Stable Theranostic and Therapeutic Nanoemulsions Using Triphilic Semifluorinated Amphiphiles
20200101014 ยท 2020-04-02
Assignee
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
A61K49/18
HUMAN NECESSITIES
Abstract
The present invention provides therapeutic formulations, including therapeutic nanoemulsions, and related methods for the in vivo delivery of hydrophobic compounds. Formulations and methods of the invention include semifluorinated block copolymers and an imaging compound to form a theranostic nanoemulsion, capable of forming a stable nanoemulsion. In certain embodiments, emulsion-based formulations are provided that are capable of formulating, delivering and releasing amounts of hydrophobic drugs effective for a range of clinical applications, including treating cancer and fungal infections in patients. In certain embodiments, emulsion-based formulations are provided that are capable of supporting controlled release, for example, over a range of rates useful for clinical applications including sustained release.
Claims
1. An emulsion for delivery of a therapeutic agent and/or an imaging agent, said emulsion comprising an oil in water emulsion comprising: a hydrophobic liquid; an aqueous solution; semi-fluorinated block copolymers; wherein each of said semi-fluorinated block copolymers independently comprises a hydrophilic block, a hydrophobic block and a fluorophilic block; wherein said fluorophilic block of each of said semi-fluorinated block copolymers is provided between said hydrophobic block and said hydrophilic block; wherein said hydrophobic liquid is capable of solubilizing the therapeutic agent; and wherein said fluorinated block is capable of solubilizing the imaging agent.
2. The emulsion of claim 1, wherein said hydrophobic liquid is lipophilic.
3. The emulsion of claim 1, wherein said hydrophobic liquid is one or more oils.
4. The emulsion of claim 3, wherein said hydrophobic liquid is one or more glycerides.
5. The emulsion of claim 3, wherein said hydrophobic liquid is one or more medium-chain triglycerides.
6. The emulsion of any of claims 1-5, wherein said semi-fluorinated block copolymers have a concentration selected from the range of 5 mg L.sup.1 to 50 mg L.sup.1, or a concentration selected from the range of 5 to 30 mM.
7. The emulsion of any of claims 1-6, wherein said hydrophobic block of each of said semi-fluorinated block copolymers is a polymer terminating group.
8. The emulsion of any of claims 1-7, wherein said hydrophilic block of each of said semi-fluorinated block copolymers is a polymer terminating group.
9. The emulsion of any of claims 1-8, wherein said hydrophobic block of each of said semi-fluorinated block copolymers is directly linked to said fluorophilic block.
10. The emulsion of any of claims 1-9, wherein said fluorophilicblock of each of said semi-fluorinated block copolymers is directly linked to said hydrophilic block.
11. The emulsion of any of claims 1-10, wherein said hydrophobic block, said hydrophilic block or both are independently linked to said fluorophilic block via a linking moiety selected from the group consisting of an ether group, a carbamate group, an amide group, a carboxyl group, an ester group, an alkyl group, an alkylene group, an amino group or any combination of these.
12. The emulsion of any of claims 1-11, wherein each of said fluorophilic blocks of said semi-fluorinated block copolymers is independently a fluorocarbon moiety having between 3 to 32 carbon-fluorine bonds.
13. The emulsion of any of claims 1-12, wherein each of said fluorophilic blocks of said semi-fluorinated block copolymers is independently a fluorinated alkyl group having a length of 6 to 16 carbons.
14. The emulsion of any of claims 1-13, wherein each of said fluorophilic blocks of said semi-fluorinated block copolymers is independently a perfluorinated alkyl group having a length of 6 to 16 carbons.
15. The emulsion of any of claims 1-14, wherein said hydrophilic blocks of said semi-fluorinated block copolymers is independently selected from the group consisting of a polyoxygenated polymer block, a poly(vinylpyrrolidone) block, a poly(acrylic) block, a polyacrylamide block, a polyoxazoline block, a polysaccharide block and a chitosan derivative block.
16. The emulsion of any of claims 1-15, wherein each of said hydrophilic blocks of said semi-fluorinated block copolymers is a poly(ethylene glycol) block having an average molecular weight selected over the range of 1000 g mol.sup.1 to 40, 000 g mol.sup.1.
17. The emulsion of any of claims 1-16, wherein each said hydrophobic blocks of said semi-fluorinated block copolymers is independently selected from the group consisting of a substituted or unsubstituted C.sub.5-C.sub.27 alkyl block, substituted or unsubstituted C.sub.5-C.sub.27 alkylene block, a poly (-caprolactone) block, a poly(lactic acid) block; a poly(propylene glycol) block; a poly(amino acid) block; a poly(ester) block and poly(lactic-co-glycolic acid), wherein the block may be linear or branched.
18. The emulsion of any of claims 1-17, wherein each of said hydrophobic blocks of said semi-fluorinated block copolymers is independently an unsubstituted C.sub.12-C.sub.20 alkyl group.
19. The emulsion of any of claims 1-18, wherein each of said hydrophobic blocks of said semi-fluorinated block copolymers is independently a C.sub.16 alkyl group.
20. The emulsion of any of claims 1-19, wherein each of said semi-fluorinated block copolymers independently has the formula (FX1): ##STR00006## wherein A is the hydrophilic block, B is the fluorophilic block and D is the hydrophobic block; wherein L.sup.1 and L.sup.2 are each independently a linking group; and wherein m is 0 or 1 and n is 0 or 1.
21. The emulsion of claim 20, wherein each of said semi-fluorinated block copolymers independently has the formula (FX2): ##STR00007## wherein q is a integer selected from the range of 10 to 300, o is an integer selected from the range of 6 to 16, and p is an integer selected from the range of 10 to 27; wherein R.sup.1 is hydrogen, methyl, C.sub.1-C.sub.10 alkyl, C.sub.3-C.sub.10 cycloalkyl, 0.sub.5-C.sub.10 aryl, C.sub.5-C.sub.10 heteroaryl, C.sub.1-C.sub.10 alkoxy or C.sub.1-C.sub.10 acyl; wherein R.sup.2 is hydrogen or C.sub.1-C.sub.5 alkyl; wherein each of L.sup.1 and L.sup.2 is independently null, O, O(CH.sub.2).sub.e, (CH.sub.2).sub.e, (CH.sub.2).sub.eO(CH.sub.2).sub.f, (CH.sub.2).sub.eS(CH.sub.2).sub.f, (CH.sub.2).sub.eNR.sup.11(CH.sub.2).sub.f, (CH.sub.2).sub.eOCONR.sup.12(CH.sub.2).sub.f, (CH.sub.2).sub.eCONR.sup.13(CH.sub.2).sub.f, (CH.sub.2).sub.eNR.sup.14COO(CH.sub.2).sub.f, (CH.sub.2).sub.eNR.sup.15CO(CH.sub.2).sub.f or (CH.sub.2).sub.eNR.sup.16CONR.sup.17(CH.sub.2).sub.f; wherein each of R.sup.11-R.sup.17 is independently hydrogen, methyl, or C.sub.1-C.sub.5 alkyl; and wherein each of e and f is independently an integer selected from the range of 0 to 5.
22. The emulsion of claim 21, wherein each of said semi-fluorinated block copolymers independently has the formula (FX3A) or (FX3B): ##STR00008##
23. The emulsion of claim 21, wherein each of said semi-fluorinated block copolymers independently has the formula (FX4A) or (FX4B): ##STR00009##
24. The emulsion of any of claims 1-23, wherein each of said semi-fluorinated block copolymers independently has a molecular weight selected from the range 1100 Da to 14000 Da.
25. The emulsion of any of claims 1-24, further comprising a perhalogenated fluorous compound.
26. The emulsion of claim 25, wherein said perhalogenated fluorous compound is 3% to 40% by volume of said emulsion.
27. The emulsion of claim 25, wherein said perhalogenated fluorous compound is selected from the group consisting of perfluorooctyl bromide, perfluorononyl bromide, perfluorodecyl bromide, perfluorodecalin, perfluorodichlorooctane, bis-perfluorobutyl ethylene and perfluoro(methyldecalin).
28. The emulsion of any of claims 1-27, further comprising a therapeutic agent.
29. The emulsion of claim 28, wherein the therapeutic agent has a concentration selected from the range of 0.1 mg mL.sup.1 to 50 mg mL.sup.1 relative to the hydrophobic liquid of said emulsion.
30. The emulsion of any of claims 28-29, wherein said therapeutic agent is a hydrophobic compound and is noncovalently associated with the hydrophobic block of said semi-fluorinated block copolymers.
31. The emulsion of any of claims 28-30, wherein said therapeutic agent is characterized by a solubility in water of equal to or less than 20 mM.
32. The emulsion of any of claims 28-31, wherein said therapeutic agent is an anticancer agent or antifungal agent.
33. The emulsion of claim 32, wherein said therapeutic agent is selected from the group consisting of paclitaxel, doxorubicin, retinoic acid series, camptothecin, docetaxel, tamoxifen, anasterozole, topotecan, belotecan, irinotecan, gleevec and vincristine.
34. The emulsion of claim 33, wherein the therapeutic agent is paclitaxel and said paclitaxel has a concentration of 0.1 mg mL.sup.1 to 50 mg mL.sup.1 relative to the hydrophobic liquid in said emulsion.
35. The emulsion of any of claims 1-34 further comprising an imaging agent.
36. The emulsion of claim 35, wherein the imaging agent is a nuclear magnetic resonance imaging contrast agent.
37. The emulsion of claim 35, wherein the imaging agent is physically associated with the fluorophilic blocks of semi-fluorinated block copolymers.
38. The emulsion of claim 36, wherein the imaging agent is a selected from the group consisting of a perfluorinated compound selected from the group consisting of perfluoroalkanes, perfluoroalkylamines, perfluoro-crown-ethers, perfluorinated alcohols, perfluorohaloalkanes, perfluorinated carboxylic acids, perfluorinated acrylates, and perfluorinated esters.
39. The emulsion of claim 38, wherein the imaging agent is perfluoropolyether, perfluoro-15-crown-5-ether, sulfur hexafluoride, hexafluoroethane, or perfluoropropane.
40. The emulsion of claim 35, wherein said imaging agent is perfluoro-15-crown-5-ether and has a concentration of either 5 mg mL.sup.1 to 750 mg mL.sup.1 or of from about 5 to 35% v/v in said emulsion.
41. The emulsion of any of claims 28-40, wherein the therapeutic agent has a concentration selected from the range of 0.1 mg mL.sup.1 to 50 mg mL.sup.1 relative to the hydrophobic liquid in said emulsion; and wherein the semi-fluorinated block copolymers have a concentration selected from the range of 5 mg mL.sup.1 to 50 mg mL.sup.1.
42. The emulsion of any of claims 1-45, wherein said aqueous solution comprises a saline solution.
43. The emulsion of any of claims 1-42, wherein said emulsions contain individual oil droplet core particles having an average diameter less than or equal to 500 nanometers.
44. The emulsion of claim 43, wherein said droplets have an average diameter less than or equal to 400 nanometers.
45. The emulsion of any of claims 42-44, wherein said droplets have a hydrophobic core comprising said hydrophobic blocks of said semi-fluorinated block copolymers.
46. The emulsion of any of claims 42-45, wherein said emulsions have a hydrophilic exterior shell comprising said hydrophilic blocks of said semi-fluorinated block copolymers.
47. The emulsion of any of claims 42-46, wherein said emulsions have a fluorophilic intermediate shell comprising said fluorophilic blocks of said semi-fluorinated block copolymers.
48. The emulsion of claim 47, wherein said therapeutic compound is noncovalently associated with said hydrophobic core.
49. The emulsion of any of claims 43-48, wherein said droplets comprise self-assembled supramolecular structures within the aqueous solution.
50. The emulsion of any of claims 1-50, wherein said emulsion is for administration to a subject in need thereof via intravenous injection.
51. The emulsion of any of claims 1-50, wherein said emulsion provides enhanced stability as compared to an emulsion in the absence of the semi-fluorinated block copolymer.
52. The emulsion of any of claims 1-50 wherein the emulsion provides an extension of the biological half-life of the therapeutic agent as compared to an emulsion in the absence of the semi-fluorinated block copolymer.
53. The emulsion of claim 52, wherein the enhanced stability is due to reduced Ostwald ripening.
54. A method of delivering an imaging agent and a therapeutic agent to a subject in need thereof, said method comprising the steps of: (a) providing an emulsion, said emulsion comprising an oil in water emulsion comprising: a hydrophobic liquid comprising a therapeutic agent; an aqueous solution; semi-fluorinated block copolymers; wherein each of said semi-fluorinated block copolymers independently comprises a hydrophilic block, a hydrophobic block and a fluorophilic block; wherein said fluorophilic block of each of said semi-fluorinated block copolymers is provided between said hydrophobic block and said hydrophilic block; and an imaging agent comprising a fluorous compound, and (b) administering said emulsion to said subject.
55. The method of claim 54, wherein said therapeutic agent is released from said emulsion after delivery to the subject.
56. A method of making a theranostic emulsion, said method comprising the steps of: (a) providing (i) a hydrophobic liquid, (ii) am aqueous solution, (iii) semi-fluorinated block copolymers; wherein each of said semi-fluorinated block copolymers independently comprises a hydrophilic block, a hydrophobic block and a fluorophilic block; wherein said fluorophilic block of each of said semi-fluorinated block copolymers is provided between said hydrophobic block and said hydrophilic block; (iv) a therapeutic agent; (v) an imaging agent comprising a fluorous compound; and (b) emulsifying said theranostic formulation to create the emulsion.
57. The method of any of claims 54-56, wherein said emulsion provides an extension of the biological half-life of the therapeutic agent as compared to an emulsion in the absence of the semi-fluorinated block copolymer and wherein said emulsion provides enhanced stability as compared to an emulsion in the absence of the semi-fluorinated block copolymer.
58. The method any of claims 54-57, wherein each of said semi-fluorinated block copolymers independently has the formula (FX4A) or (FX4B): ##STR00010##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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STATEMENTS REGARDING CHEMICAL COMPOUNDS AND NOMENCLATURE
[0086] In an embodiment, a composition or compound of the invention is isolated or purified. In an embodiment, an isolated or purified compound is at least partially isolated or purified as would be understood in the art. In an embodiment, the composition or compound of the invention has a chemical purity of 95%, optionally for some applications 99%, optionally for some applications 99.9%, optionally for some applications 99.99%, and optionally for some applications 99.999% pure.
[0087] Many of the molecules disclosed herein contain one or more ionizable groups. Ionizable groups include groups from which a proton can be removed (e.g., COOH) or added (e.g., amines) and groups which can be quaternized (e.g., amines). All possible ionic forms of such molecules and salts thereof are intended to be included individually in the disclosure herein. With regard to salts of the compounds herein, one of ordinary skill in the art can select from among a wide variety of available counterions that are appropriate for preparation of salts of this invention for a given application. In specific applications, the selection of a given anion or cation for preparation of a salt can result in increased or decreased solubility of that salt.
[0088] As used throughout the present description, the expression a group corresponding to an indicated species expressly includes a radical (including a monovalent, divalent and trivalent radical) derived from that species.
[0089] The compounds of this invention and used with the methods or emulsions of the invention can contain one or more chiral centers. Accordingly, this invention is intended to include racemic mixtures, diasteromers, enantiomers, tautomers and mixtures enriched in one or more stereoisomer. The scope of the invention as described and claimed encompasses the racemic forms of the compounds as well as the individual enantiomers and non-racemic mixtures thereof.
[0090] As used herein, the term group may refer to a functional group of a chemical compound. Groups of the present compounds refer to an atom or a collection of atoms that are a part of the compound. Groups of the present invention may be attached to other atoms of the compound via one or more covalent bonds. Groups may also be characterized with respect to their valence state. The present invention includes groups characterized as monovalent, divalent, trivalent, etc. valence states.
[0091] As used herein, the term substituted refers to a compound wherein a hydrogen is replaced by another functional group.
[0092] As is customary and well known in the art, hydrogen atoms in formulas (FX1)-(FX4) are not always explicitly shown, for example, hydrogen atoms bonded to the carbon atoms of aromatic, heteroaromatic, and alicyclic rings are not always explicitly shown in formulas (FX1)-(FX4). The structures provided herein, for example in the context of the description of formulas (FX1)-(FX4), are intended to convey to one of reasonable skill in the art the chemical composition of compounds of the methods and compositions of the invention, and as will be understood by one of skill in the art, the structures provided do not indicate the specific positions of atoms and bond angles between atoms of these compounds.
[0093] As used herein, the terms alkylene and alkylene group are used synonymously and refer to a divalent group derived from an alkyl group as defined herein. The invention includes compounds having one or more alkylene groups. Alkylene groups in some compounds function as attaching and/or spacer groups. Compounds of the invention may have substituted and/or unsubstituted C.sub.1-C.sub.20 alkylene, C.sub.1-C.sub.10 alkylene and C.sub.1-C.sub.5 alkylene groups.
[0094] As used herein, the terms cycloalkylene and cycloalkylene group are used synonymously and refer to a divalent group derived from a cycloalkyl group as defined herein. The invention includes compounds having one or more cycloalkylene groups. Cycloalkyl groups in some compounds function as attaching and/or spacer groups. Compounds of the invention may have substituted and/or unsubstituted C.sub.3-C.sub.20 cycloalkylene, C.sub.3-C.sub.10 cycloalkylene and C.sub.3-C.sub.5 cycloalkylene groups.
[0095] As used herein, the terms arylene and arylene group are used synonymously and refer to a divalent group derived from an aryl group as defined herein. The invention includes compounds having one or more arylene groups. In some embodiments, an arylene is a divalent group derived from an aryl group by removal of hydrogen atoms from two intra-ring carbon atoms of an aromatic ring of the aryl group. Arylene groups in some compounds function as attaching and/or spacer groups. Arylene groups in some compounds function as chromophore, fluorophore, aromatic antenna, dye and/or imaging groups. Compounds of the invention include substituted and/or unsubstituted C.sub.3-C.sub.30 arylene, C.sub.3-C.sub.20 arylene, C.sub.3-C.sub.10 arylene and C.sub.1-C.sub.5 arylene groups.
[0096] As used herein, the terms heteroarylene and heteroarylene group are used synonymously and refer to a divalent group derived from a heteroaryl group as defined herein. The invention includes compounds having one or more heteroarylene groups. In some embodiments, a heteroarylene is a divalent group derived from a heteroaryl group by removal of hydrogen atoms from two intra-ring carbon atoms or intra-ring nitrogen atoms of a heteroaromatic or aromatic ring of the heteroaryl group. Heteroarylene groups in some compounds function as attaching and/or spacer groups. Heteroarylene groups in some compounds function as chromophore, aromatic antenna, fluorophore, dye and/or imaging groups. Compounds of the invention include substituted and/or unsubstituted C.sub.3-C.sub.30 heteroarylene, C.sub.3-C.sub.20 heteroarylene, C.sub.1-C.sub.10 heteroarylene and C.sub.3-C.sub.5 heteroarylene groups.
[0097] As used herein, the terms alkenylene and alkenylene group are used synonymously and refer to a divalent group derived from an alkenyl group as defined herein. The invention includes compounds having one or more alkenylene groups. Alkenylene groups in some compounds function as attaching and/or spacer groups. Compounds of the invention include substituted and/or unsubstituted C.sub.2-C.sub.20 alkenylene, C.sub.2-C.sub.10 alkenylene and C.sub.2-C.sub.5 alkenylene groups.
[0098] As used herein, the terms cylcoalkenylene and cylcoalkenylene group are used synonymously and refer to a divalent group derived from a cylcoalkenyl group as defined herein. The invention includes compounds having one or more cylcoalkenylene groups. Cycloalkenylene groups in some compounds function as attaching and/or spacer groups. Compounds of the invention include substituted and/or unsubstituted C.sub.3-C.sub.20 cylcoalkenylene, C.sub.3-C.sub.10 cylcoalkenylene and C.sub.3-C.sub.5 cylcoalkenylene groups.
[0099] As used herein, the terms alkynylene and alkynylene group are used synonymously and refer to a divalent group derived from an alkynyl group as defined herein. The invention includes compounds having one or more alkynylene groups. Alkynylene groups in some compounds function as attaching and/or spacer groups. Compounds of the invention include substituted and/or unsubstituted C.sub.2-C.sub.20 alkynylene, C.sub.2-C.sub.10 alkynylene and C.sub.2-C.sub.5 alkynylene groups.
[0100] As used herein, the term halo refers to a halogen group such as a fluoro (F), chloro (Cl), bromo (Br), iodo (I) or astato (At).
[0101] The term heterocyclic refers to ring structures containing at least one other kind of atom, in addition to carbon, in the ring. Examples of such heteroatoms include nitrogen, oxygen and sulfur. Heterocyclic rings include heterocyclic alicyclic rings and heterocyclic aromatic rings. Examples of heterocyclic rings include, but are not limited to, pyrrolidinyl, piperidyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, benzoxadiazolyl, benzothiadiazolyl, triazolyl and tetrazolyl groups. Atoms of heterocyclic rings can be bonded to a wide range of other atoms and functional groups, for example, provided as substituents.
[0102] The term carbocyclic refers to ring structures containing only carbon atoms in the ring. Carbon atoms of carbocyclic rings can be bonded to a wide range of other atoms and functional groups, for example, provided as substituents.
[0103] The term alicyclic ring refers to a ring, or plurality of fused rings, that is not an aromatic ring. Alicyclic rings include both carbocyclic and heterocyclic rings.
[0104] The term aromatic ring refers to a ring, or a plurality of fused rings, that includes at least one aromatic ring group. The term aromatic ring includes aromatic rings comprising carbon, hydrogen and heteroatoms. Aromatic ring includes carbocyclic and heterocyclic aromatic rings. Aromatic rings are components of aryl groups.
[0105] The term fused ring or fused ring structure refers to a plurality of alicyclic and/or aromatic rings provided in a fused ring configuration, such as fused rings that share at least two intra ring carbon atoms and/or heteroatoms.
[0106] As used herein, the term alkoxyalkyl refers to a substituent of the formula alkyl-O-alkyl.
[0107] As used herein, the term polyhydroxyalkyl refers to a substituent having from 2 to 12 carbon atoms and from 2 to 5 hydroxyl groups, such as the 2,3-dihydroxypropyl, 2,3,4-trihydroxybutyl or 2,3,4,5-tetrahydroxypentyl residue.
[0108] As used herein, the term polyalkoxyalkyl refers to a substituent of the formula alkyl-(alkoxy).sub.n-alkoxy wherein n is an integer from 1 to 10, preferably 1 to 4, and more preferably for some embodiments 1 to 3.
[0109] Amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, asparagine, glutamine, glycine, serine, threonine, serine, rhreonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid. As used herein, reference to a side chain residue of a natural -amino acid specifically includes the side chains of the above-referenced amino acids.
[0110] Alkyl groups include straight-chain, branched and cyclic alkyl groups. Alkyl groups include those having from 1 to 30 carbon atoms. Alkyl groups include small alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length alkyl groups having from 4-10 carbon atoms. Alkyl groups include long alkyl groups having more than 10 carbon atoms, particularly those having 10-30 carbon atoms. The term cycloalkyl specifically refers to an alky group having a ring structure such as ring structure comprising 3-30 carbon atoms, optionally 3-20 carbon atoms and optionally 2-10 carbon atoms, including an alkyl group having one or more rings. Cycloalkyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring(s) and particularly those having a 3-, 4-, 5-, 6-, or 7-member ring(s). The carbon rings in cycloalkyl groups can also carry alkyl groups. Cycloalkyl groups can include bicyclic and tricycloalkyl groups. Alkyl groups are optionally substituted. Substituted alkyl groups include among others those which are substituted with aryl groups, which in turn can be optionally substituted. Specific alkyl groups include methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all of which are optionally substituted. Substituted alkyl groups include fully halogenated or semihalogenated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms. Substituted alkyl groups include fully fluorinated or semifluorinated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms. An alkoxy group is an alkyl group that has been modified by linkage to oxygen and can be represented by the formula RO and can also be referred to as an alkyl ether group. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy and heptoxy. Alkoxy groups include substituted alkoxy groups wherein the alky portion of the groups is substituted as provided herein in connection with the description of alkyl groups. As used herein MeO refers to CH.sub.3O.
[0111] Alkenyl groups include straight-chain, branched and cyclic alkenyl groups. Alkenyl groups include those having 1, 2 or more double bonds and those in which two or more of the double bonds are conjugated double bonds. Alkenyl groups include those having from 2 to 20 carbon atoms. Alkenyl groups include small alkenyl groups having 2 to 3 carbon atoms. Alkenyl groups include medium length alkenyl groups having from 4-10 carbon atoms. Alkenyl groups include long alkenyl groups having more than 10 carbon atoms, particularly those having 10-20 carbon atoms. Cycloalkenyl groups include those in which a double bond is in the ring or in an alkenyl group attached to a ring. The term cycloalkenyl specifically refers to an alkenyl group having a ring structure, including an alkenyl group having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring(s) and particularly those having a 3-, 4-, 5-, 6- or 7-member ring(s). The carbon rings in cycloalkenylgroups can also carry alkyl groups. Cycloalkenylgroups can include bicyclic and tricyclic alkenyl groups. Alkenyl groups are optionally substituted. Substituted alkenyl groups include among others those which are substituted with alkyl or aryl groups, which groups in turn can be optionally substituted. Specific alkenyl groups include ethenyl, prop-1-enyl, prop-2-enyl, cycloprop-1-enyl, but-1-enyl, but-2-enyl, cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl, pent-2-enyl, branched pentenyl, cyclopent-1-enyl, hex-1-enyl, branched hexenyl, cyclohexenyl, all of which are optionally substituted. Substituted alkenyl groups include fully halogenated or semihalogenated alkenyl groups, such as alkenyl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms. Substituted alkenyl groups include fully fluorinated or semifluorinated alkenyl groups, such as alkenyl groups having one or more hydrogen atoms replaced with one or more fluorine atoms.
[0112] Aryl groups include groups having one or more 5-, 6- or 7- member aromatic rings, including heterocyclic aromatic rings. The term heteroaryl specifically refers to aryl groups having at least one 5-, 6- or 7- member heterocyclic aromatic rings. Aryl groups can contain one or more fused aromatic rings, including one or more fused heteroaromatic rings, and/or a combination of one or more aromatic rings and one or more nonaromatic rings that may be fused or linked via covalent bonds. Heterocyclic aromatic rings can include one or more N, O, or S atoms in the ring. Heterocyclic aromatic rings can include those with one, two or three N atoms, those with one or two O atoms, and those with one or two S atoms, or combinations of one or two or three N, O or S atoms. Aryl groups are optionally substituted. Substituted aryl groups include among others those which are substituted with alkyl or alkenyl groups, which groups in turn can be optionally substituted. Specific aryl groups include phenyl, biphenyl groups, pyrrolidinyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, benzoxadiazolyl, benzothiadiazolyl, and naphthyl groups, all of which are optionally substituted. Substituted aryl groups include fully halogenated or semihalogenated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms. Substituted aryl groups include fully fluorinated or semifluorinated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more fluorine atoms. Aryl groups include, but are not limited to, aromatic group-containing or heterocylic aromatic group-containing groups corresponding to any one of the following: benzene, naphthalene, naphthoquinone, diphenylmethane, fluorene, anthracene, anthraquinone, phenanthrene, tetracene, tetracenedione, pyridine, quinoline, isoquinoline, indoles, isoindole, pyrrole, imidazole, oxazole, thiazole, pyrazole, pyrazine, pyrimidine, purine, benzimidazole, furans, benzofuran, dibenzofuran, carbazole, acridine, acridone, phenanthridine, thiophene, benzothiophene, dibenzothiophene, xanthene, xanthone, flavone, coumarin, azulene or anthracycline. As used herein, a group corresponding to the groups listed above expressly includes an aromatic or heterocyclic aromatic group, including monovalent, divalent and polyvalent groups, of the aromatic and heterocyclic aromatic groups listed herein are provided in a covalently bonded configuration in the compounds of the invention at any suitable point of attachment. In embodiments, aryl groups contain between 5 and 30 carbon atoms. In embodiments, aryl groups contain one aromatic or heteroaromatic six-membered ring and one or more additional five- or six-membered aromatic or heteroaromatic ring. In embodiments, aryl groups contain between five and eighteen carbon atoms in the rings. Aryl groups optionally have one or more aromatic rings or heterocyclic aromatic rings having one or more electron donating groups, electron withdrawing groups and/or targeting ligands provided as substituents.
[0113] Arylalkyl groups are alkyl groups substituted with one or more aryl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. Specific alkylaryl groups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups. Alkylaryl groups are alternatively described as aryl groups substituted with one or more alkyl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. Specific alkylaryl groups are alkyl-substituted phenyl groups such as methylphenyl. Substituted arylalkyl groups include fully halogenated or semihalogenated arylalkyl groups, such as arylalkyl groups having one or more alkyl and/or aryl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.
[0114] As to any of the groups described herein which contain one or more substituents, it is understood that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds. Optional substitution of alkyl groups includes substitution with one or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or aryl groups are optionally substituted. Optional substitution of alkenyl groups includes substitution with one or more alkyl groups, aryl groups, or both, wherein the alkyl groups or aryl groups are optionally substituted. Optional substitution of aryl groups includes substitution of the aryl ring with one or more alkyl groups, alkenyl groups, or both, wherein the alkyl groups or alkenyl groups are optionally substituted.
[0115] Optional substituents for any alkyl, alkenyl and aryl group includes substitution with one or more of the following substituents, among others: halogen, including fluorine, chlorine, bromine or iodine; pseudohalides, including CN;
COOR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted;
COR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted;
CON(R).sub.2 where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted; and where R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;
OCON(R).sub.2 where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted; and where R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;
N(R).sub.2 where each R, independently of each other R, is a hydrogen, or an alkyl group, or an acyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, phenyl or acetyl group, all of which are optionally substituted; and where R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;
SR, where R is hydrogen or an alkyl group or an aryl group and more specifically where R is hydrogen, methyl, ethyl, propyl, butyl, or a phenyl group, which are optionally substituted;
SO.sub.2R, or SOR where R is an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group, all of which are optionally substituted;
OCOOR where R is an alkyl group or an aryl group;
SO.sub.2N(R).sub.2 where each R, independently of each other R, is a hydrogen, or an alkyl group, or an aryl group all of which are optionally substituted and wherein R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;
OR where R is H, an alkyl group, an aryl group, or an acyl group all of which are optionally substituted. In a particular example R can be an acyl yielding
OCOR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; and
NO.SUB.2..
[0116] Specific substituted alkyl groups include haloalkyl groups, particularly trihalomethyl groups and specifically trifluoromethyl groups. Specific substituted aryl groups include mono-, di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalene groups; 3- or 4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenyl groups, 3- or 4-alkoxy-substituted phenyl groups, 3- or 4-RCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups. More specifically, substituted aryl groups include acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups, particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups, particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups, particularly 4-methylphenyl groups; and methoxyphenyl groups, particularly 4-methoxyphenyl groups.
[0117] As to any of the above groups which contain one or more substituents, it is understood that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.
[0118] Pharmaceutically acceptable salts comprise pharmaceutically-acceptable anions and/or cations. As used herein, the term pharmaceutically acceptable salt can refer to acid addition salts or base addition salts of the compounds in the present disclosure. A pharmaceutically acceptable salt is any salt which retains at least a portion of the activity of the parent compound and does not impart significant deleterious or undesirable effect on a subject to whom it is administered and in the context in which it is administered. Pharmaceutically acceptable salts include metal complexes and salts of both inorganic and organic acids. Pharmaceutically acceptable salts include metal salts such as aluminum, calcium, iron, magnesium, manganese and complex salts. Pharmaceutically acceptable salts include, but are not limited to, acid salts such as acetic, aspartic, alkylsulfonic, arylsulfonic, axetil, benzenesulfonic, benzoic, bicarbonic, bisulfuric, bitartaric, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic, 32-cilexetil, citric, edetic, edisylic, estolic, esyl, esylic, formic, fumaric, gluceptic, gluconic, glutamic, glycolic, glycolylarsanilic, hexamic, hexylresorcjnoic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, maleic, malic, malonic, mandelic, methanesulfonic, methylnitric, methylsulf uric, mucic, muconic, napsylic, nitric, oxalic, p-nitromethanesulfonic, pamoic, pantothenic, phosphoric, monohydrogen phosphoric, dihydrogen phosphoric, phthalic, polygalactouronic, propionic, salicylic, stearic, succinic, sulfamic, sulfanlic, sulfonic, sulfuric, tannic, tartaric, teoclic, toluenesulfonic, and the like. Pharmaceutically acceptable salts may be derived from amino acids, including but not limited to cysteine. Other pharmaceutically acceptable salts may be found, for example, in Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002. (ISBN 3-906390-26-8). Pharmaceutically-acceptable cations include among others, alkali metal cations (e.g., Li.sup.+, Na.sup.+, K.sup.+), alkaline earth metal cations (e.g., Ca.sup.2+, Mg.sup.2+), non-toxic heavy metal cations and ammonium (NH.sub.4.sup.+) and substituted ammonium (N(R).sub.4.sup.+, where R is hydrogen, alkyl, or substituted alkyl, i.e., including, methyl, ethyl, or hydroxyethyl, specifically, trimethyl ammonium, triethyl ammonium, and triethanol ammonium cations). Pharmaceutically-acceptable anions include among other halides (e.g., Cl.sup., Br.sup.), sulfate, acetates (e.g., acetate, trifluoroacetate), ascorbates, aspartates, benzoates, citrates, and lactate.
[0119] The compounds of this invention can contain one or more chiral centers. Accordingly, this invention is intended to include racemic mixtures, diasteromers, enantiomers, tautomers and mixtures enriched in one or more stereoisomer. The scope of the invention as described and claimed encompasses the racemic forms of the compounds as well as the individual enantiomers and non-racemic mixtures thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0120] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the invention.
[0121] Supramolecular structure refers to structures comprising an assembly of molecules. Supramolecular structures include assemblies of molecules, such as linear block copolymers having hydrophilic, fluorophilic, and hydrophobic blocks, which are selectively oriented such that hydrophilic portions of the molecules are oriented outward toward a continuous aqueous phase, hydrophobic portions form an inner core and fluorophilic portions of the molecules are oriented in between to form a fluorous shell. Supramolecular structures include, but are not limited to, droplets, emulsions, encapsulated droplets. Supramolecular structures of the present invention include self-assembled structures. Supramolecular structures may comprise the dispersed phase of a colloid, such as an emulsion or nanoemulsion.
[0122] Semi-fluorinated refers to chemical compounds having at least one fluorine atom, for example molecules having at least one carbon-fluorine bond.
[0123] Fluorocarbons as used herein refer to chemical compounds that contain at least one carbon-fluorine bond.
[0124] Perfluorinated and perfluorocarbon refers to chemical compounds that are analogs of hydrocarbons wherein all hydrogen atoms in the hydrocarbon are replaced with fluorine atoms. Perfluorinated molecules can also contain a number of other atoms, including bromine, chlorine, and oxygen. A bromine substituted perfluorocarbon is a perfluorocarbon wherein one or more of the fluorine atoms have been replaced with a bromine atom. A chlorine substituted perfluorocarbon is a perfluorocarbon wherein one or more of the fluorine atoms have been replaced with a chlorine atom. A chlorine and bromine substituted perfluorocarbon is a perfluorocarbon wherein one or more of the fluorine atoms have been replaced with a chlorine atom and wherein one or more of the fluorine atoms have been replaced with a bromine atom.
[0125] Perhalogenated fluorous compound refers to fluorophilic chemical compounds that are analogs of a substituted or unsubstituted hydrocarbon wherein the hydrogen atoms are replaced with halogen atoms, such as fluorine, chlorine and bromine. Perhalogenated fluorous compounds can also contain a number of other atoms, including oxygen, sulfur and nitrogen. Perhalogenated fluorous compounds include perfluorocarbons and substituted perfluorocarbons, such as chlorine substituted perfluorocarbons, bromine substituted perfluorocarbons and chlorine and bromine substituted perfluorocarbons.
[0126] Emulsion refers to a mixture of two or more immiscible substances, such as a mixture of two immiscible liquids. Emulsions are a type of colloid that comprise at least one dispersed phase dispersed in a continuous phase. Emulsions are broadly defined as two immiscible phases in which a first phase is dispersed within a second phase, such as a two-phase system in which one liquid is dispersed throughout a second liquid in the form of small droplets. The two phases of an emulsion are generally referred to as the continuous phase and the dispersed phase, with the dispersed phase typically present as a smaller volume percentage. A dispersion of oil in water is referred to as an oil-in-water (o/w) emulsion. For o/w emulsions the emulsifying agent is typically more soluble in the aqueous phase. The reverse emulsion, water-in-oil, is abbreviated w/o and is stabilized by surfactants that are more stable in the oil phase. In an aqueous emulsion, the continuous phase is an aqueous solution.
[0127] Emulsions are not thermodynamically stable, but the stability can be improved by additives such as surfactants. As non-equilibrium systems, the formation of nanoemulsions generally requires an input of energy. High-energy emulsification methods commonly involve the introduction of mechanical shear through such equipment as high-shear stirrers, high-pressure homogenizers, microfluidizers or ultrasound generators. A microfluidizer is the piece of equipment used in the pharmaceutical industry for the production of emulsions that works by dividing a stream of liquid into two parts, passing each through a narrow opening and then colliding the streams under high pressure. The high shear forces created by the collision provide very fine emulsions with generally narrow particle size distributions. In typical usage, a coarse emulsion (diameter >1 m) is first formed by some other method, and the size of that larger emulsion is reduced in the microfluidizer. The final droplet size and distribution shape will be dependent upon both the emulsion components (surfactant amount, oil volume percent, etc.) and the processing parameters (time, temperature, pressure etc.). As the desired droplet size decreases, the energy required for formation increases. Ultrasonic emulsification is also effective to reduce the size of emulsion droplets into the nanoscale. Emulsions can also be formed by changing the temperature of a mixture of immiscible liquids, for example by rapid cooling or heating to produce kinetically stable emulsions with small droplet sizes and narrow size distributions.
[0128] Emulsions include nanoemulsions comprising nanoscale droplets of one immiscible liquid dispersed within another. As used herein a nanoemulsion is a heterogeneous system composed of one immiscible liquid dispersed as droplets within another liquid, where the average droplet diameter is below 1000 nm.
[0129] Flocculation refers to a process in which clusters of two or more droplets behave kinetically as a unit, but individual droplets still maintain their identity. Flocculation may be reversible, or lead to coalescence, which is irreversible.
[0130] Coalescence is the collision, and subsequent irreversible fusion, of two droplets. The ultimate end of coalescence is complete phase separation. Flocculation precedes coalescence, so the same methods that are appropriate for prevention of flocculation also prevent coalescence. A thick, surfactant film adsorbed at the interface is often sufficient to prevent coalescence, whether in nano- or macro-emulsions.
[0131] Ostwald ripening refers to the growth in the size of emulsion droplets as the contents of one drop diffuse into another. The driving force for this growth is the difference in chemical potential between droplets, which is generally not substantial for droplets larger than 1 m. Therefore, Ostwald ripening primarily affects nanoemulsions, and is an important factor for nanoemulsions for therapeutic applications.
[0132] Polymer refers to a molecule comprising a plurality of repeating chemical groups, typically referred to as monomers. A copolymer, also commonly referred to as a heteropolymer, is a polymer formed when two or more different types of monomers are linked in the same polymer. Block copolymers are a type of copolymer comprising blocks or spatially segregated domains, wherein different domains comprise different polymerized monomers. In a block copolymer, adjacent blocks are constitutionally different, i.e. adjacent blocks comprise constitutional units derived from different species of monomer or from the same species of monomer but with a different composition or sequence distribution of constitutional units. Different blocks (or domains) of a block copolymer may reside on different ends of a polymer (e.g. [A][B]), or may be provided in a selected sequence ([A][B][A][B]). Diblock copolymer refers to block copolymers having two different chemical blocks. Triblock copolymer refers to block copolymers having three different chemical blocks. Polymers of the present invention include block copolymers having a first block comprising a larger polymer (e.g., 10-300) such as a PEG polymer having 10 to 270 monomers smaller polymer (e.g., 2 to 30 monomers), an intermediate block such as a fluorocarbon, including but not limited to, a fluorocarbon such as a fluorinated or perfluorinated alkane, and a third interior hydrophobic block. Block copolymers of the present invention are capable of undergoing self-assembly to make supramolecular structures, such as encapsulated droplets. As used herein, the term block copolymer includes compositions comprising a first block comprising a PEG polymer conjugated to a second block comprising perfluorinated or semifluorinated molecular domain, such as a perfluorinated or semifluorinated alkane or a perfluorinated or semifluorinated tail and further conjugated to a third block comprising a hydrophobic polymer. As used herein, the term block copolymer also includes functionalized block copolymers, such as copolymers having additional moieties for targeting a supramolecular structure to an active site, for stabilizing a supramolecular structure or for selecting the release kinetics of a supramolecular structure containing a fluorinated therapeutic compound.
[0133] As used herein hydrophilic refers to molecules and/or components (e.g., functional groups, blocks of block polymers, etc.) of molecules having at least one hydrophilic group, and hydrophobic refers to molecules and/or components (e.g., functional groups of polymers, and blocks of block copolymers etc.) of molecules having at least one hydrophobic group. Hydrophilic molecules or components thereof tend to have ionic and/or polar groups, and hydrophobic molecules or components thereof tend to have nonionic and/or nonpolar groups. Hydrophilic molecules or components thereof tend to participate in stabilizing interactions with an aqueous solution, including hydrogen bonding and dipole-dipole interactions. Hydrophobic molecules or components tend not to participate in stabilizing interactions with an aqueous solution and, thus often cluster together in an aqueous solution to achieve a more stable thermodynamic state. In the context of block copolymers of the present invention, a hydrophilic block is more hydrophilic than a hydrophobic group of an amphiphilic block copolymer, and a hydrophobic group is more hydrophobic than a hydrophilic block of an amphiphilic polymer.
[0134] As used herein fluorophilic refers to molecules and/or components (e.g., functional groups, blocks of block polymers etc.) of molecules having at least one fluorophilic group. A fluorophilic group is one that is capable of participating in stabilizing interactions with a fluorous phase. Fluorophilic groups useful in block copolymers compounds of the invention include, but are not limited to, fluorocarbon groups, perfluorinated groups and semifluorinated groups.
[0135] As used herein hydrophobic refers to molecules and/or components (e.g., functional groups, blocks of block polymers etc.) of molecules having at least one hydrophobic group. A hydrophobic group can be understood as a group that is repelled from water. Hydrophobic groups tend to be nonpolar and do not form hydrogen bonds. While hydrophobic materials are usually lipophilic, silicones and fluorocarbons are not lipophilic. In embodiments, the hydrophobic materials of the invention are lipophilic, which generally refers to the material's ability to dissolve in, or dissolve, fats, oils, lipids and hydrophobic/lipophilic compounds. Hydrophobic blocks are known in the art, and include such materials as one or more linear or branched C.sub.5-C.sub.20 alkyl block, a poly (-caprolactone) block, a poly(lactic acid) block; a poly(propylene glycol) block; a poly(amino acid) block; a poly(ester) block and poly(lactic-co-glycolic acid) block, polydimethylsiloxane (PDMS) block, poly(caprolactone (PCL) block, poly(methyl methacrylate) (PMMA), and the like.
[0136] In the context of the present invention the term patient is intended to include a mammalian subject, such as a human, as well as a subject such as an animal, such as a companion or food animal.
[0137] Before the present methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, and reagents described, as these may vary. It is also to be understood that 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 will be limited only by the appended claims.
[0138] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
[0139]
[0140] The invention may be further understood by the following non-limiting examples.
Example 1
Emulsion-Based Formulations
[0141] This example provides a description of compositions and physical properties of specific examples of emulsions useful in the present formulations and therapeutic methods, which taken together demonstrate useful properties and applications of certain embodiments of the present invention.
[0142] An example of a hydrophobic drug model is the chemotherapeutic, paclitaxel. This antimitotic drug was selected for its high potency against many cancer types including breast, ovarian, and lung cancer as well as Kaposi's carcinoma. Paclitaxel functions by the promotion of tubulin polymerization and equimolar binding to tubulin dimers, resulting in mitotic arrest. Unfortunately, this molecule exhibits very poor water solubility and pharmacokinetics in vivo. Due to these challenging characteristics, paclitaxel was first administered intravenously as Taxol in combination with Cremophor EL, a nonionic surfactant with polyoxyethylated castor oil, and ethanol. Sadly, Taxol leads to widespread hypersensitivity reactions, plasticizer leaching, and even neuropathy due to the solvent mixture. An additional paclitaxel formulation, Abraxane, was approved in 2005 involving albumin-bound paclitaxel nanoparticles. Despite improvements in side effect toxicity, this formulation still lacks high potency due to the discrepancy between injected drug and drug that actually reaches the tumor. The need for an improved delivery method for this highly cytotoxic drug is still present.
[0143] Materials and Methods
[0144] 1H,1H,10H,10H-perfluorodecane-1,10-diol was purchased from SynQuest Laboratories Inc. (Alachua, Fla.) and perfluoro-15-crown-5-ether was purchased from Oakwood Chemical (Estill, S.C.). Normal saline (AirLife sterile 0.9% sodium chloride for irrigation USP) was purchased from the University of Wisconsin Hospital Pharmacy and paclitaxel from LC Laboratories (Woburn, Mass.). Solvents and all other reagents were purchased form Sigma Aldrich Co. (Milwaukee, Wis.) and Spectrum (Gardena, Calif.) and used as received, unless otherwise mentioned. Small molecular and polymer chromatography was accomplished with Silicycle 60 SiO.sub.2 or using a Teledyne CombiFlash Rf 4x (Lincoln, NE) equipped with an evaporative light scattering detector, or ELSD, for visualization and REDI-sep Rf high performance silica or C18 columns.
[0145] .sup.1H- and .sup.19F-NMR spectra were obtained on a Varian Unity-Inova 400 or Varian Unity-Inova 500 spectrometer using CDC1.sub.3 as the solvent (unless otherwise specified) and tetramethyl silane (TMS) as the internal reference. Polymer purity was confirmed by HPLC with a Gilson 321 Pump (Middleton, Wis.) equipped with a Jordi Gel DVB 500 (Bellingham, MA) column and a Gilson Prep-ELS detector and by MALDI-MS on a Bruker Ultraflex III MALDI TOF/TOF using -cyano-4-hydroxycinnamic acid (CHCA) matrix unless otherwise specified.
[0146] Critical Micelle Concentration (CMC)Surface Tensiometry
[0147] Polymer was dissolved in Millipore Milli-Q water in a 20 mL disposable scintillation vial to the desired maximum concentrations (3 and 3.5 logM). The solutions were shaken and sonicated for 3 hours. Solutions were then allowed to equilibrate for 24 h. Serial dilutions were then prepared from these two stock solutions to achieve the desired concentrations. Each serial dilution was also sonicated 3 hours and allowed to equilibrate prior to the next dilution. Once all solutions were prepared, they were allowed to equilibrate for an additional 24 hours. Surface tensions were measured on a KSV sigma 701 tensiometer (KSV Instruments, Helsinki, Finland) equipped with a Julabo F12-MC circulator for constant temperature control. A custom round platinum rod, with a diameter of 1.034 mm and a wetted length of 3.248 mm from KSV Instruments (Helsinki, Finland), was initially cleaned with ethanol and dried in a Bunsen burner flame. Before running the experimental samples, the surface tension of Millipore Milli-Q water was measured as control to confirm vial and rod were fully cleaned and surface tension was within 2 mN m-1 of the literature value, 78.2 mN m-1. The surface tension of each sample was then measured using the Wilhelmy method, beginning with the least concentrated solution and proceeding to successively more concentrated solutions. The surface tension at each concentration was measured in quadruplicate and the average recorded. The CMC value was determined from the intersection of the slope at the crossover point of two lines: the baseline of minimal surface tension and the slope where surface tension showed linear decline. Error was determined by weighted least squares analysis.
[0148] Nanoemulsion Preparation
[0149] Aqueous polymer solutions were prepared freshly (20 mM) in sterile, normal saline and sonicated at room temperature until fully dissolved. Saline was composed of 0.9% (w/w) sodium chloride.
[0150] Paclitaxel/medium chain triglycerides (MCT) solutions were prepared freshly. Two mL of paclitaxel solution (prepared at a concentration of 7.5 mg/mL in 50:50, acetonitrile:ethanol) was dissolved in MCT followed by heating and stirring until fully solubilized. All traces of acetonitrile and ethanol were removed by vacuum.
[0151] Paclitaxel/MCT solution and perfluoro-15-crown-5-ether (PFCE) were added to the polymer solution. The homogenizer and microfluidizer were first cleaned with 100% and 70% ethanol followed by 100% and 70% methanol and finally three rinses with Millipore Milli-Q water to remove all traces of any previous nanoemulsions. The prepared mixture was then homogenized with the high-speed homogenizer (Power Gen 500, Fisher Scientific, Hampton, N.H.) for 1 minute at 21,000 rpm at room temperature. The resulting crude emulsion was then further mixed with the microfluidizer (model 110 S, Microfluidics Corp., Newton, Mass.) for 1 minute under 5,000 psi with the cooling bath kept at 0 C. The final emulsion was then filtered with a 0.45 m nylon filter and stored in a sterile, plastic centrifuge tube (Corning Inc., Corning, N.Y.) at 4 C.
[0152] Particle Size Determination Via Dynamic Light Scattering (DLS)
[0153] Long-term nanoemulsion size and stability was monitored via dynamic light scattering (NICOMP 380ZLS, Particle Sizing Systems, Santa Barbara, Calif.). The nanoemulsions were diluted at the intensity factor of 500 by adding 5 L of the nanoemulsion to 3.0 mL of Millipore Milli-Q water. Each particle size analysis was run for 5 minutes at room temperature in a quartz cuvette and repeated three times. The data was analyzed using Gaussian analysis and reported as volume weighted average diameters.
[0154] Nanoemulsion distribution and histogram data were determined via dynamic light scattering (Zetasizer Nano-ZS, Malvern Instruments, Worchestershire, UK). The nanoemulsions were diluted at the same ratio as above, 5 L nanoemulsion to 3.0 ML of Millipore Milli-Q water. Each particle analysis was run as a set of 10 scans in a semi-micro polystyrene cuvette at room temperature and repeated three times. The data were analyzed using Malvern software analysis and reported as volume weighted average diameters.
[0155] In Vitro Drug Release
[0156] The nanoemulsion was initially diluted by a factor of 20 (0.125 mL nanoemulsion plus 2.375 mL Millipore Milli-Q water). A time zero time point was established by diluting 100 L diluted nanoemulsion mixture above in 900 L acetonitrile (ACN). A 3 mL capacity SLIDE-A-LYZER Dialysis cassette (G2 2,000 MWCO from Thermo Fisher Scientific Inc., Fitchburg, Wis.) was hydrated by stirring for 12 hours in a 3 L PBS bath (300 mL 10x PBS and 2,700 mL Millipore Milli-Q water) at 37 C. After this time, the remaining diluted nanoemulsion solution (2.40 mL) was added to the cassette which was then returned to the PBS bath and allowed to stir for 1 week at 37 C.; this was performed in triplicate. Time points were taken at 0.5, 2, 3, 6, 9, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours. At each time point a long-stemmed glass pipette was used to mix the contents of the cassette three times. Then a 100 L aliquot of nanoemulsion was removed from the cassette and diluted with 900 L ACN. The nanoemulsion aliquot was then replaced in the cassette by 100 L of fresh PBS solution (1PBS). Sink conditions were maintained by replacing the 3 L PBS baths at the 3, 6, 9, and 12 hour time points and every 12 hours following.
[0157] The paclitaxel concentration remaining in the nanoemulsion was quantified by reverse phase HPLC. The HPLC system used was a Shimadzu PROMINENCE HPLC system (Shimadzu, Japan) equipped with an LC-20AT pump, SIL-20 AC HT autosampler, CTO-20 AC column over, and an SPD-M20A diode array detector. For each time point sample, 20 L was injected into a C18 column (Agilent XDB-C18, 4.6 150 mm) and eluted with an isocratic mixture of 25% water and 75% ACN. The run time was 7 minutes, the flow rate was 1.0 mL min-1, and the detection was set at 227 nm. Paclitaxel eluted at 4.15 minutes. Concentration of paclitaxel was determined by integrating the area of the peak and extrapolation from a standard calibration curve (500, 100, 50, 25, 10, 5, 2.5 g mL-1).
[0158] In Vitro Cytotoxicity
[0159] A549, a human non-small cell lung carcinoma cell line, was used to perform three day cytotoxicity studies. A549 cells were cultured in RPMI-1640 medium containing 10% FBS. The cells were plated in 96-well plates, 5,000 cells/well, and incubated at 37 C. for 24 hours. After incubation, the cells were treated with 10 L of each solution: emulsion solutions containing paclitaxel (0.04, 0.4, 4, 400, 4,000, 40,000 nM in relation to paclitaxel concentration), standard paclitaxel solutions (0.04, 0.4, 4, 40, 400, 4,000 nM in relation to paclitaxel concentration), and emulsion solutions with no paclitaxel (17 nM, 1,700 nM, 17 M in relation to polymer concentration) diluted in 90 L of fresh media and allowed to incubate for 24 hours at 37 C. On the third day, all liquid was removed from each well and 100 L of diluted CellTiter-Blue reagent (CellTiter-Blue cell viability assay, Promega) was added to each well. The cells were incubated for another three hours at 37 C. The fluorescence intensity at 560 nm was analyzed using a plate reader. The cell viability in each well was calculated relative to the untreated control wells and each type of well was averaged (n=6).
[0160] In Vivo Developmental Toxicity Study
[0161] Due to the novel nature of the M2F8H18 amphiphile, there is no preceding toxicity data. Though some predictions can be made from previous work in the Mecozzi lab, a thorough study of developmental effects related to M2F8H18 was performed using an embryo-larval zebrafish model. This animal model was selected because zebrafish eggs remain transparent from fertilization until the tissues become dense as a mature adult. Several developmental endpoints can be simultaneously monitored providing valuable developmental toxicity data for new chemicals. Following short-term exposure, survival, and hatching, non-lethal malformations were monitored including curved body axis and pericardia edemas. Mortality rate was also monitored.
[0162] Zebrafish (Danio rerio) of the AB strain were obtained from our collaborator, Dr. Michael Taylor at the University of Madison-Wisconsin School of Pharmacy, where the fish were cultured until sexual maturation for crossing. The fish were maintained in a light/dark cycle of 14:10 hat 28.5 C. in egg water (0.03% Instant Ocean, Blacksburg, Va., USA). Zebrafish were fed with live brine shrimps (Artemia nauplii) twice a day. Embryos were obtained from healthy adult fish with a ratio of 1:2 for female to male. Six breeding groups were placed in separate spawning aquariums, equipped with a mesh bottom to prevent the eggs from being cannibalized. Crossing was induced in the morning when the light was turned on. One hour later, eggs free of macroscopically discernable symptoms of infection and disease were collected, rinsed with egg water and transferred into Petri dishes until chemical exposure.
[0163] The embryo-larvae toxicity assay was carried out according to previous studies in the Taylor and Mecozzi laboratories. Briefly, 8 fertilized eggs of 2 hpf (hours post-fertilization) stages were placed into each well of a 24-well plate and each filled with 600 L egg water. Six concentrations (1 mM, 333 M, 111 M, 37 M, 12.3 M, and 4.1 M) plus two controls were plated. The plate was covered and incubated at 28.5 C. in a light/dark cycle of 14:10 throughout the 96 hpf exposure period. The observations of zebrafish development were made directly in the well using a stereomicroscope (Nikon SMZ18) every 24 hr. Endpoints including mortality, spontaneous movement, hatching success, pericardial edema, and curved body axis, were selected to monitor the effects of M2F8H18. Embryos and larvae were considered dead when no heartbeat was observed. The number of hatched embryos and a cumulative mortality tally was recorded every 24 hr from 2 hpf. The number of larvae displaying pericardial edemas or curved body axes were also recorded every 24 hr from 2 hpf. At 96 hpf, following the final observations, representative larvae were anesthetized with 0.4% tricaine mesylate solution and mounted on petri dishes using low melting point agarose. The larvae were then photographed using a high-definition color microscope camera (Nikon DS-Fi2) and finally, euthanized.
[0164] Magnetic Resonance Imaging (MRI)
[0165] Samples were prepared fully concentrated, without any dilution. The nuclear magnetic resonance (NMR) internal temperature was maintained at 25 C. The .sup.19F relaxation parameters T.sub.1 and T.sub.2, of the perfluoro-15-crown-5-ether present in the nanoemulsion particles, were measured on a Varian Unity-Inova 500 MHz (11.7 T) NMR spectrometer. The T.sub.1 was determined using an inversion recovery experiment acquired with 12 independent, quadratically spaced variable (tau) values covering a range up to 10 times the estimated T.sub.1, 0.75 s. The T.sub.2 was determined using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence experiment acquired with 12 independent, quadratically spaced variable (tau) values covering a range up to 10 times the estimated T.sub.2, 0.32 s. For T.sub.1 and T.sub.2 measurements: 90 pulse=17.1 s, nt=16, spectral width (T.sub.1)=46948.4 Hz and spectral window (T.sub.2)=46948.4 Hz.
[0166] MR images were acquired using an Agilent 4.7 T small animal horizontal bore scanner using a home-built .sup.19F quadrature volume coil with a 1.5 diameter and a 3 length. The temperature was maintained at 25 C. A nanoemulsion stock of M2F8H18 was prepared by direct dilution of the nanoemulsion to the highest phantom concentration in sterile, normal saline (0.9% (w/w) sodium chloride). Lower phantom concentrations were made as serial dilutions from the stock solution in sterile, normal saline (0.9% (w/w) sodium chloride).
[0167] Nanoemulsion formation was confirmed by DLS. Phantom nanoemulsion solutions at PFCE concentrations of 154 mM, 61 mM, 31 mM, 3 mM, and 0 mM were transferred to polystyrene micro-centrifuge tubes and .sup.1H images of the phantoms were acquired using a gradient echo pulse sequence with 0.190.192 mm.sup.3 spatial resolution, 4848 mm.sup.2 field of view (FOV), 8.82 ms TR, 4.43 ms TE, 20 degree flip angle, 195.3 Hz/voxel, 16 averages and 36.2 s imaging time. .sup.19F images of the phantoms were acquired using a fast spin echo pulse sequence with a 0.250.252.0 mm.sup.3 spatial resolution, 4848 mm.sup.2 FOV, 0.500 s TR, 16.08 ms TE, echo train length of 8 echoes, 104.2 Hz/voxel, 50 averages, and 10 min 1 s imaging time. Image background threshold was adjusted to only view above 5% voxels.
[0168] Synthesis
[0169] BnOF8OH 1H, 1H, 10H, 10H-monobenzyl-perfluorodecanol (1). To a dry roundbottom flask was added 1H,1H,10H,10H-perfluorodecane-1,10-diol (8.5 mmol, 4 g) and anhydrous dimethylformamide (40 mL). This solution was allowed to stir under argon for 10 minutes followed by the addition of sodium hydride (8.5 mmol, 0.20 g). This mixture was sonicated under argon, at room temperature for an additional 10 minutes then benzyl bromide (8.5 mmol, 1 mL) was added dropwise. The reaction was allowed to run for 3 hours under argon and sonication. The reaction was then taken up in 5% HCl then extracted with ethyl acetate, saturated aqueous NaHCO.sub.3 solution, and brine. The crude mixture was dried with MgSO.sub.4, concentrated in vacuo, and purified via silica gel column chromatography. A gradient of 0-15% ethyl acetate-hexanes was used. The separation was monitored with thin layer chromatography (TLC) and KMnO.sub.4 staining. Isolated product resulted in 1.77 grams (37.5% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.35 (m, 5H), 4.68 (s, 2H), 4.10 (td, J=15.2, 13.0, 7.6 Hz, 2H), 3.94 (t, J=13.8 Hz, 2H), 1.95 (t, J=7.5 Hz, 1 H). .sup.19F NMR (470 MHz, CDCl.sub.3) 119.36 (t, J=13.7 Hz), 121.92 (m), 122.42 (t, J=13.6 Hz), 123.31 (bs), 123.57 (bs).
[0170] H18)Ms octadecyl methanesulfonate (2). To a dry roundbottom flask was added anhydrous dichloromethane (DCM), 100 mL, 1-octadecanol (21 mmol, 5.6808 g), and triethyl amine (51 mmol, 7.10 mL). This mixture was stirred and gently heated until all solid 1-octadodecanol was dissolved. Then the flask was put in an ice bath under argon. This mixture was allowed to react for 30 minutes before methanesulfonyl chloride (27 mmol, 2.10 mL) was added dropwise. The solution turned slightly cloudy. After running overnight, the solution was diluted with more DCM and extracted with saturated aqueous NH.sub.4Cl solution. The crude mixture was then dried with MgSO.sub.4 and concentrated in vacuo. Isolated product resulted in 7.15 grams (97.7% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 4.22 (t, J=6.6 Hz, 2H), 3.00 (s, 3H), 1.75 (p, J=6.9 Hz, 2H), 1.39 (m, 3H) 1.26 (bs, 31 H), 0.88 (t, J=6.6 Hz, 3H).
[0171] BnOF8OH18 1H, 1H, 10H, 10H-monobenzyl-octadecyl-perfluorodecane (3). To a dry roundbottom flask was added anhydrous benzotrifluoride (50 mL) and 1 (3.4 mmol, 1.90 g). This mixture was flushed with argon before adding sodium hydride (14 mmol, 0.3418 g). The mixture was allowed to stir for one hour before 2 was added (4.4 mmol, 1.5611 g). The mixture was then heated to reflux for 24 hours. After running overnight, the solution was diluted with DCM and extracted with saturated aqueous NH.sub.4Cl solution. The crude mixture was then dried with MgSO.sub.4 and concentrated in vacuo. Purification was performed via silica gel chromatography using a gradient of 0-5% ethyl acetate-hexanes. The separation was monitored with thin layer chromatography and KMnO.sub.4 staining. Isolated product resulted in 2.53 grams (91.3% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.35 (m, 5H), 4.68 (s, 2H), 3.93 (q, J=13.6 Hz, 4H), 3.59 (t, J=6.6 Hz, 2H), 1.60 (p, J=6.7 Hz, 2H), 1.25 (m, 32H), 0.88 (t, J=6.8 Hz, 3H). .sup.19F NMR (470 MHz, CDCl.sub.3) 119.75 (t, J=15.7 Hz), 120.03 (t, J=15.7 Hz), 121.96 (m), 123.40 (d, J=78.8 Hz).
[0172] HOF8OH18 1H, 1H, 10H, 10H-octadecyl-perfluorodecanol (4).
[0173] Route 1) To a dry roundbottom flash was added reagent grade methanol (100 mL), anhydrous tetrahydrofuran (50 mL), and 3 (2.90 mmol, 2.34 g). This mixture was stirred and flushed with argon for one hour then palladium on carbon catalyst (3.9 mmol, 0.4100 g) was added. This mixture was flushed with argon for one more hour then a balloon of hydrogen gas was dispelled into the flask with a bubbler attached. A second balloon of hydrogen gas was then put on the reaction and it was allowed to stir and run for 24 hours under static hydrogen atmosphere. To workup the reaction, the balloon was removed and the flask flushed with argon for one hour again. The mixture was then filtered through celite with copious rising with DCM. Solvent was removed in vacuo. Isolated product resulted in 2.25 grams (96.2% recovered yield). .sup.1H NMR (500 MHz, CDCl.sub.3) 4.10 (td, J=14.0, 7.5 Hz, 2H), 3.92 (t, J=14.0 Hz, 2H), 3.59 (t, J=6.6 Hz, 2H), 1.93 (t, J=7.6 Hz, 1H), 1.60 (p, J=6.5 Hz, 2H), 1.25 (m, 24H), 0.88 (t, J=6.9 Hz, 3H). .sup.19F NMR (470 MHz, CDCl.sub.3) 119.65 (m), 121.96 (m), 122.42 (t, J=12.6 Hz), 123.51 (d, J=51.2 Hz).
[0174] (Route 2) To a dry round bottom flask was added 1H, 1H, 10H, 10H-perfluorodecane-1,10-diol (14.4 mmol, 6.6557 g) and anhydrous dimethylformamide (220 mL). The mixture was stirred and flushed with argon until fully dissolved then sodium hydride (14.5 mmol, 0.3482 g) was added. This was allowed to sonicate for 30 minutes, under argon before 2 (14.3 mmol, 5.0 g) was added. The reaction was allowed to run for 21 hours under argon and sonication. The reaction was then taken up in 5% HCl then extracted with ethyl acetate, saturated aqueous NaHCO.sub.3 solution, and brine. The crude mixture was dried with MgSO.sub.4 and concentrated in vacuo. Purification was performed using automated column chromatography, CombiFlash, using a silica gel column. A gradient of 0-20% ethyl acetate-hexanes was used. The separation was monitored using an evaporative light scattering detector (ELSD). Isolated product resulted in 1.84 grams (17.9% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) 4.10 (td, J=15.5, 13.1, 7.6 Hz, 2H), 3.92 (t, J=13.9 Hz, 2H), 3.59 (t, J=6.6 Hz, 2H), 1.96 (t, J=7.5 Hz, 1 H), 1.60 (p, J=7.5 Hz, 2H), 1.25 (m, 33H), 0.88 (t, J=6.9 Hz, 3H). .sup.19F NMR (470 MHz, CDCl.sub.3) 119.63 (t, J=13.7 Hz), 121.95 (m), 122.43 (t, 13.5 Hz), 123.53 (d, J=46.5 Hz).
[0175] M2OMs monomethyl poly(ethylene glycol) methanesulfonate (5). To a dry roundbottom flask, in an ice bath and under argon, was added anhydrous dichloromethane (50 mL), poly(ethylene glycol) monomethyl ether 2,000 (2.5 mmol, 5.0195 g), and triethyl amine (7.5 mmol, 1.50 mL). This was allowed to react for 30 minutes before methanesulfonyl chloride (6.25 mmol, 0.50 mL) was added dropwise. After running overnight, the reaction was diluted with more DCM and extracted with saturated aqueous NH.sub.4Cl solution. The crude mixture was dried with MgSO.sub.4 and concentrated in vacuo. Purification was performed on the crude mixture dissolved in minimal DCM via precipitation of pure 5 with cold ether in a dry ice/acetone bath. Isolated product resulted in 4.93 grams (94.1% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 4.38 (m, 2H), 3.64 (m, 182H), 3.38 (s, 3H), 3.09 (s, 3H).
[0176] M2F8H18 (6). To a dry round bottom flask was added 4 (3.7 mmol, 2.63 g) and anhydrous benzotrifluoride (150 mL). This mixture was cooled over ice, flushed with argon, and then sodium hydride was added (7.4 mmol, 0.1831 g). This reaction was stirred for 30 minutes then 5 was added (1.9 mmol, 3.4526 g). The flask was heated to reflux and allowed to stir and reflux for 5 days. Upon completion, the reaction was cooled, diluted with DCM, and extracted with saturated aqueous NH.sub.4Cl solution. The crude mixture was dried with MgSO.sub.4 and concentrated in vacuo. Purification was performed via automated column chromatography, CombiFlash, using a C18 column and reverse phase conditions of a 90-0% water-methanol (0.1% formic acid) to 0-100% dichloromethane-methanol gradient. Polymer purity was confirmed via NMR, HPLC, and MALDI-MS. Isolated product resulted in 2.57 grams (83.9% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 4.04 (t, J=14.1 Hz, 2H), 3.92 (t, J=14.0 Hz, 2H), 3.79 (m, 3H), 3.64 (m, 174 H), 3.47 (t, J=5.0 Hz, 1 H), 3.38 (s, 3H), 1.60 (p, J=7.0 Hz, 3H), 1.26 (m, 36H), 0.88 (t, J=6.7 Hz, 4H). .sup.19F NMR (376 MHz, CDCl3) 119.73 (dt, J=58.9, 11.9 Hz), 121.97 (m), 123.47 (bs). MALDI-MS [M+Na].sup.+ calculated for C.sub.113H.sub.212F.sub.16O.sub.44Na.sup.+: 2600.40 m/z; found: 2600.584 m/z. [M+K].sup.+ calculated for C.sub.113H.sub.212F.sub.16O.sub.44K.sup.+: 2616.37 m/z\; found: 2616.556 m/z.
[0177] Results and Discussion
[0178] Synthesis of M2F8H18 Semifluorinated Polymer
[0179] The synthesis of M2F8H18 was performed using two routes and adapted from previous work in the Mecozzi group as well as other published works. The nomenclature here will involve Mx representing poly(ethylene glycol) monomethyl ether where x is the average molecular weight in the thousands, Fy representing the fluorocarbon block where y is the number of carbons attached to fluorine atoms, and Hz representing the hydrocarbon block where z is the number of carbons attached to hydrogens. Route 1 begins with the mono-benzylation of 1H,1H,10H,10H-perfluorodecane-1,10-diol under basic conditions and sonication to yield the protected fluorous alcohol, 1. Next, 1-octadecanol was mesylated under basic conditions to isolate 2. Compounds 1 and 2 were coupled under basic conditions and reflux to the benzylated diblock intermediate, 3. Hydrogenation of 3 catalyzed by palladium on carbon resulted in the alcohol diblock intermediate, 4. Then, monomethyl poly(ethylene glycol) (mPEG) with average molecular weight of 2,000 was mesylated under basic conditions, 5. Finally, the alcohol diblock intermediate 4 was coupled to mesylated mPEG 5 under basic conditions and reflux to afford the final, desired M2F8H18 polymer, 6. Compound 6 was purified using an automated CombiFlash system resulting in very high yields of isolated amphiphile. See
[0180] Alternatively, synthetic route 2 begins with the mesylation of 1-octadeancol under basic conditions to compound 2. Next, 1H,1H,10H,10H-perfluorodecane-1,10-diol was coupled directly to 2 without prior protection steps, as seen in Scheme 1. This coupling reaction occurred under basic conditions and sonication to directly afford 4 after purification. The final two steps mimic what was accomplished in scheme 1 including mesylation of mPEG, 5, and the final coupling of 4 and 5 under basic conditions and reflux. The final M2F8H18 amphiphile, 6, was once again purified using an automated CombiFlash system. Resulting yields were lower in the initial reactions but overall synthesis length was reduced by two steps. See
[0181] Synthetic route 1 proved to be reliable and high yielding, overall, though it does involve a more extensive set of reactions. On the other hand, synthetic route 2 significantly shorted the overall reaction scheme by two steps but with a moderate loss of yield in the initial reactions.
[0182] Physiochemical Characterization of Amphiphilic Aggregates
[0183] The Critical micelle concentration (CMC) of the M2F8H18 micellar aggregates was measured using surface tensiometry. The concentration that induces aggregation for M2F8H18, 2.210.sup.6 M, is an intermediate value compared to other Mecozzi polymers M1F13 and M2H18. The standard polymer, M2DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], was also measured for reference comparison. The size of the micellar aggregates was also analyzed using DLS (Table 1). Based on aggregate size (Table 1) it can be concluded that M2F8H18 forms compact, stable particles.
TABLE-US-00001 TABLE 1 Physiochemical characteristics of aggregates. Micellar Polymer CMC (log M) Particle Size (nm) M2F8H18 5.7 0.2 16.3 4.5 M1F13 6.1 0.1 17.2 1.9 M2H18 5.1 0.1 12.2 3.1 M2DSPE 4.9 0.2 13.9 1.6
[0184] Nanoemulsion Preparation and Stability
[0185] Formulation of M2F8H18 nanoemulsions with MCT or an MCT/Paclitaxel (PTX) combination was very successful resulting in opaque, milky colloidal solutions ranging in size from 200-250 nm initially. High concentrations of paclitaxel, from 1-4 mg drug per mL nanoemulsion, were loaded into the core of the particles. See
[0186] Nanoemulsions containing the highest concentrations of paclitaxel were stable for over one year, though some drug precipitation was noted over time. As seen below (
[0187] In Vitro Drug Release Profile
[0188] The in vitro paclitaxel release profile for M2F8H18 showed slow, steady release with highly sustained half-lives. Burst release is a common issue exhibited by many nanoparticles but no trace of that is seen here. The linear M2F8H18 alone improved greatly upon reported in vitro paclitaxel release from micellar delivery systems, to a very promising 69.5 hours (
[0189] Cellular cytotoxicity was examined in vitro using A549 non-small cell lung cancer cells. The A549 cells were selected due to their robust nature and frequent use in the field. Following cell plating and incubation, the cells were treated with three solutions comprising of 10 L of each solution, diluted with 90 L of fresh media. Solutions included a M2F8H18 nanoemulsion containing 4 mg/mL paclitaxel with 4 mL MCT, a M2F8H18 nanoemulsion containing no paclitaxel with 4 mL MCT, and paclitaxel standard solutions prepared with low concentrations of dimethyl sulfoxide (DMSO). Because DMSO has been shown to be toxic to A549 cells at high concentrations DMSO cytotoxicity was also monitored in this study. Paclitaxel standard concentrations were prepared at least three magnitudes above and two below its reported IC.sub.50 for the A549 cell line, 4 nM. Following treatment and incubation, the cell viability was measured using a CellTiter-Blue cell assay. The fluorescence intensity of each well was measured by a plate reader, at 560 nm, and cell viability was then calculated in relativity to the untreated control wells. All wells treated with the same solution were averaged, n=6. As seen below (
[0190] The M2F8H18 amphiphile was also screened for initial in vivo developmental toxicity, due to the novel nature of the polymer, using a zebrafish model. Though some toxicity predictions can be made from previous work in the Mecozzi lab, a thorough study of developmental effects related to M2F8H18 was performed using an embryo-larval zebrafish model. This animal model was selected because zebrafish eggs remain transparent from fertilization until the tissues become dense as a mature adult. Several developmental endpoints can be simultaneously monitored providing valuable developmental toxicity data for new chemicals. After crossing the fish and fertilization of the eggs had occurred, the embryos were collected and plated. The embryos were then treated with M2F8H18 solutions of the following concentrations: 1 mM, 333 M, 111 M, 37 M, 12.3 M, and 4.1 M. The embryos were then incubated, monitored, and photographed over a 96-hour experiment period. Survival rates for the non-treated control fish (
[0191] .sup.19F-MR Imaging Characterization
[0192] It has now been established that the M2F8H18 nanoemulsions can be loaded with high concentrations of PFCE. The addition of the excipient fluorocarbon enhances the drug release profile of the nanoemulsions and provides further stability to the colloidal particles. Next, the imaging potential of this molecule was investigated. Preliminary .sup.19F-NMR studies at 11.7 T show an intense, single peak at 6 -91.8 ppm with CFCl.sub.3 as an internal refernce.15 This pattern is due to the twenty magnetically equivalent fluorine atoms and ring flexibility in solution of the PFCE. The .sup.19F relaxation parameters T1, longitudinal relaxation or delay between pulses, and T2, transverse relaxation or signal decay over time, were also measured to monitor potential PFCE use as an efficient contrast agent. Nanoemulsion samples were prepared fully concentrated, without any dilution. The T1 was determined using an inversion recovery experiment while T2 was determined using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence experiment. The resulting relaxation values (Table 1) show a relatively short T1 value allowing for quick recovery between pulses and increased scanning efficiency as well as a long T2 value that should avoid most signal decay over time. Based on the single resonance intensity, high payload concentration within the nanoemulsions, and good relaxation values the PFCE has potential for use as a 19F-MR contrast agent.
TABLE-US-00002 TABLE 2 Fluorous Moiety Measured T.sub.1 (sec) T.sub.2 (sec) M2F8H18/PFCE 0.781 0.002 0.163 0.006 nanoemulsion
[0193] .sup.19F relaxivity measurements for perfluoro-15-crown-5-ether at 11.7 T. PFCE exhibits favorable 19F-NMR characteristics amenable for translation to .sup.19F-MR imaging: a single 19F resonance, a relatively small T.sub.1 value, and a relatively large T.sub.2 value.
[0194] Following preliminary work to establish the relaxation parameters of PFCE, .sup.19F-MR phantom images of the PFCE loaded nanoemulsions were acquired using a 4.7 T small animal MRI instrument. The .sup.1H images (
[0195]
[0196] Conclusions
[0197] The novel, semifluorinated amphiphile M2F8H18 was synthesized and thoroughly characterized. No developmental toxicity was found for this polymer after initial in vivo studies in zebrafish. This redesigned ABC polymer forms incredibly stable, triphilic nanoemulsions, due to the intrinsic driving force of the fluorous phase, with life-times up to 1 year. Very limited Ostwald ripening is observed for these particles thus they remain well below 400 nm for their full life-time. The M2F8H18 surfactant can form therapeutic nanoemulsions where it stabilizes large MCT oil droplets containing solid, highly hydrophobic paclitaxel at concentrations up to 4 mg/mL. Drug release from the oil core of the particles is gradual and controlled due to the long, stabilizing hydrocarbon chains that penetrate the oil droplet and the intermediary fluorocarbon shell that acts as a barrier. The addition of the fluorous excipient PFCE modulates drug release even further by sealing the fluorous shell of the polymer. Upon addition of the PFCE, these nanoemulsions can be formulated into theranostic systems with dual therapeutic and diagnostic character. High concentrations of the PFCE can be loaded into the nanoemulsions resulting in strong .sup.19F-MR signal due to its intense, single resonance composed of twenty magnetically equivalent fluorine atoms, short T.sub.1, and long T.sub.2. This preliminary work with M2F8H18 showed the polymer can formulate stable nanoemulsions that carry payloads of potent chemotherapeutic drug and .sup.19F-MR contrast agent all wrapped into a powerful, theranostic system. Future studies will focus on the translation of these M2F8H18 therapeutic and theranostic nanoemulsions to in vivo models.
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STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS
[0236] All references cited throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
[0237] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.
[0238] When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, including any isomers, enantiomers, and diastereomers of the group members, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomer and enantiomer of the compound described individually or in any combination. Additionally, unless otherwise specified, all isotopic variants of compounds disclosed herein are intended to be encompassed by the disclosure. For example, it will be understood that any one or more hydrogens in a molecule disclosed can be replaced with deuterium or tritium. Isotopic variants of a molecule are generally useful as standards in assays for the molecule and in chemical and biological research related to the molecule or its use. Methods for making such isotopic variants are known in the art. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently.
[0239] It must be noted that as used herein and in the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a cell includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms a (or an), one or more and at least one can be used interchangeably herein. The expression of any of claims XX-YY (wherein XX and YY refer to claim numbers) is intended to provide a multiple dependent claim in the alternative form, and in some embodiments is interchangeable with the expression as in any one of claims XX-YY.
[0240] Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated.
[0241] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. As used herein, ranges specifically include the values provided as endpoint values of the range. For example, a range of 1 to 100 specifically includes the end point values of 1 and 100. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.
[0242] As used herein, comprising is synonymous with including, containing, or characterized by, and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms comprising, consisting essentially of and consisting of may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
[0243] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.