and 15-20 min from 100 to 40% eluent B. Finear calibration curve was established from 0 to 800 pg mF 1 for quantification. Encapsulation efficiency (EE) and loading capacity (EC) are calculated according to the following equations:
Figure imgf000057_0001
[00207] Hydrodynamic diameter and zeta potential of COVERT nanocomplexes were measured by dynamic light scattering (DLS) using Malvern Zen3600 Zetasizer (Malvern, UK). Samples were diluted to 1 mg mL 1 of verteporfm using concentrated phosphate-buffered saline (PBS) to reach final IX PBS before measurement. For stability in plasma, samples were incubated with 10% v/v pig serum in IX PBS for specified timepoint, and then diluted to 1 mg/mL of verteporfm with water prior to DLS measurement. The morphology of COVERT40 was visualized by transmission electron microscopy (TEM) without negative staining using JEOL JEM-1400 operating at 100 kV. X-ray diffraction of verteporfm power, neat CODES, and COVERT40 were detected with Bruker D2 Phaser Benchtop XRD System (Bruker USA). Turbidity of COVERT40 and VPDMSO was measured by absorbance at the wavelength (520 nm) verteporfm minimally absorbs to detect light scattering from aggregation in the formulations. In vitro release of verteporfm from COVERT40 was tested by incubating the sample at 0.25 mg mL 1 of vertepofm in water for specified timepoint, removing released drug by filtering through PD- 10 desalting column, and quantifying remaining verteporfm in COVERT40 filtrate by fluorescence (430/20 nm excitation and 690/20 nm emission) on BioTek Neo2™ plate reader.
[00208] Cell culture
[00209] 4T1 murine breast cancer cells were grown in RPMI-1640 medium with 10% FBS and
1% penicillin/streptomycin at 37 °C and 5% CO2 and 3T3 fibroblasts were grown in high-glucose Dulbecco’s modified Eagle medium (DMEM) with 10% FBS at 37 °C and 5% CO2.
[00210] Cell viability
[00211] 4T1 cells were seeded in 96-well plates at 15,000 cells per well and incubated for 24 hr to allow adherence. Media was replaced with 100 pL fresh media containing dilutions of 1) CODES from 0 to 2.5 mg mL 1, 2) stock verteporfm prepared at 3.5 mM in DMSO from 0.625 to 40 x 106 M, and 3) COVERT formulations to equivalent verteporfm concentrations from 0.625 to 40 x 106 M. Following a 3 -hr incubation, cells were washed with IX PBS and further incubated in fresh media for additional 24 hr before viability quantification using CyQUANT™ Direct Red Cell Proliferation Assay per manufacturer’s instruction (Invitrogen).
[00212] Cellular uptake and retention
[00213] 4T1 cells were seeded in 96-well plates at 15,000 cells per well and incubated for 24 hr to allow adherence. For cellular uptake study, media was replaced with 100 pF fresh media containing dilutions of 1) stock verteporfm prepared at 3.5 mM in DMSO and 2) COVERT formulations to equivalent verteporfm concentrations of 0.625, 1.25, and 2.5 x 106 M. Following a 2-hr incubation, cells were washed twice with IX PBS, trypsinized, and resuspended in IX PBS containing 2% FBS for flow cytometry (BD Fortessa™ Cell Analyzer). Fluorescence from verteporfm was detected with PerCp Cy5.5 channel (laser 488 nm with bandpass fdter 695/40). nm excitation and nm emission. For cellular retention study, cells were similarly treated with 100 pF fresh media containing dilutions of 1) stock verteporfm prepared at 3.5 mM in DMSO and 2) COVERT formulations to equivalent verteporfm concentrations of 0.625, 1.25, and 2.5 x 106 M. Following a 2-hr incubation, cells were washed twice with IX PBS and replenished with 100 pF fresh media. At predetermined timepoints, cells were again washed with IX PBS, trypsinized, and resuspended in IX PBS containing 2% v/v FBS for flow cytometry.
[00214] Three-dimensional tumor spheroid penetration
[00215] Three-dimensional tumor spheroids consisting of 4T1 cells and fibroblasts were constructed using the previously reported method [21]. Briefly, single-cell suspension was prepared with 1:5 ratio of 4T1 cells and 3T3 fibroblasts at a total cell concentration of 1 c 105 cells mF 1, and added to U-bottomed 96-well spheroid microplates (Coming life sciences) at 20,000 cells per well. Cells were then incubated at 37 °C with 5% CO2 with medium replaced every other day. Spontaneously formed tumor spheroids of 500-600 pm diameter were visible in each well at day 6. [00216] For penetration study, spheroid in each well was treated for 2 hr with 100 pF media containing COVERT40 or free verteporfm at equivalent verteporfm concentration of 2.5 x 106 M. During the last 30 min of treatment, 1 pF of Hoescht dye was added to the wells to a final concentration of 1 x 106 M. Spheroids were then washed once with IX PBS, trypsinized to achieve single cell suspension, and resuspended in IX PBS containing 2% v/v FBS for flow cytometry. DAPI channel (laser 405 nm with bandpass filter 515/20) was used to separate two layers of the spheroids based on Hoescht fluorescence intensity, FSC-H vs. SSC-H was used to separate 4T1 cells and 3T3 fibroblasts, and PerCP Cy5.5 channel was used to quantify verteporfm uptake.
[00217] For spheroid viability assay, spheroid in each well was treated for 3 hr with 100 pF media containing COVERTso, COVERT40, or free verteporfm at equivalent verteporfm concentration of 2.5 x 106 M. Spheroids were then washed with IX PBS and further incubated in fresh media for additional 48 hr before viability quantification using CellTiter-Glo® 3D Cell Viability assay.
[00218] Biodistribution
[00219] All of the experiments were performed according to the approved protocols by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Arts and Sciences (FAS), Harvard University. Female BAUB/c mice (aged 7-8 weeks) were purchased from Charles River Uaboratories. Orthotopic breast tumor was developed by injection of 1054T1 cells in IX PBS above the fourth mammary fat pad. On day 14 post inoculation, mice were intravenously injected with 5 mg kg 1 of verteporfm as either COVERT40 formulation or free verteporfm. Tumor tissue and major organs, including spleen, liver, kidneys, and heart, were harvested at 3 and 24 hr following injection, weighed, and homogenized in water to obtain lysates. Verteporfm was recovered from the lysates by extraction in methanol, and quantified by fluorescence (430/20 nm excitation and 690/20 nm emission) on BioTek Neo2 plate reader. Uinear calibration curve was established from 1 x 109 to 3 x 106 M verteporfm in each of the organ lysates for quantification.
[00220] In vivo anti-tumor efficacy
[00221] Orthotopic breast tumor was developed in female BAUB/c mice (aged 7-8 weeks) by injection of 1054T1 cells in IX PBS above the fourth mammary fat pad. When tumor size reached approximately 50 mm3 on day 6, mice were treated with four intravenous injections three days apart of 5 mg kg 1 verteporfm as either COVERT40 formulation or free verteporfm, in addition to the saline and CODES controls. Tumor volume and body weight were monitored every other day. The study was terminated when the tumors in control group reached the endpoint criteria of 1000 mm3, at which point lungs were harvested to count surface-visible metastatic nodules and to visualize them in histological sections.
[00222] In vivo toxicity
[00223] Healthy female BALB/c mice (aged 7-8 weeks) were treated with four intravenous injections three days apart of 5 mg kg 1 verteporfm as either COVERT40 formulation or free verteporfm, in addition to the saline and CODES controls. 24 hours post the fourth injection, mice were euthanized and major organs, including the spleen, kidneys, liver, lungs, and heart, were excised and fixed in paraformaldehyde. H&E-stained histological sections were obtained and imaged using Axioscan™.
[00224] Statistical analysis
[00225] Statistical comparisons were performed in GraphPad Prism 8™. All data are presented as mean ± standard deviation. Unpaired, two-tailed Student’s t-test was used for comparisons between two groups, while one-way ANOVA with individually specified post-hoc testing was performed to compare between multiple groups. Statistical significance is defined as *p < 0.05, * *p < 0.01,
***/>< 0.001. [00226] Further Discussion
[00227] This is the first report of using deep eutectic solvent to formulate a nanocomplex for systemic administration of chemotherapeutic drug. Described herein is the successful synthesis of a lipid-like variant of DES composed of choline and oleate at 2: 1 molar ratio, and formulation of nanocomplexes by simple solvation of a small molecule drug, verteporfm, with the DES. The nanocomplex efficiently overcame biological barriers, including cellular uptake and retention, 4T1 tumor spheroid penetration, and tumor tissue accumulation in vivo. Greater drug sensitivity for the nanocomplex over the free drug correlated with effective inhibition of orthotopic, primary 4T1 breast tumor growth. In addition to targeting the primary tumor, DES-based verteporfm nanocomplex is contemplated as a treatment to reduce tumor metastasis.
[00228] References
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Example 2
[00229] The following drugs were formulated in CODES as above, displaying the following characteristics:
Figure imgf000062_0001

Claims

What is claimed herein is:
1. A composition comprising: a) an ionic liquid comprising i) a quaternary ammonium cation and ii) an oleate and/or oleic acid anion; and b) an active agent.
2. The composition of claim 1, wherein the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da.
3. The composition of any one of the preceding claims, wherein the active agent comprises at least one drug with a water solubility of less than 10 mg/mL.
4. The composition of any one of the preceding claims, wherein the active agent comprises at least one drug with a water solubility of less than 10 mg/mL and a molecular weight of less than 1000 Da.
5. The composition of any one of the preceding claims, wherein the drug has a water solubility of less than 1 mg/mL.
6. The composition of any one of the preceding claims, wherein the at least one drug has a water solubility of less than 1 mg/mL and a molecular weight of less than 1000 Da.
7. The composition of any one of the preceding claims, wherein the at least one drug is selected from a photosensitizer, an anthracylcine, a taxane, or camptothecin and camptothecin analogs.
8. The composition of any one of the preceding claims, wherein the at least one drug is selected from verteporfm, gemcitabine, doxorubicin, paclitaxel, and camptothecin.
9. The composition of any one of the preceding claims, wherein the at least one drug is selected from verteporfm, doxorubicin, paclitaxel, and camptothecin.
10. The composition of any one of the preceding claims, wherein the at least one drug is selected from verteporfm, doxorubicin, and paclitaxel.
11. The composition of any one of the preceding claims, wherein the at least one drug is selected from verteporfm, paclitaxel, and camptothecin.
12. The composition of any one of the preceding claims, wherein the at least one drug comprises verteporfm.
13. The composition of any one of the preceding claims, wherein the active agent is present at a concentration of at least 1 mg/mL.
14. The composition of any one of the preceding claims, wherein the active agent is present at a concentration of at least 2 mg/mL.
15. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration of at least 20 mg/mL in water.
16. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration of 20-40 mg/mL in water.
17. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration which is 1-lOOx the concentration of the agent.
18. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration which is 5 -5 Ox the concentration of the agent.
19. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration which is 10-30x the concentration of the agent.
20. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration which is 15-20x the concentration of the agent.
21. The composition of any one of the preceding claims, wherein the cation is choline.
22. The composition of any one of the preceding claims, wherein the molar ratio of cation to anion is 2:1 to 1:4, inclusive.
23. The composition of any one of the preceding claims, wherein the molar ratio of cation to anion is 1.5:1 to 1:4, inclusive.
24. The composition of any one of the preceding claims, wherein the molar ratio of cation to anion is 1:1 to 1:4, inclusive.
25. The composition of any one of the preceding claims, wherein the molar ratio of cation to anion is 2: 1 to 1:1, inclusive.
26. The composition of any one of the preceding claims, wherein the molar ratio of cation to anion is 1.7: 1 to 2: 1, inclusive.
27. The composition of any one of the preceding claims, wherein the molar ratio of cation to anion is 1.87:1.
28. The composition of any one of the preceding claims, wherein the ionic liquid and active agent are present together in nanocomplexes.
29. A method of treating cancer in a subject in need thereof, the method comprising administering a composition of any of the preceding claims to the subject.
30. The method of claim 29, wherein the cancer is breast cancer.
31. The method of any one of claims 29-30, wherein the composition is administered intravenously.
32. A method of treating macular degeneration in a subject in need thereof, the method comprising administering a composition of any of the preceding claims to the subject.
33. The method of claim 32, wherein the at least one drug comprises verteporfm.
34. The method of any one of claims 32-33, wherein the composition is administered intraocularly.
35. A method of drug delivery, the method comprising administering a composition of any of the preceding claims to a subject in need of the active agent.
36. The method of claim 35, wherein the administration is intravenous, parenteral, or intraocular.
37. A composition of any one of claims 1-28, for use in a method of treating cancer in a subject in need thereof.
38. The composition of claim 37, wherein the cancer is breast cancer.
39. The composition of any one of claims 37-38, wherein the composition is administered intravenously.
40. A composition of any one of claims 1-28, for use in a method of treating macular degeneration in a subject in need thereof.
41. The compsition of claim 40, wherein the at least one drug comprises verteporfm.
42. The composition of any one of claims 40-41, wherein the composition is administered intraocularly.
43. A composition of any one of claims 1-28, for use in a method of drug delivery.
44. The composition of claim 43, wherein the administration is intravenous, parenteral, or intraocular.
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