WO2009056955A1 - Amine-bearing phospholipids (abps), their synthesis and use - Google Patents

Amine-bearing phospholipids (abps), their synthesis and use Download PDF

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Publication number
WO2009056955A1
WO2009056955A1 PCT/IB2008/002908 IB2008002908W WO2009056955A1 WO 2009056955 A1 WO2009056955 A1 WO 2009056955A1 IB 2008002908 W IB2008002908 W IB 2008002908W WO 2009056955 A1 WO2009056955 A1 WO 2009056955A1
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group
abps
amine
abp
substituted
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PCT/IB2008/002908
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French (fr)
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Andreas ZUMBÜHL
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University Of Basel
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • This invention is directed at a new class of phospholipids, namely amine-bearing phospholipids (herein referred to as ABPs).
  • ABPs amine-bearing phospholipids
  • two amines replace the ester moieties in naturally ocurring phospholipids.
  • This new class of phospholipids has properties that find a wide variety of applications, in particular in the field of drug delivery such as in the production of vesicles for particle-based drug delivery, and in the production polymers for polymer surfaces, including biocompatible and antimicrobial surfaces and artificial cell membranes (cell membrane mimics).
  • the biological membrane is a complex barrier that regulates the expensive imbalance between an outside and an inside (Bloom et al, 1995). It comprises primarily proteins and lipids in a mixture adapted to the current local environment (Seeling et al., 2001 ). By nature's design, the phospholipids of a biological membrane serve primarily structural and signaling purposes.
  • Natural phospholipids generally comprise a polar hydrophilic head group, an uncharged hydrophobic portion and an interface-region based on a phosphoric acid substituted glycerol.
  • Phospholipids have been crosslinked either in the alkyl-chain or the head-group region of the molecule to provide polymerization products for a variety of uses.
  • natural phospholipids have been cross-linked to form nanometer sized particles.
  • the scaffold of natural phospholipids does not allow for chemical modifications without severely affecting and/or destroying their structural integrity, thus limiting their usage.
  • U.S. Patent 5,540,935 (EP 0 657 463) describes phospholipid derivatives that are modified either at the polar hydrophilic head group or at the uncharged hydrophobic portion.
  • the phospholipids are employed in reactive vesicles and functional substance-fixed vesicles as well as a drug delivery vesicles.
  • amine-containing lipids have been described.
  • WO 2006/138380 describes nitrogen-containing lipids prepared by conjugate addition of amines to acrylates, acrylamides, or other carbon-carbon double bonds conjugated to electron- withdrawing groups.
  • these lipids have a hydrophobic half and a hydrophilic half.
  • the hydrophobic portion is typically provided by fatty acid moieties attached to the acrylate, and the hydrophilic portion is provided by the esters, amines, and side chains of the amine.
  • These lipids may be prepared by the addition of a primary amine to a double bond conjugated with an electron withdrawing groups such as a carbonyl moiety. They may serve the delivery of nucleic acids in gene therapy as well as in the packaging and/or delivery of diagnostic, therapeutic, and prophylactic agents.
  • lipid-analogs have focused on head-group modifications, acyl chain modifications, and ester-to-amide or ester-to-ether modifications (see e.g. www.avantilipids.com). Also, a great variety of new cationic lipids have been synthesized that lack the phosphate ester motive altogether (Blagbrough et al, 2003; Akinc, 2008).
  • the invention is, in one embodiment, directed towards an amine-bearing phospholipid (ABP) having: a head group, e.g., a polar head group, an hydrophobic portion, e.g. an uncharged hydrophobic portion, and an interface-region comprising an amine group substituted propanol backbone, in particular a 2,3-diaminopropan-1 -ol (also commonly referred to as just "1 ,2-diaminopropanol”) or a 1 ,3- diaminopropan-2-ol (also commonly referred to as just "1 ,3-diaminopropanol”) backbone.
  • ABSP amine-bearing phospholipid
  • a typical ABP according to the present invention can be depicted as follows:
  • the headgroup while shown to be at a 180 degree angle relative the hydrophobic portion, may also be at or almost at a right angle to the hydrophobic moieties of the hydrophobic portion.
  • the amine-bearing phospholipid may correspond to any of the formulas referred to herein. As the person skilled in the art will readily understand any 2,3- diaminopropan-1 -ol shown or described can be replaced by a 1 ,3- diaminopropan-2- ol and vice versa.
  • the present invention is directed at an amine-bearing phospholipid (ABP) comprising: an interface region comprising an amine group substituted propanol backbone, preferably a 2,3-diaminopropan-1-ol or a 1 ,3- diaminopropan-2-ol, providing at least three attachment regions, a hydrophilic head group, a first and second hydrophobic portion, preferably a first and second uncharged hydrophobic portion, wherein the head group comprises and is covalently attached via a phosphate ester bond to a first one of the attachment regions, and the first hydrophobic portion comprises two hydrophobic moieties each being covalently attached to an amine of a second one of the attachment regions, the second hydrophobic portion comprises two hydrophobic moieties each being covalently attached to an amine of a third one of the attachment regions, wherein, in an aqueous surrounding, the head group extends from the interface region into a first direction and the hydrophobic portions extends from
  • the ABPs of the present invention may, in particular, have the following formulas: (I)
  • head group A may be one of the following: a phosphatic acid or a phosphate ester substituted group such as: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, phosphate ester substituted electrophile, such as an activated ester including N- hydroxysuccinimic ester, or an acid chloride or an halogenide, a phosphate ester substituted nucleophile such as a thiol or an amine, a phosphate ester substituted fluorescent group such as fluoresceine, a phosphate ester substituted radioactively labeled group, a phosphate ester substituted alkyne, or a phosphate ester substituted
  • hydrophobic moieties may be Y 1 , Y 2 , Z 1 and Z 2 , wherein,
  • W may be a stereocenter that is R, S or racemic;
  • Y 1 may be:
  • a sterol preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group or a linear alkyl group or an aryl group;
  • Y 2 may be:
  • Z 1 may be:
  • a sterol preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group;
  • Z 2 may be:
  • a sterol preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group; or
  • CH CH 2 ; with the proviso that at least one, preferably two or three of Y 1 , Y 2 , Z 1 and Z 2 are unequal H.
  • Y , Y , Z and Z may be selected from one or more of the Table III moieties.
  • the ABPs of the present invention may, in particular have the following formulas: (III)
  • X 1 may be:
  • alkyl- preferably methyl-, ethyl-, propyl-, isopropyl-;
  • a fluorescent reporter group which may, as any other fluorescent reporter group set forth herein, preferably be fluorescein, alexafluor 488, Tokio green, wherein the reporter group is bound to the amine of the interface region directly via an amide bond or via a spacer group, preferably an alkyl-, aryl- or PEG spacer group;
  • a non-fluorescent reporter group preferably a spin label or a radioactive label
  • an electrophilic or nucleophilic group preferably an ⁇ /-hydroxysuccinimide, an anhydride, or an /V-methylisatoic anhydride, a methylisothiocyanate, a methylisocyanate, or methylthiourea, 3-[4-(trimercapto)phenyl]propionyl, 4- benzyloxybenzaldehyde, 2-chlorotrityl chloride, sulfonic acid, morpholinomethyl, piperidinomethyl, piperazinomethyl, acrylate, methyl- acrylate, styrene, acrylamide, or methyl-acrylamide;
  • a metal complexing group preferably ethylenediamine tetraacetate, wherein the group is bound to the amine N 1 either directly or via a linker, preferably an alkyl chain or a polyethylene glycol chain;
  • X 2 may be:
  • alkyl- preferably methyl-, ethyl-, propyl- or isopropyl-;
  • alkyl- such as, but not limited to, methyl-, ethyl-, or propyl-; or
  • W may be as above;
  • n may be as above;
  • Y 1 , Y 2 , Z 1 and Z 2 may be as above;
  • V may be
  • W may be as above;
  • n may be as above;
  • Y 1 , Y 2 , Z 1 and Z 2 may be as above.
  • the sugar phosphate ester substituted sugar may be a simple sugar or may be part of a simple glucose series or a globo, ganglio, lacto, neolacto, isoglobo, mollu, arthro or gala series.
  • the head group of said ABP may be labeled, preferably fluorescently, may be deuterated, radioactively labeled or spin labled.
  • One or more positions of the sugars set forth above may be substituted, preferably with a sugar including a substituted sugar, even more preferably with a simple sugar.
  • a dimer or polymer as described herein may be a bolaamphiphile in which the ABPs are linked to one another via one or both their hydrophobic portions.
  • a dimer or polymer as described herein may comprise one or more linkers, in particular sugar or polysugar linkers.
  • the ABPs may be crosslinked, preferably via a linker, most preferably one or more of the following linkers:
  • X may be O or N
  • Y may be O or N
  • Z may be H or CH 3 ;
  • n may be an integer from 0 to 10; or any of the crosslinker(s) set forth elsewhere herein.
  • Any of the dimers set forth herein may be a heterodimer.
  • the present invention is also directed at a transfection method comprising providing at least one ABP as set forth herein; admixing the at least one ABP with at least one nucleic acid; and transfecting a cell, in vitro or in vivo.
  • the nucleic acid may be at least one siRNA, miRNA or pDNA.
  • the cell may comprise a gene whose expression is targeted by, e.g., said siRNA.
  • the ABP may be part of a micro- or nanoparticle.
  • the ABPs may form degradable or nondegradable cationic polymers, preferably cationic polymers as set forth herein.
  • the invention is also directed at polymeric surfaces comprising ABPs as set forth herein and polymerized as set forth herein.
  • the polymeric surface is preferably biocompatible, even more preferably antimicrobial.
  • the polymeric surface may comprise an agent covalently attached to a reactive group of a ABP, or a reactive group of a further molecule, wherein an attachment is a pre- or postpolymerization attachment.
  • the agent may be at least one antimicrobial agent, preferably a peptidic antimicrobial agent.
  • the invention is also directed at the producing of such a polymeric surface:
  • a carboxylic acid, an activated carboxylic acid (active ester), an aldehyde, a ketone, an amine, a hydrazine, an azide or an alkine may be admixed prior to polymerization to a solution comprising one or more types of ABPs.
  • the invention is also directed at a method for linking one or more types of ABPs:
  • the ABPs are reacted to comprise one or more, preferably two, acrylamide and/or methylacrylamide groups, and the resulting acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs are polymerized via UV light, optionally in presence of a polymerization initiator, preferably a radical initiator or by heat.
  • the ABPs may be reacted to comprise one or more, preferably two, acrylamide and/or methylacrylamide groups to provide acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs, comprising reacting terminal double bonds of said acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs with a further amine, and polymerizing the reaction product.
  • the amines may be primary or secondary amines, preferably an amine selected from (1) to (94) of Table I:
  • the ABPs form linear polymers, 2d-sheets or 3d polymers.
  • the ABPs may be chemically modified after polymerization. Prior to polymerization proteins and/or natural lipids, in particular phospholipids, may be admixed.
  • the ABPs of the present invention including the dimers and polymers created therefrom have a wide variety of uses in particular in the medical field, but also for research and development. They can, for example, be employed as the production of drug delivery vesicles and/or vehicles, in particular nucleic acid transfection vesicles and/or vehicle or vesicles for non nucleic acid drug delivery. However, a wide variety of other uses are contemplated including, but not limited to, uses as cell- membrane mimetic materials, responsive surfaces, fluorescence markers, MRI/PET markers, consumer products, liquid crystals and in electronics.
  • Fig. 1 shows in A a naturally occurring phospholipid and in B a amine-bearing phospholipid (ABP) according to the present invention.
  • B a amine-bearing phospholipid
  • the Figure shows the integration of an APB into a phospholipids bilayer.
  • Fig. 2 depicts an example of a synthesis path for an ABP.
  • Fig. 3 depicts an alternative synthesis path for an ABP.
  • Fig. 4 depicts a further synthesis path for an ABP.
  • Fig. 5 depicts a synthetic route for the establishment of a ABP library.
  • Fig. 6 shows under I to III reactions that lead to the polymerization of APBs into different polymerization products.
  • Fig. 7 depicts under IV to Vl reactions that lead to the polymerization of APBs into further polymerization products.
  • the amine-bearing phospholipids of the present invention and their polymers may address one or more of said needs and/or other needs which will become apparent from the following disclosure.
  • the amine-bearing phospholipids presented herein are a new class of phospholipids bearing at least two free amines instead of ester moieties of natural phospholipids in the interface region.
  • Fig. 1 B shows a natural occurring phospholipid.
  • An "amine bearing phospholipid” according to the present invention comprises an interface region that is covalently attached to a head group and at least one, preferably two hydrophobic portion.
  • the ABP also contains a phosphate ester bond, which provides the link between the interface region to a head group.
  • the head group extends, in an aqueous surrounding, from the interface region in one direction and the hydrophobic moiety/(ies) of the hydrophobic portion(s) into a second direction.
  • the angles between the headgroups and hydrophilic portions preferably include about 180 degrees, about 150 degrees, about 120 degrees, about 100 degrees or about 90 degrees. The angles may vary over time and may alter in different surroundings.
  • the ABPs of the present invention have many properties and/or functionalities of naturally occurring phospholipids, such as the ability to self assemble, but provide additional properties and/or functionalities discussed in more detail herein.
  • a “head group” according to the present invention find is counterpart in what is generally referred to as a "head” in context of natural phospholipids (also referred to herein as naturally occurring phospholipids), both functionally and structurally.
  • the head group is generally hydrophilic so that it can interact with an aqueous surrounding.
  • the head group comprises and is attached to the so called “interface region" via a phosphate ester bond.
  • the head group often comprises an elongated molecule that may be branched or unbranched.
  • the head group may also contain a ring structure that is substituted, preferably with hydrophilic groups and, often with amine or amide groups. However, it may, for example, be a short branched, generally hydrophilic molecule.
  • headgroups include phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol, a phosphatic acid substituted sugar, or a phosphate ester substituted polyethyleneglycol, or a phosphate ester substituted electrophile such as an activated ester like ⁇ /-hydroxysuccinimic ester, or an acid chloride or a halogenide, or a phosphate ester substituted nucleophile such as a thiol, or an amine, or a phosphate ester substituted fluorescent group such as fluoresceine or a radioactively labeled group, or a phosphate ester substituted alkyne, or a phosphate ester azide and headgroups described in Fahy et al.
  • GalNAc ⁇ 1-3Gal ⁇ 1 -4Gal ⁇ 1 -4Glc- (globo series)
  • GalNAc ⁇ 1-4Gal ⁇ 1 -4Glc- (ganglio series)
  • GalNAc ⁇ 1-3Gal ⁇ 1 -3Gal ⁇ 1 -4Glc- isoglobo series
  • GaI- gallium
  • poly refers to more than one that is 2, 3, 4, 5, 6, 7, 8, 9, 10, about 20, about 30, about 40, about 50, about 100 or more.
  • hydrophobic portion finds it counterpart in naturally occurring phospholipids in what is generally referred to as a “hydrophobic portion or lipophilic portion” in this context, both functionally and also structurally.
  • the hydrophobic portion of the present invention is covalently linked to the interface region via an amine/amide bond.
  • the hydrophobic portion can interact, with other hydrophobic molecules such as transmembrane proteins and hydrophobic portions of phospholipids and/or other ABPs.
  • it contains reactive hydrophobic groups so that polymerization of ABPs via the hydrophobic portion is possible.
  • the hydrophobic portion also allows the ABP to integrate into existing membranes or to form membranes and/or membrane like structures.
  • the hydrophobic portion often contains, as hydrophobic moiety, chains of hydrophobic molecules, which may contain one or more double and triple bonds.
  • These hydrophobic moieties are a hydrophobic tails or just tails.
  • the tails are unbranched.
  • the chains contain at least 5 carbon atoms, preferably at least 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, more preferably 16, 17, 18, 19, 20, 25 or 30 carbon atoms, wherein an even number of carbon atoms is preferred.
  • one, two, three, four, five or more double or triple bonds are present.
  • molecules such as a cholesterol or ergosterol may constitute or be part of the hydrophobic moiety.
  • moieties may be attached to the amine via a linker such as a linear alkyl or aryl group.
  • a linker such as a linear alkyl or aryl group.
  • a hydrophobic portion contains two hydrophobic moieties. These moieties are covalently attached to the amine of the interface region.
  • different hydrophobic moieties may be combined within one hydrophobic portion or an ABPs.
  • hydrophobic moieties which take the form of hydrophobic chains are depicted in Table III: Table III. Examples of hydrophobic chains in hydrophobic portions are depicted in Table III: Table III. Examples of hydrophobic chains in hydrophobic portions
  • the "interface region” finds its counterpart in the interface region in naturally occurring phospholipids, namely the phosphorylated diglyceride in phospholipids.
  • it comprises an amine group substituted backbone molecule, most preferably it comprises an aminopropanol, preferably a diaminopropanol, namely a 2, 3- diaminopropan-1-ol or a 1 , 3- diaminopropan-2-ol backbone.
  • the phosphate group provides a first attachment region in the context of the present invention, generally to the head group, while the one or two amine groups provide a second and third attachment region for the hydrophobic portion, which comprises hydrophobic moieties as described above.
  • alkyl refers to saturated, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
  • alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert- butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.
  • Alkene and alkylene refers to counterparts having at least one double or triple bond.
  • aryl and heteroaryl refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted.
  • Substituents include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound.
  • aryl refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.
  • heteroaryl refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
  • substituted refers to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained.
  • substituent may be either the same or different at every position.
  • the substituents may also be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted with fluorine at one or more positions).
  • a substituted sugar according to the present invention refers to a sugar wherein one of the hydroxyl groups is substituted by an amine, an alkyl-amine or an alkyl-amide.
  • a substituted sugar also refers to a sugar linked via a glycosidic bond to another sugar.
  • a “bolaamphiphile” is, according to the present invention, an amphiphilic molecule that has a hydrophilic group at two ends. This introduction of a “second” head group generally induces a higher solubility in water, an increase in the critical micelle concentration (cmc), and a decrease in aggregation number.
  • the aggregate morphologies of bolaamphiphiles include spheres, cylinders, disks, and vesicles.
  • Bolaamphiphiles and other lipids including other ABPs may be be admixed according to the present invention.
  • label is intended to mean that a compound has at least one element, isotope, or chemical compound attached to enable the detection of the compound.
  • labels typically fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes, including, but not limited to, 2 H (D,
  • photophores can be employed, most relying on photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (See, Bayley, H., Photogenerated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam).
  • the photoaffinity labels employed are o-, m- and p- azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to 4-azido-2,3,5,6- tetrafluorobenzoic acid.
  • nitroxyl radical (N-O) is incorporated into a heterocyclic ring.
  • Labels according to the present invention also include "caged groups", that are labels that are protected and can be deprotected under certain conditions, e.g. pH change or a light pulse, e.g. inside the animal (including human) body for most in vivo uses or on in a cell for in vitro and in vivo.
  • damaged groups that are labels that are protected and can be deprotected under certain conditions, e.g. pH change or a light pulse, e.g. inside the animal (including human) body for most in vivo uses or on in a cell for in vitro and in vivo.
  • a “pre-polymer group” allows for polymerization of molecules, e.g., in light or by heat, optionally with a radical initiator.
  • "By heat” refers preferably, though not exclusively, to heat ranges of between about 30 0 C and 100 0 C, including between about 35 °C and 100 0 C, between about 40 0 C and 100°C, between about 50 0 C and 100 0 C, between about 60 0 C and 100 0 C and between about 80 0 C and 100 °C.
  • racemic mixture is a mixture that contains left (S) - and right (R) - handed enatiomer of a chiral molecule.
  • transfection is the process of introducing nucleic acids into cells by non-viral methods.
  • ABP mediated transfection includes in partular the intoduction of the nucleic acid using the ABPs as “carriers” or “vehicles” across cell membranes. Included are in particular the formation of ABP containing vesciles which can fuse with cell membranes to carry the nucleic acids across the cell membrane.
  • the nucleic acid is a siRNA, a miRNA or a pDNA.
  • liposome refers to an aqueous lipid-containing suspension of multi- layered (consisting of at least a double layer of lipid) generally spherical accumulations of lipid molecules which are formed by mechanically mixing a dry lipid in water.
  • Lipoplexes Complexes comprising cationic lipids and nucleic acid molecules are referred to as "lipoplexes" and form part of the present invention. Lipoplexes can spontaneously form in the presence of negatively charged pDNA (plasmid DNA). ABPs allow for the attachment of, e.g., cell specific targeting molecules and thus target delivery of, e.g. the pDNA.
  • pDNA plasmid DNA
  • ABPs can form vesicles for drug delivery, which includes delivery of nucleic acid based drugs and non-nucleic acid based drugs. Such vesicles generally have a rounded shape and form discrete drug delivery packets. The process is referred to herein as “vesicle based drug delivery.”
  • a "vescile” which inlcudes the term “nanoparticle” or “miicroparticle”, is, in the context of the present invention, a selfassembled and/or polymerized, in an aqueous solution rounded, generally spherical structure that engulfs, e.g., a drug and thus becomes a carrier of the drug.
  • Selfassembled vesciles can deliver a drug into a cell by merging with its cell membrane.
  • Targeting molecules for, e.g., specific cell surface receptors may be for example be attached directly or indirectly to a secondary amine of the ABP.
  • a vesicle may, on the one hand, display the targeting molecules on its surface and, on the other hand, shield the drug from its surrounding and vice versa.
  • the targeting molecule will interact, e.g. bind, its target, e.g., a surface antigen expressed on the target cell and the vesicle will fuse with the cell mebrane of the target cell to release the drug into the lumen of the target cell.
  • Drug delivery of drugs that are subject to degradation and highly toxic drugs are of particular interest in this context.
  • Drug delivery according to the present invention to an individual, in particular a peson in need thereof can be by any route, including intravenously, parenterally, orally, intramuscularly, intrathecal ⁇ or as an aerosol.
  • the mode of delivery will depend on the ABP vehicle/vesicle used, the drug delivered and the desired effect.
  • a skilled artisan will readily know the best route of administration for a particular treatment in accordance with the present invention.
  • the effective amount will depend on the route of administration and the treatment indicated, and can readily be determined by a skilled artisan in view of current treatment protocols.
  • Non-nucleic acid based drugs that can be encapsulated by the vesicles of the present invention include, but are not limited to pharmacological agents, and therapeutic agents.
  • non-nucleic acid based drugs suitable for use in the present invention include, but are not limited to, peptides, and particularly small peptides; hormones, and particularly hormones which are susceptible to chemical cleavage by acids and enzymes in the gastrointestinal tract; polysaccharides, and particularly mixtures of muco-polysaccharides; carbohydrates; lipids; or any combination thereof.
  • Further examples include, but are not limited to, human growth hormones; bovine growth hormones; growth releasing hormones; interferons; interleukin-1 ; insulin; heparin, and particularly low molecular weight heparin; calcitonin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropin releasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium or disodium chromoglycate); vancomycin or desferrioxamine (DFO).
  • human growth hormones bovine growth hormones
  • growth releasing hormones interferons
  • interleukin-1 insulin
  • insulin heparin, and particularly low molecular weight heparin
  • calcitonin erythropoietin
  • atrial naturetic factor antigens
  • monoclonal antibodies somatostatin
  • a “nucleic acid” is any molecule composed of chains of monomeric nucleotides and includes desoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both single standed (ss) as well as double stranded (ds).
  • DNA desoxyribonucleic acid
  • RNA ribonucleic acid
  • pDNA plasmid DNA
  • RNAi RNA interference
  • the latter include, for example, small interfering RNAs (siRNAs), which usually comprise 20 to 25 nucleotide long double-stranded RNA molecules.
  • miRNAs microRNAs
  • miRNAs are an abundant class of small single-stranded non-coding RNAs (19-30 nucleotides long) that serve widespread functions in post-transcriptional gene silencing.
  • biocompatible means that the ABP containing compound or structure, when introduced into a human patient, will not result in any severe degree of unacceptable toxicity, including allergic responses and inflammation.
  • the ABP containing compounds or structures are inert.
  • a “biocompatible surface” according to the present invention is a surface that can be employed in cell and molecular biology without substantially affecting biological processes, e.g., as a surface for cell, e.g. stem cell, growth. Such a surface may comprise releasable growth factors.
  • a “antimicrobial agent” refers herein as an agent having an activity which is capable of killing or inhibiting growth of microbial cells.
  • the term “antimicrobial” is intended to mean that there is a bactericidal and/or a bacteriostatic ("antibacterial") and/or fungicidal and/or fungistatic (antifungal") effect and/or a virucidal (“antiviral”) effect.
  • Bacteriostatic antibacterial
  • fungicidal and/or fungistatic (antifungal) effect and/or a virucidal (“antiviral") effect.
  • Peptidic antimicrobial agents whose main functional moiety is a peptide or protein, are preferred.
  • Classes of antifungal agents include, but are not limited to, allylamines, azoles, pyrimidines, tetraenes, thiocarbamates, sulfonamides, glucan synthesis inhibitors. Allylamines include, for example, amorolfine, butenafine, naftifine and terbinafine.
  • Azoles include, for example, ketoconazole, fluconazole, elubiol, econazole, econaxole, itraconazole, isoconazole, imidazole, miconazole, sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole, terconazole, butoconazole, thiabendazole, voriconazole, saperconazole, sertaconazole, fenticonazole, posaconazole, bifonazole, flutrimazole.
  • Polyenes include, for example, nystatin, pimaricin and amphotericin B.
  • Pyrimidines include, for example, flucytosine. Tetraenes include, for example, natamycin. Thiocarbamates include, for example, tolnaftate. Sulfonamides include, for example, mafenidine. Other antifungal drugs include ciclopirox and ciclopirox olamine.
  • Antibacterial agents include but are not limited to aminoglycosides, ⁇ lactam agents, cephalosporins, macrolides, penicillins, quinolones, sulfonamides, and tetracyclines.
  • Examples of anti-bacterial agents include but are not limited to: Acedapsone, Acetosulfone Sodium, Alamecin, Alexidine, Amdinocillin Clavulanate Potassium, Amdinocillin, Amdinocillin Pivoxil, Amicycline, Amifloxacin, Amifloxacin Mesylate, Amikacin, Amikacin Sulfate, Aminosalicylic acid, Aminosalicylate sodium, Amoxicillin, Amphomycin, Ampicillin, Ampicillin Sodium, Apalcillin Sodium, Apramycin,
  • Fosfomycin Tromethamine Fumoxicillin, Furazolium Chloride, Furazolium Tartrate, Fusidate Sodium, Fusidic Acid, Gatifloxacin, Genifloxacin, Gentamicin Sulfate, Gloximonam, Gramicidin, Haloprogin, Hetacillin, Hetacillin Potassium, Hexedine, Ibafloxacin, Imipenem, Isoconazole, Isepamicin, Isoniazid, Josamycin, Kanamycin Sulfate, Kitasamycin, Levofloxacin, Levofuraltadone, Levopropylcillin Potassium, Lexithromycin, Lincomycin, Lincomycin Hydrochloride, Linezolid, Lomefloxacin, Lomefloxacin Hydrochloride, Lomefloxacin Mesylate, Loracarbef, Mafenide, Meclocycline, Meclocycline Subsal
  • Anti-viral agents useful in the invention include but are not limited to: immunoglobulins, amantadine, interferons, nucleotide analogues, and protease inhibitors.
  • Specific examples of anti-virals include but are not limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium; Ganciclovir; Ganci
  • a “degradable” ABP polymer as used herein means that the polymer can be readily broken down enzymatically.
  • a biodegradable polymer according to the present invention is 80% broken down within preferably one week to six months, including about one, two, three, four or five months.
  • non-degradable ABP polymer as used herein means that the polymer can be not broken down enzymatically.
  • a non-biodegradable polymer according to the present invention will be substantially intact (max. 5% degradation) for one week to 3 years including about 6 months, 1 year or 2 years.
  • the degradabily of the ABP can be adjusted by the linkers used in the polymerisation.
  • a "linker” for the polymerization of ABPs according to the present invention is any molecule that can react with an ABP and can be used to covalently link one ABP to another to create a dimer, trimer or polymer.
  • linkers can be employed in the polymerization reactions. Those include, but are not limited to, epichlorohydrine, divinylbenzene, diisocyanate and styrenes.
  • the cross-linkers are selected from the group of crosslinking acrylates; crosslinking methyl acrylates, crosslinking acrylamides, crosslinking methyl acrylamide or combinations thereof.
  • Crosslinkers that are within the scope of the present invention include in partiular Dithiobis(succinimidylpropionate) (DSP), 3,3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP), Dissucinimidyl suberate (DSS), Bis(sulfosuccinimidyl)suberate (BS 3 ), Disuccinimidyl tartrate (DST), Disulfosuccinimidyl tartrate (Sulfo-DST), Bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), Bis[2- (sulfosuccinimidooxycarbonyloxyjethyllsulfone (Sulfo-BSCOES), Ethylene glycolbis(succinimidylsuccinate) (EGS), Ethylene glycolbis(sulfosuccinimidylcuccinate) (Sul
  • the ABPs form homo-bilayers and incorporate into natural membranes and behave like natural phospholipids.
  • the ABPs of the present invention bear "functional handle(s)" at the interface-region.
  • These secondary amines of the ABPs allow substitution reactions at the interface region. Such substitution reactions include polymerization, which permits the formation of cell-surface like materials.
  • the substitution reactions render the ABPs partially positively charged, allowing for non- covalent interactions with negatively charged DNA and RNA.
  • the secondary amines of the ABP may disturb the natural dipole-moment of the phospholipids rendering the molecule partially positively charged, allowing, e.g., for non-covalent interactions with negatively charged DNA and siRNA.
  • the structure of the ABPs in particular the presence of these "functional handles", improves the structural integrity of the phospholipids, in particular subsequent to polymerization, opening the door for a wide variety of applications, only a few of which will be discussed in the following.
  • the person skilled in the art will, with the guidance provided herein, readily be able to envision and execute further uses of the modified phospholipids of the present invention.
  • ABSPs amine-bearing phospholipids
  • Fig. 1 shows in (A) a classic phospholipid built from a glycerophosphate backbone.
  • B an amine-bearing phospholipids (ABP) with a 2,3-diaminopropyl phosphate backbone produced by de novo synthesis is shown. Positions sn1 , sn2 and sn3 are indicated.
  • C the ABPs can incorporate into natural phospholipid bilayers.
  • the structure of the ABPs of the present invention allow for a wide variety of uses, including the non-covalent complexation of nucleic acids such as plasmid DNA (pDNA) or siRNA generally followed by the transfection of cells.
  • nucleic acids such as plasmid DNA (pDNA) or siRNA generally followed by the transfection of cells.
  • pDNA plasmid DNA
  • siRNA siRNA
  • the ABPs serve as carriers without necessarily physically sequestering the nucleic acid from its surroundings.
  • the ABPs can also form vesicles (also referred to herein as nanoparticles) that engulf a nucleic acid or drug.
  • ABPs may be used as or as part of a cationic transfection lipids to form lipoplexes which may serve as transfection vectors, which may or may not be polymerized.
  • Such complexes may also incorporate target specific molecules at the interface region of the phospholipids, this allowing targeted delivery of a non-nucleic acid drug and/or a nucleic acid, including DNA and RNA, in particular pDNA, siRNA miRNA (mirco RNA).
  • a non-nucleic acid drug and/or a nucleic acid including DNA and RNA, in particular pDNA, siRNA miRNA (mirco RNA).
  • the structure of the ABPs also allow for the formation of cell-membrane-like polymers and the incorporation of antimicrobial peptides to form antimicrobial surfaces.
  • Polymeric ABPs can serve as a new core material for antimicrobial surfaces, including antifungal and antibacterial surfaces, and for coatings, in particular biocompatible coatings and surfaces.
  • antimicrobial peptides may be immobilized on the polymeric ABPs. These surface can be used on a wide variety of medical devices, e.g., catheters that are prone to contamination.
  • cell-membrane mimics and cell-membrane-like polymers which integrate (trans- membrane) proteins and cellular components and which are based on ABPs provide a wide array of applications for basic and applied research.
  • ABPs Amine-Bearing Phospholipids
  • ABPs Amine-Bearing Phospholipids
  • Such amine-bearing phospholipids represent, as discussed above, a versatile platform in biology and biophysics.
  • the synthesis of ABPs can be accomplished by various synthetic routes. A number of non-limiting approaches are provided in the following:
  • amine-bearing phospholipids can start from L-serine (3), drawing from the natural chiral pool to install the stereocenter at sn2 (see, FIG. 2). Following the protocol of McKillop et al., the methyl ester is formed for solubility purposes at later stages of the synthesis (McKillop et al., 1994). ⁇ /-Acylation with the commercially available palmitoylchloride indroduces the first hydrophobic tail of the molecule (4).
  • the second alkyl chain is attached following a methodology developed by Solladie-Cavallo et al., who showed that lithium aluminum amides (formed from a simple substitution of lithium aluminum hydride and an amine) react readily with esters to form an amide (Solladie-Cavallo et al., 1992). Simultaneous reduction of both amides (5) to amines (6) followed by BOC-protection finishes the hydrophobic part of the molecule (7). The free hydroxyl group (7) is transformed into the phosphate (8) and then into the phosphocholate head-group (9) as described by Harbison and Griffin (Harbison et al., 1984).
  • the free hydroxyl group (7) is reacted with chloro 2-cyanoethyl (N,N- diisopropyl)phosphoramidite followed by reaction with e.g. choline tosylate (Bay et al. 2004), followed by the proper oxidation and deprotection of the headgroup.
  • the free hydroxyl group (7) is reacted with 1 -choline 2-cyanoethyl (N,N- diisopropyl) phosphoramidite, followed by the proper oxidation and deprotection of the headgroup.
  • the oxazolidinone is opened by a catalytic reaction with caesium carbonate, following a similar protocol reported by Sibi et al. (Sibi et al., 1999). Both secondary amines are now BOC- protected (7) which completes the hydrophobic part of the molecule. The free hydroxyl group (7) is transformed into the phosphate (8) and then into the phosphocholate head-group (9) as described by Harbison and Griffin (Harbison et al., 1984). BOC-deprotection under acidic conditions will free both amines (2).
  • phosphoramidite chemistry can be employed and the free hydroxyl group (7) is reacted with chloro 2-cyanoethyl ( ⁇ /, ⁇ /-diisopropyl)phosphoramidite followed by reaction with e.g. choline tosylate (Bay et al., 2004), followed by the proper oxidation and deprotection of the headgroup.
  • the free hydroxyl group (7) can be reacted with 1 -choline 2-cyanoethyl ( ⁇ /, ⁇ /-diisopropyl)phosphoramidite, followed by the proper oxidation and deprotection of the headgroup.
  • lipid with an 1 ,3-diaminopropan-2-ol backbone may also follow another route (see FIG. 4): Starting with a double nucleophilic attack of a long-alkyl chain amine to racemic epichlorohydrin (14). The resulting lipid, can be protected in the form of a cyclic urea (15) in the same flask similar to a procedure reported by Enders using bis(4-nitrophenyl)carbonate and thus avoiding a kinetically favored five- membered ring carbamate (Enders et al., 1999).
  • the free hydroxyl group (7) is reacted with 1 -choline 2-cyanoethyl ( ⁇ /, ⁇ /-diisopropyl)phosphoramidite, followed by the proper oxidation and deprotection of the headgroup.
  • 1 -choline 2-cyanoethyl ( ⁇ /, ⁇ /-diisopropyl)phosphoramidite followed by the proper oxidation and deprotection of the headgroup.
  • ABP libraries The synthesis of ABP libraries is designed to be flexible and highly modular. In particular, it allows the introduction of i) the enantiomeric form at sn2, ii) different chain-lengths at sn2 and sn1 , independently of each other, iii) unsaturation in one or both alkyl chains, iv) different head-groups containing free amines, free or activated carboxylic acids or thiols, and v) unusual phospholipid tails such as e.g. cholesterol (see FIG. 5).
  • FIG. 5 shows that the ABPs are highly modular, and libraries of structurally different molecules can be synthesized.
  • Various scientific questions can be targeted with individual, optimized combinations.
  • libraries of amine-bearing phospholipids are synthesized. This approach allows the preparation of lipids with optimized properties, e.g. deuterated ABPs for NMR- structural studies.
  • ABPs ABPs with natural membrane components, i.e. the phospholipids POPC, POPG, and also cholesterol is tested through isothermal titration calorimetry. Physical characteristics are measured such as binding affinities K 3 , enthalpy changes ⁇ H as well as Gibbs free energy changes ⁇ G and entropy changes ⁇ S (Heerklotz et al., 2000). Measuring the surface-pressure to area isotherm in a Langmuir-Blodgett trough reveals the ABPs cross-sectional area A covered at the surface.
  • a thin film of ABPs is rehydrated and freeze-thawed, followed by extrusion through 400 and/or 200 and/or100 and/or 50 nm tracked-edge membrane filters (Walde, 2004), light scattering experiments, and freeze-fracture cryo transmission electron microscopy.
  • the Synthesis of Phospholipid-Polymers Polymers that mimic natural cell-membranes are highly attractive targets in biomedical engineering (Akimoto et al., 1981).
  • the amine-bearing phospholipids may be polymerized via, e.g., one of the following three routes (see FIG. 6), which shows the synthesis of three types (l.-lll.) of poly ⁇ -amino phospholipids, which shall serve as non-limiting examples).
  • the secondary amines of an 2, 3 diaminopropan-1-ol of an ABP (2) react with a bis-acrylate (18) to form a poly( ⁇ -amino ester) (19).
  • This reaction depending on the melting properties of ABPs, is, in certain embodiments of the invention, performed under solvent-free conditions so that the products can be used directly without need for solvent removal (Zugates et al., 2007).
  • Polymers formed from diacrylates may generally be degradable under conditions found in the body, polymers formed from bis-acryl-amides may be non-degradable.
  • the amine-bearing phospholipids may be reacted with acryloyl chloride to create ABP-bis-acrylamides (20). These molecules may, in certain embodiments of the invention, react with primary or secondary amines (21) to form polymers.
  • the groups thus introduced may, e.g., carry carboxylic acids as shown that can be modified after the polymerization.
  • ABP-bis-acrylamides (20) may undergo photopolymerization to form homo- polymers (23). This re-action can be performed by ad-mixing other reactive molecules such as e.g. cholesteryl acrylate or PEG-acrylates, producing hetero- polymers.
  • the 1 ,3 diaminopropan-2-ol (or 2,3 diaminopropan-1 -ol) based amine-bearing phospholipid type contains two reactive secondary amines.
  • structure (24) contains two reactive secondary amines.
  • the amine-bearing phospholipid (24) are reacted with a diacrylate (25) in solvent free conditions at 90 0 C over night or in the presence of an appropriate solvent such as but not limited to dimethyl sulfoxide, to produce a linear poly( ⁇ -amino ester) (26).
  • a library of linear polymers is so synthesized.
  • Bis-acrylamide phospholipids (27) can be transformed into polymers (29) by reaction with either primary or secondary amines (28).
  • acrylate-containing molecules can be UV-polymerized in the presence of e.g. the lipophilic radical initiator 2,2'-azobis(2-methylpropionitrile) (AIBN) (Nijst et al., 2007). Aliquots of liquid bis-acrylamide phospholipids (27) can be polymerized using, e.g., a built-in ballast ultraviolet lamp.
  • AIBN 2,2'-azobis(2-methylpropionitrile)
  • the pre-polymer can, e.g., be introduced between two microscopy slides spaced, e.g., 1 mm, away from each other (Zumbuehl et al., 2007) or polymeric sponges are produced (Chen et al., 2002).
  • the porogen leaching method involves the casting of a mixture of polymer solution and porogen in a mold, drying the mixture, followed by a leaching out of the porogen with water to generate the pores.
  • water soluble particulates such as salts and carbohydrates are used as the porogen materials.
  • the pore structures can be readily manipulated by controlling the property(ies) and/or fraction of the porogen. Such a process can be readily reproduced.
  • the polymers can be tested for their biocompatibility by seeding the surfaces with primary human foreskin fibroblasts, as we have done with other polymeric materials (Nijst et al., 2007). 2. APPLICATIONS OF THE ABPS IN BIOMEDICAL ENGINEERING
  • ABSPs amine-bearing phospholipids
  • ABP containing cationic ABPs or cationic vesicles (nanoparticles) for RNA interference therapy ABP cationic polymers for gene- therapy
  • ABP-cell membrane like materials onto which antimicrobial peptides are grafted as antimicrobial material ABP-cell membrane like materials onto which antimicrobial peptides are grafted as antimicrobial material.
  • RNA interference has enormous potential for the sequence-specific reduction of gene expression in medicine (Behlke 2006).
  • the synthesis of 1 ,200 structurally diverse lipid-like molecules (lipidoids) and their successful application in vitro and in vivo as vectors for siRNA has been reported (Akinc et al., 2008).
  • 53 members of this library were able to mediate high levels of uptake of small interfering RNA (siRNA against Firefly Luciferase) molecules into HeLa cells, surpassing the state-of-the-art transfection vector Lipofectamine2000TM.
  • siRNA against Firefly Luciferase small interfering RNA
  • Lipofectamine2000TM small interfering RNA
  • apolipoprotein B-specific siRNA nanoparticulate formulation resulted in a 30 day, up to 50% reduction of serum LDL cholesterol levels.
  • the lipidoid system may be advanced to carry both anti-VEGF (Vascular Endothelial Growth Factor) and anti- KSP (Kinesin Spindle Protein) siRNAs to treat solid liver tumors into clinical Phase I.
  • VEGF Vascular Endothelial Growth Factor
  • KSP Keratin Spindle Protein
  • the lipidoid nanoparticle system is one of the few if not the only existing systems for efficacious in vivo RNA interference therapy; others systems include another lipid-based system (Zimmerman et al., 2006) and a ⁇ -cyclodextrin-based polymer (Bartlett et al., 2007).
  • siRNA transfection vectors using the ABPs of the present invention may improve the toxicity profile, transfection efficiency and/or targeting of different tissues.
  • ABPs of certain embodiments of the invention are particularly well suited for siRNA transfection: in particular those embodiments that have the following features:
  • ABPs may be designed to be cationic or become cationic under acidic conditions (e.g., due to the two secondary amines at sn1 and sn2, through a proposed disturbance of the dipole-moment of the phosphocholine headgroup that may be parallel to the bilayer membrane normal instead of nearly perpendicular as depicted in Fig. 1 ), and/or through the introduction of cationic headgroups (Seeling et al., 2001).
  • ABPs may also be net cationic or anionic due to the different headgroups used (Zimmermann et al., 2006).
  • Cationic lipid-like molecules are classic DNA and siRNA delivery- vectors (Blagbrough et al., 2003; Miller, 1998). Cationic ABPs may form non-covalent complexes with negatively charged siRNAs, protecting the payload from harsh conditions found in blood.
  • ABPs have structures similar to natural phospholipids. As a result toxicity profile may be improved over existing structures such as the lipidoids discussed above.
  • the ABPs used for siRNA transfection are, e.g., tested using a standard protocol (Akinc et al., 2008):
  • the ABPs are, e.g., reconstituted in NaOAc buffer and added to a solution of siLUC (targeting luciferase expression, Alnylam Pharmaceuticals, USA) in the same buffer.
  • siLUC targeting luciferase expression, Alnylam Pharmaceuticals, USA
  • This complex is diluted in medium and added to HeLa cells stably transfected with firefly and renilla luciferase (Alnylam Pharmaceuticals).
  • the invention is also directed at formulating nanoparticles, e.g. containing siRNAs against multi-drug resistance transporters.
  • the ABPs may condense siRNAs and/or transfect cancer cells in vitro and/or in vivo.
  • Example II Gene Therapy- cationic polymeric ABPs
  • plasmid DNA is preferentially mediated by polymeric vectors
  • siRNA is preferably transfected by lipid-like materials (an effect that is possibly based on the size- difference between DNA and a 21mer RNA).
  • Polymeric materials for plasmid DNA (pDNA) delivery e.g. a series of poly( ⁇ -amino esters) that are transfecting COS-7 cells at levels of viral transfection have been developed (Zugates et al., 2007).
  • the amine-bearing phospholipids may, e.g., be transformed into cationic polymers, and may serve as vectors for pDNA transfection. Their cell-membrane mimicking properties improve, in certain embodiments of the invention, the toxicity profile over existing vectors such as poly(ethylene imine) or poly( ⁇ -amino esters) (Zugates et al., 2007).
  • the ABP polymers may be designed to be degradable (using diacrylates) or nondegradable (using diacrylamides). Hundreds of different diacrylates and diacrylamides are commercially available or can be rapidly synthesized. A combinatorial library of poly( ⁇ -amino phospholipids) is build to test each as a potential pDNA vector.
  • a standard high-throughput screening protocol is used (Zugates et al., 2007): Reconstituted ABPs in NaOAc buffer is preferably added to a buffer-solution of pDNA encoding Luciferase (pCMV-Luc; Elim Biopharmeceuticals, Hayward, CA). This lipoplex is added to COS-7 cells (ATCC, Manassas, VA) pre-plated in a 96-well format in DMEM containing 10% serum. The cells are incubated for 1 h, washed and left for 3 days. The transfection efficiency is analyzed using a Bright-Glo kit (Promega, Madison, Wl). In parallel, hits may be screened by FACS, MTT toxicity assay, light scattering (size) and zeta potential (net charge). The new non-viral materials have wide uses in basic biology as well as in medical applications.
  • Example III Antimicrobial Surfaces- grafting of polymeric surfaces
  • the material is biocompatible in vivo and does not cause hemolysis in human blood. Amphogel inoculated with Candida albicans and implanted in mice completely prevents fungal infections and mitigates biofilm formation.
  • Polymeric ABPs may serve in this context, as well as other contexts, as new core material that are preferably biocompatible and tunable, e.g. by incorporation of degradable ester bonds.
  • Antimicrobial peptides may be immobilized onto ABP surfaces. Antimicrobial peptides are potent small proteins used by a host's immune system to combat bacterial infections in multicellular eukaryotes and have the potential to be a important next generation of therapeutic agents (Loose et al., 2006).
  • polymeric ABPs due to their versatility and cell-membrane- similarity, form a platform for biomedical surfaces and coatings. The amine-bearing phospholipids are transformed into polymers, e.g.
  • Antimicrobial peptides bearing e.g., a C-terminal cysteine may be reacted with the activated acid.
  • these surfaces are antimicrobial, anti-biofouling, and/or non-hemolytic.
  • phospholipids and methods of the instant invention can be incorporated in the form of a variety of embodiments, such as pharmaceutical nanoparticles, cell-membrane mimetic materials, responsive surfaces, fluorescence markers, MRI/PET markers, consumer products, liquid crystals and electronics to name only a few in addition to those which are disclosed herein. It will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

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Abstract

Disclosed herein are amine-bearing phospholipids (ABP). The ABPs display many properties and functionalities of naturally occurring phospholipids, but also new properties and functionalities, such as the ability to polymerize while maintaining structural integrity, that may supplement or expand the functionalities of naturally occurring phospholipids.

Description

AMINE-BEARING PHOSPHOLIPIDS (ABPS), THEIR SYNTHESIS AND USE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application 60/983,930, filed October 30, 2007, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
This invention is directed at a new class of phospholipids, namely amine-bearing phospholipids (herein referred to as ABPs). Generally, in the ABPs of the present invention, two amines replace the ester moieties in naturally ocurring phospholipids. This new class of phospholipids has properties that find a wide variety of applications, in particular in the field of drug delivery such as in the production of vesicles for particle-based drug delivery, and in the production polymers for polymer surfaces, including biocompatible and antimicrobial surfaces and artificial cell membranes (cell membrane mimics).
BACKGROUND
The biological membrane is a complex barrier that regulates the expensive imbalance between an outside and an inside (Bloom et al, 1995). It comprises primarily proteins and lipids in a mixture adapted to the current local environment (Seeling et al., 2001 ). By nature's design, the phospholipids of a biological membrane serve primarily structural and signaling purposes.
Natural phospholipids generally comprise a polar hydrophilic head group, an uncharged hydrophobic portion and an interface-region based on a phosphoric acid substituted glycerol.
The publications and other materials, including patents, used herein to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated herein by reference. For convenience, the publications referenced in the following text by author and date, are listed alphabetically in the appended Bibliography.
Phospholipids have been crosslinked either in the alkyl-chain or the head-group region of the molecule to provide polymerization products for a variety of uses. For example, natural phospholipids have been cross-linked to form nanometer sized particles. See, e.g., U.S. Patent 5,560,960. However, the scaffold of natural phospholipids does not allow for chemical modifications without severely affecting and/or destroying their structural integrity, thus limiting their usage. U.S. Patent 5,540,935 (EP 0 657 463) describes phospholipid derivatives that are modified either at the polar hydrophilic head group or at the uncharged hydrophobic portion. The phospholipids are employed in reactive vesicles and functional substance-fixed vesicles as well as a drug delivery vesicles.
Certain amine-containing lipids have been described. In particular, WO 2006/138380 describes nitrogen-containing lipids prepared by conjugate addition of amines to acrylates, acrylamides, or other carbon-carbon double bonds conjugated to electron- withdrawing groups. Generally, these lipids have a hydrophobic half and a hydrophilic half. The hydrophobic portion is typically provided by fatty acid moieties attached to the acrylate, and the hydrophilic portion is provided by the esters, amines, and side chains of the amine. These lipids may be prepared by the addition of a primary amine to a double bond conjugated with an electron withdrawing groups such as a carbonyl moiety. They may serve the delivery of nucleic acids in gene therapy as well as in the packaging and/or delivery of diagnostic, therapeutic, and prophylactic agents.
The synthesis of lipid-analogs has focused on head-group modifications, acyl chain modifications, and ester-to-amide or ester-to-ether modifications (see e.g. www.avantilipids.com). Also, a great variety of new cationic lipids have been synthesized that lack the phosphate ester motive altogether (Blagbrough et al, 2003; Akinc, 2008).
There is a need for phospholipids that overcome shortcomings of known phospholipids.
In particular, there is a need for drug delivery systems, in particular, for target-specific gene delivery systems that can overcome systemic and/or cellular barriers. There is also a need for biocompatible surfaces, such as antimicrobial surfaces for the medical industry, e.g., for use on invasive devices such as catheters. SUMMARY OF THE INVENTION
The invention is, in one embodiment, directed towards an amine-bearing phospholipid (ABP) having: a head group, e.g., a polar head group, an hydrophobic portion, e.g. an uncharged hydrophobic portion, and an interface-region comprising an amine group substituted propanol backbone, in particular a 2,3-diaminopropan-1 -ol (also commonly referred to as just "1 ,2-diaminopropanol") or a 1 ,3- diaminopropan-2-ol (also commonly referred to as just "1 ,3-diaminopropanol") backbone.
A typical ABP according to the present invention can be depicted as follows:
Figure imgf000004_0001
The headgroup, while shown to be at a 180 degree angle relative the hydrophobic portion, may also be at or almost at a right angle to the hydrophobic moieties of the hydrophobic portion.
The amine-bearing phospholipid may correspond to any of the formulas referred to herein. As the person skilled in the art will readily understand any 2,3- diaminopropan-1 -ol shown or described can be replaced by a 1 ,3- diaminopropan-2- ol and vice versa.
In particular, the present invention is directed at an amine-bearing phospholipid (ABP) comprising: an interface region comprising an amine group substituted propanol backbone, preferably a 2,3-diaminopropan-1-ol or a 1 ,3- diaminopropan-2-ol, providing at least three attachment regions, a hydrophilic head group, a first and second hydrophobic portion, preferably a first and second uncharged hydrophobic portion, wherein the head group comprises and is covalently attached via a phosphate ester bond to a first one of the attachment regions, and the first hydrophobic portion comprises two hydrophobic moieties each being covalently attached to an amine of a second one of the attachment regions, the second hydrophobic portion comprises two hydrophobic moieties each being covalently attached to an amine of a third one of the attachment regions, wherein, in an aqueous surrounding, the head group extends from the interface region into a first direction and the hydrophobic portions extends from the interface region into a second direction.
The ABPs of the present invention may, in particular, have the following formulas: (I)
Figure imgf000005_0001
(II)
Figure imgf000005_0002
wherein head group A may be one of the following: a phosphatic acid or a phosphate ester substituted group such as: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, phosphate ester substituted electrophile, such as an activated ester including N- hydroxysuccinimic ester, or an acid chloride or an halogenide, a phosphate ester substituted nucleophile such as a thiol or an amine, a phosphate ester substituted fluorescent group such as fluoresceine, a phosphate ester substituted radioactively labeled group, a phosphate ester substituted alkyne, or a phosphate ester substituted azide;
and the hydrophobic moieties may be Y1, Y2 , Z1 and Z2 , wherein,
W may be a stereocenter that is R, S or racemic; Y1 may be:
• H;
• optionally substituted C1 -30 alkyl; • an optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
• an optionally substituted C1-30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds; or
• a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group or a linear alkyl group or an aryl group;
Y2 may be:
• H;
• optionally substituted C1 -30 alkyl;
• an optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
• an optionally substituted C1-30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds;
• a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group; or • a pre-polymer group polymerizable in light or by heat, optionally with a radical initiator, wherein the group is preferably -C(O)-C(CH3)=CH2 or -C(O)- CH=CH2;
Z1 may be:
• H;
• optionally substituted C1 -30 alkyl;
• an optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds; • an optionally substituted C1-30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds; or
• a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group; and
Z2 may be:
• H;
• optionally substituted C1 -30 alkyl;
• optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds; • optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds;
• a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group; or
• a pre-polymer group polymerizable in light or by heat, optionally with a radical initiator, wherein the group is preferably -C(O)-C(CH3)=CH2 or -C(O)-
CH=CH2; with the proviso that at least one, preferably two or three of Y1, Y2 , Z1 and Z2 are unequal H.
Y , Y , Z and Z may be selected from one or more of the Table III moieties.
The ABPs of the present invention may, in particular have the following formulas: (III)
Figure imgf000008_0001
(VII)
Figure imgf000009_0001
(Xl)
Figure imgf000010_0001
(XIII)
Figure imgf000010_0002
(XV)
Figure imgf000011_0001
(XIX)
Figure imgf000012_0001
wherein,
W may be as above; n may be an integer between n=0 and n=6;
X1 may be:
• H;
• alkyl- , preferably methyl-, ethyl-, propyl-, isopropyl-;
• aryl-;
• a fluorescent reporter group which may, as any other fluorescent reporter group set forth herein, preferably be fluorescein, alexafluor 488, Tokio green, wherein the reporter group is bound to the amine of the interface region directly via an amide bond or via a spacer group, preferably an alkyl-, aryl- or PEG spacer group;
• a non-fluorescent reporter group, preferably a spin label or a radioactive label;
• an electrophilic or nucleophilic group, preferably an Λ/-hydroxysuccinimide, an anhydride, or an /V-methylisatoic anhydride, a methylisothiocyanate, a methylisocyanate, or methylthiourea, 3-[4-(trimercapto)phenyl]propionyl, 4- benzyloxybenzaldehyde, 2-chlorotrityl chloride, sulfonic acid, morpholinomethyl, piperidinomethyl, piperazinomethyl, acrylate, methyl- acrylate, styrene, acrylamide, or methyl-acrylamide;
• CH3-[CH2Jn-SH with n=0-6;
• CH3-[CH2Jn-NH2 with n=0-6; • CH3-[CH2Jn-NH-CH2-[CHg]0-NH2 with n=0-6; 0=0-6;
• CH3-[CH2Jn-NH-CH2-[CH2]O-NH-CH3-[CH2Jp-NH2 with n=0-6; o=0-6; p= 0-6;
• CH3-[CH2Jn-NH-CH3-[CH2]O-NH-CH2-[CH2Jp-NH-CH2-[CH2Jq-NH2 with n=0-6; o=0-6; p= 0-6; q=0-6;
• CH3-[CH2Jn-OH with n=0-6; • CH3-[CH2Jn-(O-CH2-[CH2J0) P-OH with n=0-6; o=0-6; p= 0-200;
• CH3-[CH2Jn-(O-CH2-[CH2J0) P-O-CH3 with n=0-6; o=0-6; p= 0-200;
• CH3-[CH2Jn-(O-CH2-[CH2J0) P-O-CH2-CH3 with n=0-6 ; o=0-6 ; p= 0-200 ;
• CH3-[CH2Jn-C-C-CH with n=0-6;
• =N=N; • =C=S;
• =C=O; or
• a metal complexing group, preferably ethylenediamine tetraacetate, wherein the group is bound to the amine N1 either directly or via a linker, preferably an alkyl chain or a polyethylene glycol chain;
X2 may be:
• H;
• alkyl-, preferably methyl-, ethyl-, propyl- or isopropyl-;
• aryl-; • CH3-[CH2Jn-SH with n=0-6;
• CH3-[CH2Jn-NH2 with n=0-6;
• CH3-[CH2Jn-NH-CH2-[CH2J0-NH2 with n=0-6 and o=0-6;
• CH3-[CH2Jn-NH-CH2-[CH2J0-NH-CH2-[CH2Jp-NH2 with n=0-6, o=0-6 and p= 0-6;
• CH3-[CH2Jn-NH-CH2-[CH2J0-NH-CH2-[CH2Jp-NH-CH2-[CH2Jq-NH2 with n=0-6, o=0-6, p= 0-6 and q=0-6;
• CH3-[CH2Jn-OH with n=0-6;
• CH3-[CH2Jn-(O-CH2-[CH2J0) p-OH with n=0-6, o=0-6 and p= 0-200;
• CH3-[CH2Jn-(O-CH2-[CH2J0) p-O-CH3 with n=0-6, o=0-6 and p= 0-200;
• CH3-[CH2Jn-(O-CH2-[CH2J0) P-O-CH2-CH3 with n=0-6, o=0-6 and p= 0-200; or • CH3-[CH2Jn-C-C-CH with n=0-6. • X3 may be, if present, selected from:
• alkyl- such as, but not limited to, methyl-, ethyl-, or propyl-; or
• aryl-;
or may have one of the following formulas:
(XXI)
Figure imgf000014_0001
(XXII)
Figure imgf000014_0002
(XXIII)
(XXIV)
Figure imgf000015_0001
(XXV)
Figure imgf000015_0002
(XXVI)
Figure imgf000016_0001
wherein: W may be as above;
n may be as above;
Y1, Y2, Z1 and Z2 may be as above;
V may be
• a hydroxyl group;
• an amine; or
• an amide;
and X may be:
• a thiol;
• an ether;
• an ester;
• an amide;
• an alkene; or
• an alkyne;
or may have one of the following formulas: (XXVII)
Figure imgf000017_0001
(XXVIII)
Figure imgf000017_0002
Figure imgf000017_0003
Figure imgf000018_0001
wherein,
W may be as above;
m may be an integer between n=0 and n=6;
n may be as above;
Y1, Y2, Z1 and Z2 may be as above.
The sugar phosphate ester substituted sugar may be a simple sugar or may be part of a simple glucose series or a globo, ganglio, lacto, neolacto, isoglobo, mollu, arthro or gala series.
The head group of said ABP may be labeled, preferably fluorescently, may be deuterated, radioactively labeled or spin labled.
One or more positions of the sugars set forth above may be substituted, preferably with a sugar including a substituted sugar, even more preferably with a simple sugar.
A dimer or polymer as described herein may be a bolaamphiphile in which the ABPs are linked to one another via one or both their hydrophobic portions.
A dimer or polymer as described herein may comprise one or more linkers, in particular sugar or polysugar linkers. The ABPs may be crosslinked, preferably via a linker, most preferably one or more of the following linkers:
• an epichlorohydrin
• a divinylbenzene • a diisocyanate
• a styrene
Figure imgf000019_0001
- wherein X may be O or N;
- wherein Y may be O or N;
- wherein Z may be H or CH3; and
- wherein n may be an integer from 0 to 10; or any of the crosslinker(s) set forth elsewhere herein.
Any of the dimers set forth herein may be a heterodimer.
The present invention is also directed at a transfection method comprising providing at least one ABP as set forth herein; admixing the at least one ABP with at least one nucleic acid; and transfecting a cell, in vitro or in vivo.
The nucleic acid may be at least one siRNA, miRNA or pDNA. The cell may comprise a gene whose expression is targeted by, e.g., said siRNA. The ABP may be part of a micro- or nanoparticle. The ABPs may form degradable or nondegradable cationic polymers, preferably cationic polymers as set forth herein.
The invention is also directed at polymeric surfaces comprising ABPs as set forth herein and polymerized as set forth herein. The polymeric surface is preferably biocompatible, even more preferably antimicrobial. The polymeric surface may comprise an agent covalently attached to a reactive group of a ABP, or a reactive group of a further molecule, wherein an attachment is a pre- or postpolymerization attachment. The agent may be at least one antimicrobial agent, preferably a peptidic antimicrobial agent.
The invention is also directed at the producing of such a polymeric surface: Here a carboxylic acid, an activated carboxylic acid (active ester), an aldehyde, a ketone, an amine, a hydrazine, an azide or an alkine may be admixed prior to polymerization to a solution comprising one or more types of ABPs.
The invention is also directed at a method for linking one or more types of ABPs: Here the ABPs are reacted to comprise one or more, preferably two, acrylamide and/or methylacrylamide groups, and the resulting acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs are polymerized via UV light, optionally in presence of a polymerization initiator, preferably a radical initiator or by heat.
The ABPs may be reacted to comprise one or more, preferably two, acrylamide and/or methylacrylamide groups to provide acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs, comprising reacting terminal double bonds of said acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs with a further amine, and polymerizing the reaction product. The amines may be primary or secondary amines, preferably an amine selected from (1) to (94) of Table I:
Table I:
Figure imgf000021_0001
The ABPs form linear polymers, 2d-sheets or 3d polymers. The ABPs may be chemically modified after polymerization. Prior to polymerization proteins and/or natural lipids, in particular phospholipids, may be admixed.
The ABPs of the present invention including the dimers and polymers created therefrom have a wide variety of uses in particular in the medical field, but also for research and development. They can, for example, be employed as the production of drug delivery vesicles and/or vehicles, in particular nucleic acid transfection vesicles and/or vehicle or vesicles for non nucleic acid drug delivery. However, a wide variety of other uses are contemplated including, but not limited to, uses as cell- membrane mimetic materials, responsive surfaces, fluorescence markers, MRI/PET markers, consumer products, liquid crystals and in electronics.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows in A a naturally occurring phospholipid and in B a amine-bearing phospholipid (ABP) according to the present invention. In C, the Figure shows the integration of an APB into a phospholipids bilayer. Fig. 2 depicts an example of a synthesis path for an ABP. Fig. 3 depicts an alternative synthesis path for an ABP. Fig. 4 depicts a further synthesis path for an ABP. Fig. 5 depicts a synthetic route for the establishment of a ABP library. Fig. 6 shows under I to III reactions that lead to the polymerization of APBs into different polymerization products.
Fig. 7 depicts under IV to Vl reactions that lead to the polymerization of APBs into further polymerization products.
DETAILED DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS OF THE INVENTION
The amine-bearing phospholipids of the present invention and their polymers may address one or more of said needs and/or other needs which will become apparent from the following disclosure. The amine-bearing phospholipids presented herein are a new class of phospholipids bearing at least two free amines instead of ester moieties of natural phospholipids in the interface region. One example is shown in Fig. 1 B, while Fig. 1 A shows a natural occurring phospholipid.
An "amine bearing phospholipid" (ABP) according to the present invention comprises an interface region that is covalently attached to a head group and at least one, preferably two hydrophobic portion. The ABP also contains a phosphate ester bond, which provides the link between the interface region to a head group. The head group extends, in an aqueous surrounding, from the interface region in one direction and the hydrophobic moiety/(ies) of the hydrophobic portion(s) into a second direction. The angles between the headgroups and hydrophilic portions preferably include about 180 degrees, about 150 degrees, about 120 degrees, about 100 degrees or about 90 degrees. The angles may vary over time and may alter in different surroundings.
The ABPs of the present invention have many properties and/or functionalities of naturally occurring phospholipids, such as the ability to self assemble, but provide additional properties and/or functionalities discussed in more detail herein.
A "head group" according to the present invention find is counterpart in what is generally referred to as a "head" in context of natural phospholipids (also referred to herein as naturally occurring phospholipids), both functionally and structurally. The head group is generally hydrophilic so that it can interact with an aqueous surrounding. As in a phospholipid, the head group comprises and is attached to the so called "interface region" via a phosphate ester bond. The head group often comprises an elongated molecule that may be branched or unbranched. The head group may also contain a ring structure that is substituted, preferably with hydrophilic groups and, often with amine or amide groups. However, it may, for example, be a short branched, generally hydrophilic molecule. Examples of suitable headgroups include phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol, a phosphatic acid substituted sugar, or a phosphate ester substituted polyethyleneglycol, or a phosphate ester substituted electrophile such as an activated ester like Λ/-hydroxysuccinimic ester, or an acid chloride or a halogenide, or a phosphate ester substituted nucleophile such as a thiol, or an amine, or a phosphate ester substituted fluorescent group such as fluoresceine or a radioactively labeled group, or a phosphate ester substituted alkyne, or a phosphate ester azide and headgroups described in Fahy et al. (Fahy et al. 2005), whose disclosure of headgroups is specifically incorporated herein by reference. In particular, the sugars as found in headgroups as described by Fahy et al.'s Table 7 and reflected in Table I herein are incorporated herein by reference:
Table II. Examples for sugars (including substituted sugars) in headgroups
"Simple sugars" such as GIu
"Simple glucose series" (GlcCer, LacCer, etc.)
GalNAcβ1-3Galα1 -4Galβ1 -4Glc- ("globo series")
GalNAcβ1-4Galβ1 -4Glc- ("ganglio series")
Galβ1-3GlcNAcβ1 -3Galβ1 -4Glc- ("lacto series")
Galβ1-4GlcNAcβ1 -3Galβ1 -4Glc- ("neolacto series")
GalNAcβ1-3Galα1 -3Galβ1 -4Glc- ("isoglobo series")
GlcNAcβ1 -2Manα1 -3Manβ1-4Glc- ("mollu series")
GalNAcβ1-4GlcNAcβ1 -3Manβ1 -4Glc- ("arthro series")
GaI- ("gala series")
The prefix "poly" as used herein in the context of sugars refers to more than one that is 2, 3, 4, 5, 6, 7, 8, 9, 10, about 20, about 30, about 40, about 50, about 100 or more.
The terms "admixed" and mixed are used interchangeably herein.
A "hydrophobic portion" according to the present invention finds it counterpart in naturally occurring phospholipids in what is generally referred to as a "hydrophobic portion or lipophilic portion" in this context, both functionally and also structurally. However, the hydrophobic portion of the present invention is covalently linked to the interface region via an amine/amide bond. Functionally, the hydrophobic portion can interact, with other hydrophobic molecules such as transmembrane proteins and hydrophobic portions of phospholipids and/or other ABPs. In certain embodiments, it contains reactive hydrophobic groups so that polymerization of ABPs via the hydrophobic portion is possible. The hydrophobic portion also allows the ABP to integrate into existing membranes or to form membranes and/or membrane like structures. Structurally, the hydrophobic portion often contains, as hydrophobic moiety, chains of hydrophobic molecules, which may contain one or more double and triple bonds. These hydrophobic moieties are a hydrophobic tails or just tails. In a preferred embodiment, the tails are unbranched. In another preferred embodiment, the chains contain at least 5 carbon atoms, preferably at least 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, more preferably 16, 17, 18, 19, 20, 25 or 30 carbon atoms, wherein an even number of carbon atoms is preferred. In certain embodiments, one, two, three, four, five or more double or triple bonds are present. Alternatively or additionally, rather than chains, molecules such as a cholesterol or ergosterol may constitute or be part of the hydrophobic moiety. These moieties may be attached to the amine via a linker such as a linear alkyl or aryl group. However, in certain embodiments, hydrophobic moieties, in particular -C(O)-C(CH3)=CH2 or -C(O)-CH=CH2, that may be polymerized in the presence of light or by heat, in the presence or absence of a radical initiator, may be employed. In a preferred embodiment, a hydrophobic portion contains two hydrophobic moieties. These moieties are covalently attached to the amine of the interface region. As the person skilled in the art will readily recognize, different hydrophobic moieties may be combined within one hydrophobic portion or an ABPs.
Some examplatory hydrophobic moieties which take the form of hydrophobic chains are depicted in Table III: Table III. Examples of hydrophobic chains in hydrophobic portions
Figure imgf000025_0001
The "interface region" finds its counterpart in the interface region in naturally occurring phospholipids, namely the phosphorylated diglyceride in phospholipids. Preferably, it comprises an amine group substituted backbone molecule, most preferably it comprises an aminopropanol, preferably a diaminopropanol, namely a 2, 3- diaminopropan-1-ol or a 1 , 3- diaminopropan-2-ol backbone. The phosphate group provides a first attachment region in the context of the present invention, generally to the head group, while the one or two amine groups provide a second and third attachment region for the hydrophobic portion, which comprises hydrophobic moieties as described above. The term "alkyl" as used herein refers to saturated, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert- butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl. "Alkene" and "alkylene" refers to counterparts having at least one double or triple bond.
The terms "aryl" and "heteroaryl", as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl", as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
The terms "substituted," whether preceded by the term "optionally" or not, and substituent, as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents may also be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted with fluorine at one or more positions). A substituted sugar according to the present invention refers to a sugar wherein one of the hydroxyl groups is substituted by an amine, an alkyl-amine or an alkyl-amide. A substituted sugar also refers to a sugar linked via a glycosidic bond to another sugar.
A "bolaamphiphile" is, according to the present invention, an amphiphilic molecule that has a hydrophilic group at two ends. This introduction of a "second" head group generally induces a higher solubility in water, an increase in the critical micelle concentration (cmc), and a decrease in aggregation number. The aggregate morphologies of bolaamphiphiles include spheres, cylinders, disks, and vesicles. Bolaamphiphiles and other lipids including other ABPs may be be admixed according to the present invention.
Any reference to, e.g., n=0-6 or n=0-10 m=0-6, o=0-6, p=0-6 shall be understood to specifically include n/m/o/p= 1 , 2, 3, 4 or 5 and 6, 7, 8 or 9, respectively, while a reference to p=0-200 shall be interpreted accordingly, but ranges are also included, in particular, 40-200, 40-100, 80-200, 130-200 and 160-200.
As used herein, the term "labeled" is intended to mean that a compound has at least one element, isotope, or chemical compound attached to enable the detection of the compound. In general, labels typically fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes, including, but not limited to, 2H (D,
"deuterated"), 3H, 32P, 35S, 67Ga, 99mTc (Tc-99m), 111In, 1231, 1251, 169Yb and 186Re; b) immune labels, which may be antibodies or antigens, or aptamers, which may be bound to enzymes (such as horseradish peroxidase) that produce detectable agents; and c) colored, luminescent, phosphorescent, or fluorescent dyes. It will be appreciated that the labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound that is being detected. In certain embodiments of the invention, photoaffinity labeling is utilized for the direct elucidation of intermolecular interactions in biological systems. A variety of known photophores can be employed, most relying on photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (See, Bayley, H., Photogenerated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam). In certain embodiments of the invention, the photoaffinity labels employed are o-, m- and p- azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to 4-azido-2,3,5,6- tetrafluorobenzoic acid. In preferred spin labels according to the present invention nitroxyl radical (N-O) is incorporated into a heterocyclic ring. Labels according to the present invention also include "caged groups", that are labels that are protected and can be deprotected under certain conditions, e.g. pH change or a light pulse, e.g. inside the animal (including human) body for most in vivo uses or on in a cell for in vitro and in vivo.
A "pre-polymer group" allows for polymerization of molecules, e.g., in light or by heat, optionally with a radical initiator. "By heat" refers preferably, though not exclusively, to heat ranges of between about 300C and 1000C, including between about 35 °C and 1000C, between about 400C and 100°C, between about 500C and 1000C, between about 600C and 1000C and between about 800C and 100 °C.
"Radical Initators" include, but are not limted to, organic peroxide molecules, or other molecules containing an 0-0 single bond or by reacting oxygen with ethene, such as the pre-polymer group -C(O)-C(CH3)=CH2, -C(O)-CH=CH2.
A racemic mixture, or "racemate", is a mixture that contains left (S) - and right (R) - handed enatiomer of a chiral molecule.
As used herein "transfection" is the process of introducing nucleic acids into cells by non-viral methods. ABP mediated transfection includes in partular the intoduction of the nucleic acid using the ABPs as "carriers" or "vehicles" across cell membranes. Included are in particular the formation of ABP containing vesciles which can fuse with cell membranes to carry the nucleic acids across the cell membrane. As the person skilled in the art will appreciate, any kind of nucleic acid can be transported using this method. In certain preferred embodiments, the nucleic acid is a siRNA, a miRNA or a pDNA.
The term "liposome" refers to an aqueous lipid-containing suspension of multi- layered (consisting of at least a double layer of lipid) generally spherical accumulations of lipid molecules which are formed by mechanically mixing a dry lipid in water.
Complexes comprising cationic lipids and nucleic acid molecules are referred to as "lipoplexes" and form part of the present invention. Lipoplexes can spontaneously form in the presence of negatively charged pDNA (plasmid DNA). ABPs allow for the attachment of, e.g., cell specific targeting molecules and thus target delivery of, e.g. the pDNA.
ABPs can form vesicles for drug delivery, which includes delivery of nucleic acid based drugs and non-nucleic acid based drugs. Such vesicles generally have a rounded shape and form discrete drug delivery packets. The process is referred to herein as "vesicle based drug delivery." A "vescile" which inlcudes the term "nanoparticle" or "miicroparticle", is, in the context of the present invention, a selfassembled and/or polymerized, in an aqueous solution rounded, generally spherical structure that engulfs, e.g., a drug and thus becomes a carrier of the drug. Selfassembled vesciles can deliver a drug into a cell by merging with its cell membrane. Targeting molecules for, e.g., specific cell surface receptors may be for example be attached directly or indirectly to a secondary amine of the ABP. A vesicle may, on the one hand, display the targeting molecules on its surface and, on the other hand, shield the drug from its surrounding and vice versa. The targeting molecule will interact, e.g. bind, its target, e.g., a surface antigen expressed on the target cell and the vesicle will fuse with the cell mebrane of the target cell to release the drug into the lumen of the target cell. Drug delivery of drugs that are subject to degradation and highly toxic drugs are of particular interest in this context. Drug delivery according to the present invention to an individual, in particular a peson in need thereof, can be by any route, including intravenously, parenterally, orally, intramuscularly, intrathecal^ or as an aerosol. The mode of delivery will depend on the ABP vehicle/vesicle used, the drug delivered and the desired effect. A skilled artisan will readily know the best route of administration for a particular treatment in accordance with the present invention. The effective amount will depend on the route of administration and the treatment indicated, and can readily be determined by a skilled artisan in view of current treatment protocols.
"Non-nucleic acid based drugs" that can be encapsulated by the vesicles of the present invention include, but are not limited to pharmacological agents, and therapeutic agents. For example, non-nucleic acid based drugs suitable for use in the present invention include, but are not limited to, peptides, and particularly small peptides; hormones, and particularly hormones which are susceptible to chemical cleavage by acids and enzymes in the gastrointestinal tract; polysaccharides, and particularly mixtures of muco-polysaccharides; carbohydrates; lipids; or any combination thereof. Further examples include, but are not limited to, human growth hormones; bovine growth hormones; growth releasing hormones; interferons; interleukin-1 ; insulin; heparin, and particularly low molecular weight heparin; calcitonin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropin releasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium or disodium chromoglycate); vancomycin or desferrioxamine (DFO).
A "nucleic acid" according to the present invention is any molecule composed of chains of monomeric nucleotides and includes desoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both single standed (ss) as well as double stranded (ds). Particular preferred is plasmid DNA (pDNA) that can, e.g., be used for gene therapy and RNAs that can be employed for RNA interference (RNAi). The latter include, for example, small interfering RNAs (siRNAs), which usually comprise 20 to 25 nucleotide long double-stranded RNA molecules. Also included microRNAs (miRNAs) are an abundant class of small single-stranded non-coding RNAs (19-30 nucleotides long) that serve widespread functions in post-transcriptional gene silencing.
The term "biocompatible" as used herein, means that the ABP containing compound or structure, when introduced into a human patient, will not result in any severe degree of unacceptable toxicity, including allergic responses and inflammation. Preferably the ABP containing compounds or structures are inert.
A "biocompatible surface" according to the present invention is a surface that can be employed in cell and molecular biology without substantially affecting biological processes, e.g., as a surface for cell, e.g. stem cell, growth. Such a surface may comprise releasable growth factors.
A "antimicrobial agent" refers herein as an agent having an activity which is capable of killing or inhibiting growth of microbial cells. In the context of the present invention the term "antimicrobial" is intended to mean that there is a bactericidal and/or a bacteriostatic ("antibacterial") and/or fungicidal and/or fungistatic (antifungal") effect and/or a virucidal ("antiviral") effect. "Peptidic" antimicrobial agents, whose main functional moiety is a peptide or protein, are preferred.
Classes of antifungal agents include, but are not limited to, allylamines, azoles, pyrimidines, tetraenes, thiocarbamates, sulfonamides, glucan synthesis inhibitors. Allylamines include, for example, amorolfine, butenafine, naftifine and terbinafine. Azoles include, for example, ketoconazole, fluconazole, elubiol, econazole, econaxole, itraconazole, isoconazole, imidazole, miconazole, sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole, terconazole, butoconazole, thiabendazole, voriconazole, saperconazole, sertaconazole, fenticonazole, posaconazole, bifonazole, flutrimazole. Polyenes include, for example, nystatin, pimaricin and amphotericin B. Pyrimidines include, for example, flucytosine. Tetraenes include, for example, natamycin. Thiocarbamates include, for example, tolnaftate. Sulfonamides include, for example, mafenidine. Other antifungal drugs include ciclopirox and ciclopirox olamine. The polyene macrolide amphotericin B (AmB), is particularly preferred.
Antibacterial agents include but are not limited to aminoglycosides, β lactam agents, cephalosporins, macrolides, penicillins, quinolones, sulfonamides, and tetracyclines. Examples of anti-bacterial agents include but are not limited to: Acedapsone, Acetosulfone Sodium, Alamecin, Alexidine, Amdinocillin Clavulanate Potassium, Amdinocillin, Amdinocillin Pivoxil, Amicycline, Amifloxacin, Amifloxacin Mesylate, Amikacin, Amikacin Sulfate, Aminosalicylic acid, Aminosalicylate sodium, Amoxicillin, Amphomycin, Ampicillin, Ampicillin Sodium, Apalcillin Sodium, Apramycin,
Aspartocin, Astromicin Sulfate, Avilamycin, Avoparcin, Azithromycin, Azlocillin, Azlocillin Sodium, Bacampicillin Hydrochloride, Bacitracin, Bacitracin Methylene Disalicylate, Bacitracin Zinc, Bambermycins, Benzoylpas Calcium, Berythromycin, Betamicin Sulfate, Biapenem, Biniramycin, Biphenamine Hydrochloride, Bispyrithione Magsulfex, Butikacin, Butirosin Sulfate, Capreomycin Sulfate, Carbadox, Carbenicillin Disodium, Carbenicillin lndanyl Sodium, Carbenicillin Phenyl Sodium, Carbenicillin Potassium, Carumonam Sodium, Cefaclor, Cefadroxil, Cefamandole, Cefamandole Nafate, Cefamandole Sodium, Cefaparole, Cefatrizine, Cefazaflur Sodium, Cefazolin, Cefazolin Sodium, Cefbuperazone, Cefdinir, Cefditoren Pivoxil, Cefepime, Cefepime Hydrochloride, Cefetecol, Cefixime, Cefinenoxime Hydrochloride, Cefinetazole, Cefinetazole Sodium, Cefonicid Monosodium, Cefonicid Sodium, Cefoperazone Sodium, Ceforanide, Cefotaxime, Cefotaxime Sodium, Cefotetan, Cefotetan Disodium, Cefotiam Hydrochloride, Cefoxitin, Cefoxitin Sodium, Cefpimizole, Cefpimizole Sodium, Cefpiramide, Cefpiramide Sodium, Cefpirome Sulfate, Cefpodoxime Proxetil, Cefprozil, Cefroxadine, Cefsulodin Sodium, Ceftazidime, Ceftazidime Sodium, Ceftibuten, Ceftizoxime Sodium, Ceftriaxone Sodium, Cefuroxime, Cefuroxime Axetil, Cefuroxime Pivoxetil, Cefuroxime Sodium, Cephacetrile Sodium, Cephalexin, Cephalexin Hydrochloride, Cephaloglycin, Cephaloridine, Cephalothin Sodium, Cephapirin Sodium, Cephradine, Cetocycline Hydrochloride, Cetophenicol, Chloramphenicol, Chloramphenicol Palmitate, Chloramphenicol Pantothenate Complex, Chloramphenicol Sodium Succinate, Chlorhexidine Phosphanilate, Chloroxylenol, Chlortetracycline Bisulfate,
Chlortetracycline Hydrochloride, Cilastatin, Cinoxacin, Ciprofloxacin, Ciprofloxacin Hydrochloride, Cirolemycin, Clarithromycin, Clavulanate Potassium, Clinafloxacin Hydrochloride, Clindamycin, Clindamycin Dextrose, Clindamycin Hydrochloride, Clindamycin Palmitate Hydrochloride, Clindamycin Phosphate, Clofazimine, Cloxacillin Benzathine, Cloxacillin Sodium, Cloxyquin, Colistimethate, Colistimethate Sodium, Colistin Sulfate, Coumermycin, Coumermycin Sodium, Cyclacillin, Cycloserine, Dalfopristin, Dapsone, Daptomycin, Demeclocycline, Demeclocycline Hydrochloride, Demecycline, Denofungin, Diaveridine, Dicloxacillin, Dicloxacillin Sodium, Dihydrostreptomycin Sulfate, Dipyrithione, Dirithromycin, Doxycycline, Doxycycline Calcium, Doxycycline Fosfatex, Doxycycline Hyclate, Doxycycline
Monohydrate, Droxacin Sodium, Enoxacin, Epicillin, Epitetracycline Hydrochloride, Ertapenem, Erythromycin, Erythromycin Acistrate, Erythromycin Estolate, Erythromycin Ethylsuccinate, Erythromycin Gluceptate, Erythromycin Lactobionate, Erythromycin Propionate, Erythromycin Stearate, Ethambutol Hydrochloride, Ethionamide, Fleroxacin, Floxacillin, Fludalanine, Flumequine, Fosfomycin,
Fosfomycin Tromethamine, Fumoxicillin, Furazolium Chloride, Furazolium Tartrate, Fusidate Sodium, Fusidic Acid, Gatifloxacin, Genifloxacin, Gentamicin Sulfate, Gloximonam, Gramicidin, Haloprogin, Hetacillin, Hetacillin Potassium, Hexedine, Ibafloxacin, Imipenem, Isoconazole, Isepamicin, Isoniazid, Josamycin, Kanamycin Sulfate, Kitasamycin, Levofloxacin, Levofuraltadone, Levopropylcillin Potassium, Lexithromycin, Lincomycin, Lincomycin Hydrochloride, Linezolid, Lomefloxacin, Lomefloxacin Hydrochloride, Lomefloxacin Mesylate, Loracarbef, Mafenide, Meclocycline, Meclocycline Subsalicylate, Megalomicin Potassium Phosphate, Mequidox, Meropenem, Methacycline, Methacycline Hydrochloride, Methenamine, Methenamine Hippurate, Methenamine Mandelate, Methicillin Sodium, Metioprim, Metronidazole Hydrochloride, Metronidazole Phosphate, Mezlocillin, Mezlocillin Sodium, Minocycline, Minocycline Hydrochloride, Mirincamycin Hydrochloride, Monensin, Monensin Sodium, Moxifloxacin Hydrochloride, Nafcillin Sodium, Nalidixate Sodium, Nalidixic Acid, Natamycin, Nebramycin, Neomycin Palmitate, Neomycin Sulfate, Neomycin Undecylenate, Netilmicin Sulfate, Neutramycin,
Nifuradene, Nifuraldezone, Nifuratel, Nifuratrone, Nifurdazil, Nifurimide, Nifurpirinol, Nifurquinazol, Nifurthiazole, Nitrocycline, Nitrofurantoin, Nitromide, Norfloxacin, Novobiocin Sodium, Ofloxacin, Ormetoprim, Oxacillin Sodium, Oximonam, Oximonam Sodium, Oxolinic Acid, Oxytetracycline, Oxytetracycline Calcium, Oxytetracycline Hydrochloride, Paldimycin, Parachlorophenol, Paulomycin, Pefloxacin, Pefloxacin Mesylate, Penamecillin, Penicillin G Benzathine, Penicillin G Potassium, Penicillin G Procaine, Penicillin G Sodium, Penicillin V, Penicillin V
Benzathine, Penicillin V Hydrabamine, Penicillin V Potassium, Pentizidone Sodium, Phenyl Aminosalicylate, Piperacillin, Piperacillin Sodium, Pirbenicillin Sodium, Piridicillin Sodium, Pirlimycin Hydrochloride, Pivampicillin Hydrochloride, Pivampicillin Pamoate, Pivampicillin Probenate, Polymyxin B Sulfate, Porfiromycin, Propikacin, Pyrazinamide, Pyrithione Zinc, Quindecamine Acetate, Quinupristin, Racephenicol, Ramoplanin, Ranimycin, Relomycin, Repromicin, Rifabutin, Rifametane, Rifamexil, Rifamide, Rifampin, Rifapentine, Rifaximin, Rolitetracycline, Rolitetracycline Nitrate, Rosaramicin, Rosaramicin Butyrate, Rosaramicin Propionate, Rosaramicin Sodium Phosphate, Rosaramicin Stearate, Rosoxacin, Roxarsone, Roxithromycin, Sancycline, Sanfetrinem Sodium, Sarmoxicillin, Sarpicillin, Scopafungin, Sisomicin, Sisomicin Sulfate, Sparfloxacin, Spectinomycin Hydrochloride, Spiramycin, Stallimycin Hydrochloride, Steffimycin, Sterile Ticarcillin Disodium, Streptomycin Sulfate, Streptonicozid, Sulbactam Sodium, Sulfabenz, Sulfabenzamide, Sulfacetamide, Sulfacetamide Sodium, Sulfacytine, Sulfadiazine, Sulfadiazine Sodium, Sulfadoxine, Sulfalene, Sulfamerazine, Sulfameter, Sulfamethazine,
Sulfamethizole, Sulfamethoxazole, Sulfamonomethoxine, Sulfamoxole, Sulfanilate Zinc, Sulfanitran, Sulfasalazine, Sulfasomizole, Sulfathiazole, Sulfazamet, Sulfisoxazole, Sulfisoxazole Acetyl, Sulfisoxazole Diolamine, Sulfomyxin, Sulopenem, Sultamicillin, Suncillin Sodium, Talampicillin Hydrochloride, Tazobactam, Teicoplanin, Temafloxacin Hydrochloride, Temocillin, Tetracycline, Tetracycline Hydrochloride, Tetracycline Phosphate Complex, Tetroxoprim, Thiamphenicol, Thiphencillin Potassium, Ticarcillin Cresyl Sodium, Ticarcillin Disodium, Ticarcillin Monosodium, Ticlatone, Tiodonium Chloride, Tobramycin, Tobramycin Sulfate, Tosufloxacin, Trimethoprim, Trimethoprim Sulfate, Trisulfapyrimidines, Troleandomycin, Trospectomycin Sulfate, Trovafloxacin, Tyrothricin, Vancomycin, Vancomycin Hydrochloride, Virginiamycin and Zorbamycin.
Anti-viral agents useful in the invention include but are not limited to: immunoglobulins, amantadine, interferons, nucleotide analogues, and protease inhibitors. Specific examples of anti-virals include but are not limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine; Zidovudine; and Zinviroxime.
A "degradable" ABP polymer as used herein means that the polymer can be readily broken down enzymatically. A biodegradable polymer according to the present invention is 80% broken down within preferably one week to six months, including about one, two, three, four or five months.
A "non-degradable" ABP polymer as used herein means that the polymer can be not broken down enzymatically. A non-biodegradable polymer according to the present invention will be substantially intact (max. 5% degradation) for one week to 3 years including about 6 months, 1 year or 2 years.
The degradabily of the ABP can be adjusted by the linkers used in the polymerisation.
A "linker" for the polymerization of ABPs according to the present invention is any molecule that can react with an ABP and can be used to covalently link one ABP to another to create a dimer, trimer or polymer.
As the person skilled in the art will appreciate, a wide array of linkers can be employed in the polymerization reactions. Those include, but are not limited to, epichlorohydrine, divinylbenzene, diisocyanate and styrenes. In a preferred embodiment, the cross-linkers are selected from the group of crosslinking acrylates; crosslinking methyl acrylates, crosslinking acrylamides, crosslinking methyl acrylamide or combinations thereof.
Crosslinkers that are within the scope of the present invention include in partiular Dithiobis(succinimidylpropionate) (DSP), 3,3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP), Dissucinimidyl suberate (DSS), Bis(sulfosuccinimidyl)suberate (BS3), Disuccinimidyl tartrate (DST), Disulfosuccinimidyl tartrate (Sulfo-DST), Bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), Bis[2- (sulfosuccinimidooxycarbonyloxyjethyllsulfone (Sulfo-BSCOES), Ethylene glycolbis(succinimidylsuccinate) (EGS), Ethylene glycolbis(sulfosuccinimidylcuccinate) (Sulfo-EGS), Disuccinimidyl glutarate (DSG), N,N'-Disuccinimidyl Carbonate (DSC), Dimethyl adipimidate dihydrochloride (DMA), Dimethyl pimelimidate dihydrochloride (DMP), Dimethyl suberimidate dihydrochloride (DMS), Dimethyl-3,3'-dithiobispropionimidate dihydrochloride (DTBP), 1 ,4-di-[3'-(2'- pyridyldithio)propionamido]butane (DPDPB), Bismaleimidohexane (BMH), 1 ,5- Difluoro-2,4-dinitrobenzene (DFDNB), 4,4'-Difluoro-3,3'-dinitrodiphenylsulfone (DFDNPS), Bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), Formaldehyde, Gluaraldehyde, 1 ,4-Butanediol Diglycidyl Ether, Adipic Acid Dihydrazide, Carbohydrazide, bis-Diazotized o-Tolidine, Bis-Diazotized Benzidine, N,N'-Ethylene- bis(iodoacetamide), N,N'-Hexamethylene-bis(iodoacetamide), N, N'- Undecamethylene-bis(iodoacetamide), α,α'-Diiodo-p-xylene sulfonic acid, Tri(2- chloroethyl)amine, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), LC-SPDP, Sulfo-LC-SPDP, 4-Succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), Sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (Sulfo- LC-SMPT), Succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), Sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (Sulfo-SMCC), m-Maleimidobenzoyl-N-hydroxy-succinimide ester (MBS), m-Maleimidobenzoyl-N- hydroxy-sulfosuccinimide ester (Sulfo-MBS), N-Succinimidyl(4-iodoacetyl)- aminobenzoate (SIAB), Sulfo-succinimidyl(4-iodoacetyl)-aminobenzoate (Sulfo- SIAB), 4-(p-maleimidophenyl)butyrate (SMPB), Sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate (Sulfo-SMPB), N-γ-Maleimidobutyryl-oxysuccinimide ester (GMBS), N-γ-Maleimidobutyryl-oxysulfosuccinimide ester (Sulfo-GMBS), Succinimidyl 6[6-(((iodoacetyl)amino)-hexanoyl)amino]hexanoate (SIAXX), Succinimidyl 6-[(iodoacetyl)-amino]hexanoate (SIAX), Succinimidyl 6-[6- (((iodoacetyl)amino)-hexanoyl)amino]hexanoate (SIAXX), Succinimidyl 6- [(iodoacetyl)-amino]hexanoate (SIAX), p-Nitrophenyl iodoacetate (NPIA), 4-(4-N- Maleimidophenyl)butyric acid hydrazide hydrochloride (MPBH), 4-(N- Maleimidomethyljcyclohexane-i-carboxyl-hydrazide hydrochloride (M2C2H), 3-(2- Pyridyldithio)propionyl hydrazide (PDPH), N-Hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), N-Hydroxysolfosuccinimidyl-4-azidosalicylic acid (Sulfo-NHS-ASA), Sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (Sulfo-NHS-LC-ASA),
Sulfosuccinimidyl-2-(p-azido-salicylamido)ethyl-1 ,3'-dithiopropionate (SASD), N- Hydroxysuccinimidyl-4-azidobenzoate (HSAB), N-Hydroxysulfosuccinimidyl-4- azidobenzoate (SuIf o-HSAB), N-Succinimidyl-6-4'-azido-2'- nitrophenylamino)hexanoate (SANPAH), Sulfosuccinimidyl-6-(4'-azido-2'- πitrophenylamino)hexanoate (Sulfo-SANPAH), N-5-Azido-2-nitrobenzoyloxy- succinimide (ANB-NOS), Sulfosuccinimidyl-2-(m-azido-o-nitro-benzamido)-ethyl-1 ,3'- dithiopropionate (SAND), N-Succinimidyl(4-azidophenyl)-1 ,3'-dithiopropionate (SADP), N-Sulfosuccinimidyl(4-azidophenyl)-1 ,3'-dithiopropionate (Sulfo-SADP), Sulfosuccinimidyl-4-(p-azidophenyl)butyrate (Sulfo-SAPB), Sulfosuccinimidyl-2-(7- azido-4-methyl coumarin-3-acetamide)ethyl-1 ,3'-dithiopropionate (SAED), Sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate (SuIf o-SAMCA), p-Nitrophenyl diazopyruvate (pNDP), p-Nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1- (p-Azidosalicylamido)-4-(iodoacetamido)butane (ASIB), N-[(4-p- Azidosalicylamido)butyl]3'-(2'-pyridyldithio)propionamide (APDP), Benzophenone-4- iodoacetamide, Benzophenone-4-maleimide, p-Azidobenzoyl hydrazide (ABH), 4-(p- Azidosalicylamido)butylamide (ASBA), p-Azidophenyl glyoxal (APG), 4-Azido-2- nitrophenylbiocytin-4-nitrophenyl ester, (SuIf osuccinimidyl-2-[6-(biotinamido)-2-(p- azidobenzamido) hexanoamido]ethyl-1 ,3'-dithiopropionate (Sulfo-SBED), Methanethiosulfonate-azidotetrafluoro-Biotin (Mts-Atf-Biotin), Methanethiosulfonate- azidotetrafluoro-LongChain-Biotin (Mts-Atf-LC-Biotin), Tris(hydroxymethyl) phosphine propionic acid (THPP), Tris(hydroxymethyl)phosphine (THP).
As the person skilled in the art will readily understand, many other cross linkers are possible and form part of the present invention.
TABLES IV to VII show selected preferred linkers for polymerization.
TABLE IV: Examples of crosslinking acrylates
Figure imgf000037_0001
TABLE V: Examples of crosslinking methyl acrylates
Figure imgf000037_0002
TABLE Vl: Examples of crosslinking aery lam ides
Figure imgf000038_0001
TABLE VII: Examples of crosslinking methyl acrylamides
Figure imgf000038_0002
In certain embodiments of the invention, the ABPs form homo-bilayers and incorporate into natural membranes and behave like natural phospholipids. The ABPs of the present invention bear "functional handle(s)" at the interface-region. These secondary amines of the ABPs allow substitution reactions at the interface region. Such substitution reactions include polymerization, which permits the formation of cell-surface like materials. In certain preferred embodiments, the substitution reactions render the ABPs partially positively charged, allowing for non- covalent interactions with negatively charged DNA and RNA. Alternatively or additionally, the secondary amines of the ABP may disturb the natural dipole-moment of the phospholipids rendering the molecule partially positively charged, allowing, e.g., for non-covalent interactions with negatively charged DNA and siRNA.
In certain embodiments of the invention, the structure of the ABPs, in particular the presence of these "functional handles", improves the structural integrity of the phospholipids, in particular subsequent to polymerization, opening the door for a wide variety of applications, only a few of which will be discussed in the following. However, the person skilled in the art will, with the guidance provided herein, readily be able to envision and execute further uses of the modified phospholipids of the present invention.
In one embodiment of the invention two secondary amines instead of ester moieties are introduced to yield amine-bearing phospholipids (ABPs, e.g., Figure 1 b).
Fig. 1 shows in (A) a classic phospholipid built from a glycerophosphate backbone. In (B) an amine-bearing phospholipids (ABP) with a 2,3-diaminopropyl phosphate backbone produced by de novo synthesis is shown. Positions sn1 , sn2 and sn3 are indicated. As shown in (C) the ABPs can incorporate into natural phospholipid bilayers.
The structure of the ABPs of the present invention allow for a wide variety of uses, including the non-covalent complexation of nucleic acids such as plasmid DNA (pDNA) or siRNA generally followed by the transfection of cells. In a simple complexation, the ABPs serve as carriers without necessarily physically sequestering the nucleic acid from its surroundings. However, the ABPs can also form vesicles (also referred to herein as nanoparticles) that engulf a nucleic acid or drug. ABPs may be used as or as part of a cationic transfection lipids to form lipoplexes which may serve as transfection vectors, which may or may not be polymerized. Such complexes may also incorporate target specific molecules at the interface region of the phospholipids, this allowing targeted delivery of a non-nucleic acid drug and/or a nucleic acid, including DNA and RNA, in particular pDNA, siRNA miRNA (mirco RNA).
The structure of the ABPs also allow for the formation of cell-membrane-like polymers and the incorporation of antimicrobial peptides to form antimicrobial surfaces.
Polymeric ABPs can serve as a new core material for antimicrobial surfaces, including antifungal and antibacterial surfaces, and for coatings, in particular biocompatible coatings and surfaces. As will be discussed in more detail below, antimicrobial peptides may be immobilized on the polymeric ABPs. These surface can be used on a wide variety of medical devices, e.g., catheters that are prone to contamination.
Also, cell-membrane mimics and cell-membrane-like polymers which integrate (trans- membrane) proteins and cellular components and which are based on ABPs provide a wide array of applications for basic and applied research.
EXAMPLES:
1. SYNTHESIS, POLYMERIZATION AND CHARACTERIZATION OF ABPS
A. Synthesis of Amine-Bearing Phospholipids (ABPs) Presented herein is an ester-to-amine modification of natural phospholipids resulting in the ABPs of the present invention. Such amine-bearing phospholipids represent, as discussed above, a versatile platform in biology and biophysics. The synthesis of ABPs can be accomplished by various synthetic routes. A number of non-limiting approaches are provided in the following:
The synthesis of amine-bearing phospholipids can start from L-serine (3), drawing from the natural chiral pool to install the stereocenter at sn2 (see, FIG. 2). Following the protocol of McKillop et al., the methyl ester is formed for solubility purposes at later stages of the synthesis (McKillop et al., 1994). Λ/-Acylation with the commercially available palmitoylchloride indroduces the first hydrophobic tail of the molecule (4). The second alkyl chain is attached following a methodology developed by Solladie-Cavallo et al., who showed that lithium aluminum amides (formed from a simple substitution of lithium aluminum hydride and an amine) react readily with esters to form an amide (Solladie-Cavallo et al., 1992). Simultaneous reduction of both amides (5) to amines (6) followed by BOC-protection finishes the hydrophobic part of the molecule (7). The free hydroxyl group (7) is transformed into the phosphate (8) and then into the phosphocholate head-group (9) as described by Harbison and Griffin (Harbison et al., 1984).
In a modification of the scheme shown in FIG. 2, employing phosphoamidite chemistry, the free hydroxyl group (7) is reacted with chloro 2-cyanoethyl (N,N- diisopropyl)phosphoramidite followed by reaction with e.g. choline tosylate (Bay et al. 2004), followed by the proper oxidation and deprotection of the headgroup. Alternatively, the free hydroxyl group (7) is reacted with 1 -choline 2-cyanoethyl (N,N- diisopropyl) phosphoramidite, followed by the proper oxidation and deprotection of the headgroup.
BOC-deprotection under acidic conditions to free both amines (2) [structure not shown]. The person skilled in the art will appreciate that alternative synthetic pathways are possible, e.g., pathway that include Garner's oxazolidines (McKillop, 1994).
Another synthesis follow a similar route: This synthesis starts also from L-serine (3, see FIG. 3). Following the protocol of McKillop et al., the methyl ester is formed to ensure solubility later on (McKillop, 1994). According to a protocol reported by Sibi et al. the oxazolidinone (10) is provided (Sibi et al., 1999). /V-Alkylation with the commercially available 1 -bromohexadecane introduces the first hydrophobic tail of the molecule (11). The second alkyl chain is attached by reductive amination following the reduction of the methyl ester to the aldehyde (12). The oxazolidinone is opened by a catalytic reaction with caesium carbonate, following a similar protocol reported by Sibi et al. (Sibi et al., 1999). Both secondary amines are now BOC- protected (7) which completes the hydrophobic part of the molecule. The free hydroxyl group (7) is transformed into the phosphate (8) and then into the phosphocholate head-group (9) as described by Harbison and Griffin (Harbison et al., 1984). BOC-deprotection under acidic conditions will free both amines (2). Equally, phosphoramidite chemistry can be employed and the free hydroxyl group (7) is reacted with chloro 2-cyanoethyl (Λ/,Λ/-diisopropyl)phosphoramidite followed by reaction with e.g. choline tosylate (Bay et al., 2004), followed by the proper oxidation and deprotection of the headgroup. Alternatively, the free hydroxyl group (7) can be reacted with 1 -choline 2-cyanoethyl (Λ/,Λ/-diisopropyl)phosphoramidite, followed by the proper oxidation and deprotection of the headgroup.
The synthesis of a lipid with an 1 ,3-diaminopropan-2-ol backbone may also follow another route (see FIG. 4): Starting with a double nucleophilic attack of a long-alkyl chain amine to racemic epichlorohydrin (14). The resulting lipid, can be protected in the form of a cyclic urea (15) in the same flask similar to a procedure reported by Enders using bis(4-nitrophenyl)carbonate and thus avoiding a kinetically favored five- membered ring carbamate (Enders et al., 1999). Using a one-batch-procedure reported by Bittman, the phosphocholine headgroup (16) is introduced (Byun et al., 1996). Deprotection with sodium hydroxide would lead to the desired phospholipid (17). Phosphoramidite chemistry can also be employed: Here the free hydroxyl group (7) is reacted with chloro 2-cyanoethyl (Λ/,Λ/-diisopropyl)phosphoramidite followed by reaction with e.g. choline tosylate (Bay et al., 2004), followed by the proper oxidation and deprotection of the headgroup (compare FIG 3.). Alternatively, the free hydroxyl group (7) is reacted with 1 -choline 2-cyanoethyl (Λ/,Λ/-diisopropyl)phosphoramidite, followed by the proper oxidation and deprotection of the headgroup. However, as the person skilled in the art will readily understand further routes of synthesis are possible and within the scope of the present invention.
B. Synthesis of ABP-Libraries
The synthesis of ABP libraries is designed to be flexible and highly modular. In particular, it allows the introduction of i) the enantiomeric form at sn2, ii) different chain-lengths at sn2 and sn1 , independently of each other, iii) unsaturation in one or both alkyl chains, iv) different head-groups containing free amines, free or activated carboxylic acids or thiols, and v) unusual phospholipid tails such as e.g. cholesterol (see FIG. 5).
FIG. 5 shows that the ABPs are highly modular, and libraries of structurally different molecules can be synthesized. Various scientific questions can be targeted with individual, optimized combinations. Thus, depending on the scientific questions, libraries of amine-bearing phospholipids are synthesized. This approach allows the preparation of lipids with optimized properties, e.g. deuterated ABPs for NMR- structural studies. C. Biophysical Characterization of ABPs
The interaction of ABPs with natural membrane components, i.e. the phospholipids POPC, POPG, and also cholesterol is tested through isothermal titration calorimetry. Physical characteristics are measured such as binding affinities K3, enthalpy changes ΔH as well as Gibbs free energy changes ΔG and entropy changes ΔS (Heerklotz et al., 2000). Measuring the surface-pressure to area isotherm in a Langmuir-Blodgett trough reveals the ABPs cross-sectional area A covered at the surface. Also, the possible formation of liposomes is recorded: A thin film of ABPs is rehydrated and freeze-thawed, followed by extrusion through 400 and/or 200 and/or100 and/or 50 nm tracked-edge membrane filters (Walde, 2004), light scattering experiments, and freeze-fracture cryo transmission electron microscopy.
D. The Synthesis of Phospholipid-Polymers Polymers that mimic natural cell-membranes are highly attractive targets in biomedical engineering (Akimoto et al., 1981). The amine-bearing phospholipids may be polymerized via, e.g., one of the following three routes (see FIG. 6), which shows the synthesis of three types (l.-lll.) of poly β-amino phospholipids, which shall serve as non-limiting examples).
I. The secondary amines of an 2, 3 diaminopropan-1-ol of an ABP (2) react with a bis-acrylate (18) to form a poly(β-amino ester) (19). This reaction, depending on the melting properties of ABPs, is, in certain embodiments of the invention, performed under solvent-free conditions so that the products can be used directly without need for solvent removal (Zugates et al., 2007). Polymers formed from diacrylates may generally be degradable under conditions found in the body, polymers formed from bis-acryl-amides may be non-degradable.
II. The amine-bearing phospholipids may be reacted with acryloyl chloride to create ABP-bis-acrylamides (20). These molecules may, in certain embodiments of the invention, react with primary or secondary amines (21) to form polymers. The groups thus introduced may, e.g., carry carboxylic acids as shown that can be modified after the polymerization.
III. ABP-bis-acrylamides (20) may undergo photopolymerization to form homo- polymers (23). This re-action can be performed by ad-mixing other reactive molecules such as e.g. cholesteryl acrylate or PEG-acrylates, producing hetero- polymers.
The 1 ,3 diaminopropan-2-ol (or 2,3 diaminopropan-1 -ol) based amine-bearing phospholipid type, exemplified by structure (24) contains two reactive secondary amines. Thus, three different types of polymerizations that can be performed using the bulk lipid, the lipid in monolayers and/or the lipid in vesicles (see FIG. 7).
IV. The amine-bearing phospholipid (24) are reacted with a diacrylate (25) in solvent free conditions at 90 0C over night or in the presence of an appropriate solvent such as but not limited to dimethyl sulfoxide, to produce a linear poly(β-amino ester) (26). A library of linear polymers is so synthesized.
V. Bis-acrylamide phospholipids (27) can be transformed into polymers (29) by reaction with either primary or secondary amines (28).
Vl. It was shown that acrylate-containing molecules can be UV-polymerized in the presence of e.g. the lipophilic radical initiator 2,2'-azobis(2-methylpropionitrile) (AIBN) (Nijst et al., 2007). Aliquots of liquid bis-acrylamide phospholipids (27) can be polymerized using, e.g., a built-in ballast ultraviolet lamp.
As the person skilled in the art will appreciate, an array of techniques used in biomedical engineering can be used to effect the polymerization. To name just a few, the pre-polymer can, e.g., be introduced between two microscopy slides spaced, e.g., 1 mm, away from each other (Zumbuehl et al., 2007) or polymeric sponges are produced (Chen et al., 2002). The porogen leaching method involves the casting of a mixture of polymer solution and porogen in a mold, drying the mixture, followed by a leaching out of the porogen with water to generate the pores. Usually, water soluble particulates such as salts and carbohydrates are used as the porogen materials. The pore structures can be readily manipulated by controlling the property(ies) and/or fraction of the porogen. Such a process can be readily reproduced. The polymers can be tested for their biocompatibility by seeding the surfaces with primary human foreskin fibroblasts, as we have done with other polymeric materials (Nijst et al., 2007). 2. APPLICATIONS OF THE ABPS IN BIOMEDICAL ENGINEERING
The amine-bearing phospholipids (ABPs) of the present invention provide an ideal platform for numerous applications in the field of biomedical engineering, such as, but not limited to, providing ABP containing cationic ABPs or cationic vesicles (nanoparticles) for RNA interference therapy, ABP cationic polymers for gene- therapy, and ABP-cell membrane like materials onto which antimicrobial peptides are grafted as antimicrobial material.
Example I: RNA Interference Therapy- cationic ABPs
RNA interference has enormous potential for the sequence-specific reduction of gene expression in medicine (Behlke 2006). The synthesis of 1 ,200 structurally diverse lipid-like molecules (lipidoids) and their successful application in vitro and in vivo as vectors for siRNA has been reported (Akinc et al., 2008). 53 members of this library were able to mediate high levels of uptake of small interfering RNA (siRNA against Firefly Luciferase) molecules into HeLa cells, surpassing the state-of-the-art transfection vector Lipofectamine2000™. Several of these lipidoid molecules were also capable of acting as an siRNA vector in vivo. In collaboration with Alnylam Pharmaceuticals (USA and GER) high knockdown of several proteins in mice, rats as well as non-human primates have been achieved. One single bolus injection of the apolipoprotein B-specific siRNA nanoparticulate formulation resulted in a 30 day, up to 50% reduction of serum LDL cholesterol levels. The lipidoid system may be advanced to carry both anti-VEGF (Vascular Endothelial Growth Factor) and anti- KSP (Kinesin Spindle Protein) siRNAs to treat solid liver tumors into clinical Phase I. Currently, the lipidoid nanoparticle system is one of the few if not the only existing systems for efficacious in vivo RNA interference therapy; others systems include another lipid-based system (Zimmerman et al., 2006) and a β-cyclodextrin-based polymer (Bartlett et al., 2007).
siRNA transfection vectors using the ABPs of the present invention may improve the toxicity profile, transfection efficiency and/or targeting of different tissues. ABPs of certain embodiments of the invention are particularly well suited for siRNA transfection: in particular those embodiments that have the following features:
A) ABPs may be designed to be cationic or become cationic under acidic conditions (e.g., due to the two secondary amines at sn1 and sn2, through a proposed disturbance of the dipole-moment of the phosphocholine headgroup that may be parallel to the bilayer membrane normal instead of nearly perpendicular as depicted in Fig. 1 ), and/or through the introduction of cationic headgroups (Seeling et al., 2001). ABPs may also be net cationic or anionic due to the different headgroups used (Zimmermann et al., 2006).
B) Cationic lipid-like molecules are classic DNA and siRNA delivery- vectors (Blagbrough et al., 2003; Miller, 1998). Cationic ABPs may form non-covalent complexes with negatively charged siRNAs, protecting the payload from harsh conditions found in blood.
C) ABPs have structures similar to natural phospholipids. As a result toxicity profile may be improved over existing structures such as the lipidoids discussed above.
As the person skilled in the art will appreciate, the ABPs used for siRNA transfection are, e.g., tested using a standard protocol (Akinc et al., 2008): The ABPs are, e.g., reconstituted in NaOAc buffer and added to a solution of siLUC (targeting luciferase expression, Alnylam Pharmaceuticals, USA) in the same buffer. This complex is diluted in medium and added to HeLa cells stably transfected with firefly and renilla luciferase (Alnylam Pharmaceuticals). In this assay, toxicity or other nonspecific effects results in reduction of expression of both luciferase proteins, while noncytotoxic, specific silencing results in reduction of only firefly luciferase. In vivo transfections of the ABP-formulated siRNAs and Antagomir-RNAs are performed. Furthermore in vitro transfections in HeLa cells demonstrate the usefulness of the system.
The invention is also directed at formulating nanoparticles, e.g. containing siRNAs against multi-drug resistance transporters. In certain embodiment of the invention, the ABPs may condense siRNAs and/or transfect cancer cells in vitro and/or in vivo. Example II: Gene Therapy- cationic polymeric ABPs
Over 4,000 human diseases are estimated to be caused by a single defect on the genome (Kinnon et al., 2002). Treatment of these and other illnesses through the application of nucleotide-based drugs such as DNA and siRNA therefore has the potential to revolutionize the medical field. The transfection of plasmid DNA is preferentially mediated by polymeric vectors, whereas siRNA is preferably transfected by lipid-like materials (an effect that is possibly based on the size- difference between DNA and a 21mer RNA). Polymeric materials for plasmid DNA (pDNA) delivery, e.g. a series of poly(β-amino esters) that are transfecting COS-7 cells at levels of viral transfection have been developed (Zugates et al., 2007). This of course allows transfections in the absence of virus, and experiments can be performed in standard biological laboratories. There is still an urgent need for new materials with improved toxicology, transfection efficiencies and/or organ targeting. The amine-bearing phospholipids may, e.g., be transformed into cationic polymers, and may serve as vectors for pDNA transfection. Their cell-membrane mimicking properties improve, in certain embodiments of the invention, the toxicity profile over existing vectors such as poly(ethylene imine) or poly(β-amino esters) (Zugates et al., 2007). The ABP polymers may be designed to be degradable (using diacrylates) or nondegradable (using diacrylamides). Hundreds of different diacrylates and diacrylamides are commercially available or can be rapidly synthesized. A combinatorial library of poly(β-amino phospholipids) is build to test each as a potential pDNA vector.
Preferably, a standard high-throughput screening protocol is used (Zugates et al., 2007): Reconstituted ABPs in NaOAc buffer is preferably added to a buffer-solution of pDNA encoding Luciferase (pCMV-Luc; Elim Biopharmeceuticals, Hayward, CA). This lipoplex is added to COS-7 cells (ATCC, Manassas, VA) pre-plated in a 96-well format in DMEM containing 10% serum. The cells are incubated for 1 h, washed and left for 3 days. The transfection efficiency is analyzed using a Bright-Glo kit (Promega, Madison, Wl). In parallel, hits may be screened by FACS, MTT toxicity assay, light scattering (size) and zeta potential (net charge). The new non-viral materials have wide uses in basic biology as well as in medical applications.
Example III: Antimicrobial Surfaces- grafting of polymeric surfaces
Hospital infections are becoming increasingly costly and difficult to treat due to the spread of drug resistant bacteria (Wisplinghoff et al., 2004). Despite efforts to improve the sterility of surgical procedures, infection remains common. These infections are often associated with medical devices. Skin penetrating (invasive) devices, such as central venous catheters, as well as urinary catheters, provide a route for bacteria to enter the body and implanted materials may form surfaces favorable for bacterial growth in the form of biofilms (O'Toole et al., 2000). Amphogel, a dextran-based hydrogel into which the antifungal agent amphotericin B is absorbed has been described (Zumbuehl et al., 2007). Amphogel kills fungi within 2 hrs. or contact and that the same material can be reused for at least 15 days without losing its effectiveness against Candida albicans. The material is biocompatible in vivo and does not cause hemolysis in human blood. Amphogel inoculated with Candida albicans and implanted in mice completely prevents fungal infections and mitigates biofilm formation.
Polymeric ABPs may serve in this context, as well as other contexts, as new core material that are preferably biocompatible and tunable, e.g. by incorporation of degradable ester bonds. Antimicrobial peptides may be immobilized onto ABP surfaces. Antimicrobial peptides are potent small proteins used by a host's immune system to combat bacterial infections in multicellular eukaryotes and have the potential to be a important next generation of therapeutic agents (Loose et al., 2006). In certain embodiments, polymeric ABPs, due to their versatility and cell-membrane- similarity, form a platform for biomedical surfaces and coatings. The amine-bearing phospholipids are transformed into polymers, e.g. poly(ethylene-glycol) carrying headgroups substituted with terminal carboxylic acids. Antimicrobial peptides bearing, e.g., a C-terminal cysteine may be reacted with the activated acid. In certain embodiments of the invention, these surfaces are antimicrobial, anti-biofouling, and/or non-hemolytic.
It will be appreciated that the phospholipids and methods of the instant invention can be incorporated in the form of a variety of embodiments, such as pharmaceutical nanoparticles, cell-membrane mimetic materials, responsive surfaces, fluorescence markers, MRI/PET markers, consumer products, liquid crystals and electronics to name only a few in addition to those which are disclosed herein. It will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.
BIBLIOGRAPHY
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Claims

What I claim is:
1. An amine-bearing phospholipid (ABP) comprising: an interface region comprising an amine group substituted propanol backbone, preferably a 2,3-diaminopropan-1-ol or a 1 ,3- diaminopropan-2-ol, providing at least three attachment regions, a hydrophilic head group, a first and second hydrophobic portion, preferably a first and second uncharged hydrophobic portion, wherein the head group comprises and is covalently attached via a phosphate ester bond to a first one of the attachment regions, and the first hydrophobic portion comprises two hydrophobic moieties each being covalently attached to an amine of a second one of the attachment regions, the second hydrophobic portion comprises two hydrophobic moieties each being covalently attached to an amine of a third one of the attachment regions, wherein, in an aqueous surrounding, the head group extends from the interface region into a first direction and the hydrophobic portions extends from the interface region into a second direction.
2. The ABP of claim 1 having the following formula
(I)
Figure imgf000050_0001
(H)
Figure imgf000050_0002
wherein headgroup A is one of the following: a phosphatic acid or a phosphate ester substituted group such as: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, phosphate ester substituted electrophile, such as an activated ester including N- hydroxysuccinimic ester, or an acid chloride or an halogenide, a phosphate ester substituted nucleophile such as a thiol or an amine, a phosphate ester substituted fluorescent group such as fluoresceine, a phosphate ester substituted radioactively labeled group, a phosphate ester substituted alkyne, or a phosphate ester substituted azide;
and the hydrophobic moieties are Y1, Y2 , Z1 and Z2 , wherein,
W is a stereocenter that is R, S or racemic; Y1 Is:
• H;
• optionally substituted C1-30 alkyl;
• an optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
• an optionally substituted C1-30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds; or
• a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group or a linear alkyl group or an aryl group;
Y2 is:
H; optionally substituted C1-30 alkyl; an optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds; an optionally substituted C 1-30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds; a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group; or • 'a pre-polymer group polymerizable in light or by heat, optionally with a radical initiator, wherein the group is preferably -C(O)-C(CH3)=CH2 or -C(O)-CH=CH2;
Z1 is:
• H;
• optionally substituted C1 -30 alkyl;
• an optionally substituted C 1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
• an optionally substituted C1-30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds; or
• a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group; and
Z2 Is:
• H;
• optionally substituted C1-30 alkyl;
• an optionally substituted C 1 -30 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
• an optionally substituted C1 -30 alkyl group with 1 , 2, 3, 4, or 5 triple bonds;
• a sterol, preferably cholesterol or ergosterol, wherein the sterol is attached via a short linker, preferably a carbonyl group, a linear alkyl group or an aryl group; or
• a pre-polymer group polymerizable in light or by heat, optionally with a radical initiator, wherein the group is preferably -C(O)-C(CH3)=CH2 or -C(O)-CH=CH2; with the proviso that at least one, preferably two or three of Y1, Y2 , Z1 and Z2 are unequal H.
3. The ABP of claim 2, wherein Y1, Y2, Z1 and Z2 is selected from one or more of the following:
X
1-29
Figure imgf000053_0001
4. The ABP of any one of the preceding claims having one of the following formulas: (III)
Figure imgf000054_0001
(VII)
Figure imgf000055_0001
(Xl)
Figure imgf000056_0001
(XIII)
Figure imgf000056_0002
(XV)
Figure imgf000057_0001
(XIX)
Figure imgf000058_0001
wherein,
W is as defined in claim 2; n is an integer between n=0 and n=6;
X1 is:
H; alkyl- , preferably methyl-, ethyl-, propyl-, isopropyl-; aryl-; a fluorescent reporter group, preferably fluorescein, alexafluor 488, Tokio green, wherein the reporter group is bound to the amine of the interface region directly via an amide bond or via a spacer group, preferably an alkyl-, aryl- or PEG spacer group; a non-fluorescent reporter group, preferably a spin label or a radioactive label; an electrophilic or nucleophilic group, preferably an Λ/-hydroxysuccinimide, an anhydride, or an /V-methylisatoic anhydride, a methylisothiocyanate, a methylisocyanate, or methylthiourea, 3-[4-(trimercapto)phenyl]propionyl, 4- benzyloxybenzaldehyde, 2-chlorotrityl chloride, sulfonic acid, morpholinomethyl, piperidinomethyl, piperazinomethyl, acrylate, methyl-acrylate, styrene, acrylamide, or methyl-acrylamide;
CH3-[CH2Jn-SH with n=0-6;
CH3-[CHj]n-NH2 with n=0-6;
CH3-[CH2]n-NH-CH2-[CH2]o-NH2 with n=0-6; o=0-6; CH3-[CH2In-NH-CH2-[CH2]O-NH-CH3-[CH2]P-NH2 with n=0-6; o=0-6; p= 0-6;
CH3-[CH2]n-NH-CH3-[CH2]0-NH-CH2-[CH2]p-NH-CH2-[CH2]q-NH2 with n=0-6; o=0-6; p=
0-6; q=0-6;
CH3-[CH2Jn-OH with n=0-6;
CH3-[CH2]n-{O-CH2-[CH2]o} p-OH with n=0-6; o=0-6; p= 0-200;
CH3-[CH2]n-{O-CH2-[CH2]o} P-O-CH3 with n=0-6; o=0-6; p= 0-200;
CH3-[CH2]n-[O-CH2-[CH2]o} P-O-CH2-CH3 with n=0-6; o=0-6; p= 0-200;
CH3-[CH2Jn-C-C-CH with n=0-6;
=N=N;
=C=S;
=C=O; or a metal complexing group, preferably ethylenediamine tetraacetate, wherein the group is bound to the amine N1 either directly or via a linker, preferably an alkyl chain or a polyethylene glycol chain;
X2 is:
• H;
• alkyl-, preferably methyl-, ethyl-, propyl- or isopropyl-;
• aryl-;
• CH3-[CH2Jn-SH with n=0-6;
• CH3-[CH2Jn-NH2 with n=0-6;
• CH3-[CH2Jn-NH-CH2-[CH2J0-NH2 with n=0-6 and o=0-6;
• CH3-[CH2Jn-NH-CH2-[CH2J0-NH-CH2-[CH2Jp-NH2 with n=0-6, o=0-6 and p= 0-6;
• CH3-[CH2]n-NH-CH2-[CH2]0-NH-CH2-[CH2]p-NH-CH2-[CH2]q-NH2 with n=0-6, o=0-6, p= 0-6 and q=0-6;
• CH3-[CH2Jn-OH with n=0-6;
• CH3-[CH2Jn-(O-CH2-[CH2J0J p-OH with n=0-6, o=0-6 and p= 0-200;
• CH3-[CH2Jn-(O-CH2-[CH2J0) P-O-CH3 with n=0-6, o=0-6 and p= 0-200;
• CH3-[CH2Jn-(O-CH2-[CH2J0J P-O-CH2-CH3 with n=0-6, o=0-6 and p= 0-200; or
• CH3-[CH2Jn-C-C-CH with n=0-6.
X3 is, if present, selected from:
• alkyl- such as, but not limited to, methyl-, ethyl-, or propyl-; or
• aryl-; or having one of the following formulas:
(XXI)
Figure imgf000060_0001
(XXII)
Figure imgf000060_0002
(XXIII)
Figure imgf000060_0003
(XXIV)
Figure imgf000061_0001
Figure imgf000061_0002
(XXVI)
Figure imgf000061_0003
wherein:
W is as defined in claim 2; n is as defined in claim 4;
Y1, Y2, Z1 and Z2 are as defined in claim 2; V is:
• a hydroxyl group;
• an amine; or
• an amide;
and X is:
• a thiol;
• an ether;
• an ester;
• an amide;
• an alkene; or
• an alkyne;
or having one of the following formulas: (XXVII)
Figure imgf000062_0001
(XXVIII)
Figure imgf000062_0002
Figure imgf000063_0001
Figure imgf000063_0002
wherein,
W is as defined in claim 2; m is an integer between n=0 and n=6; n is as defined in claim 4;
Y1, Y2, Z1 and Z2 are as defined in claim 2.
5. The ABP according to any one of the preceding, wherein the sugar phosphate ester substituted sugar is a simple sugar or is part of a simple glucose series or a globo, ganglio, lacto, neolacto, isoglobo, mollu, arthro or gala series.
6. The ABP according to any one of the preceding claims, wherein the head group of said ABP is labeled, preferably fluorescently, is deuterated, radioactively labeled or spin labled.
7. The ABP of claim 4, wherein one or more positions of the sugars (XXV), (XXVI), (XXIX) or (XXX) is substituted, preferably with a sugar including a substituted sugar, even more preferably with a simple sugar.
8. A dimer or polymer of the ABPs of any one of the preceding claims, wherein the dimer or polymer is a bolaamphiphile in which the ABPs are linked to one another via one or both their hydrophobic portions.
9. A dimer or polymer of ABPs of any one of the preceding claims comprising one or more linkers, in particular sugar or polysugar linkers.
10. A dimer or polymer of the ABPs of any one of the preceding claims, wherein the ABPs are crosslinked, preferably via a linker, most preferably one or more of the following linkers:
• an epichlorohydrin
• a divinylbenzene
• a diisocyanate
• a styrene
Figure imgf000064_0001
- wherein X is O or N;
- wherein Y is O or N;
- wherein Z is H or CH3; and
- wherein n is an integer from 0 to 10;
or via cross-linkers selected from the group of crosslinking acrylates; crosslinking methyl acrylates, crosslinking acrylamides, crosslinking methyl acrylamide or combinations thereof.
11. The dimer or polymer of claim 10, wherein the crosslinking acrylates are selected from (A) to (BB):
Figure imgf000065_0001
(ii) crossliπking methyl acrylates are selected from (A) to (BB):
Figure imgf000066_0001
and (iv) crosslinking methyl acrylamides are selcted from (A) to (PP):
Figure imgf000067_0002
Figure imgf000067_0001
Figure imgf000067_0003
12. The dimer or polymer of any one of claims 8 to 11 , wherein the dimer is a heterodimer.
13. A transfection method comprising providing at least one ABP according to any one of claims 1 to 7; admixing the at least one ABP with at least one nucleic acid; and transfecting a cell, in vitro or in vivo.
14. The transfection method of claim 13, wherein the nucleic acid is at least one siRNA and wherein the cell comprises a gene whose expression is targeted by said siRNA.
15. The transfection method of claims 13 or 14, wherein said at least one ABP is part of a micro- or nanoparticle.
16. The transfection method of claim 13, wherein more than one ABP form a degradable or nondegradable cationic polymer, preferably a cationic polymer according to any one of claims 8 to 12, and wherein the nucleic acid is pDNA.
17. A polymeric surface comprising a polymeric ABP according to any one of claims 8 to 12.
18. The polymeric surface according to claim 17, further comprising an agent covalently attached to (i) a reactive group of said ABP, or
(ii) a reactive group of a further molecule, wherein an attachment is a pre- or postpolymerization attachment.
19. The polymeric surface of claim 18, wherein said agent is at least one antimicrobial agent, preferably a peptidic antimicrobial agent.
20. A method for producing the polymeric surface according to any one of claims 17 to 19, wherein a carboxylic acid, an activated carboxylic acid, an aldehyde, a ketone, an amine, a hydrazine, an azide or an alkine is admixed prior to polymerization to a solution comprising one or more types of ABPs.
21. A method for linking one or more types of ABPs of any one of claims 1 to 7, wherein the ABPs are reacted to comprise one or more, preferably two, acrylamide and/or methylacrylamide groups, and polymerizing the resulting acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs via UV light, optionally in presence of a polymerization initiator, preferably a radical initiator or by heat.
22. A method for linking one or more types of ABPs of any one of the preceding claims, wherein the ABPs are reacted to comprise one or more, preferably two, acrylamide and/or methylacrylamide groups to provide acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs, comprising reacting terminal double bonds of said acrylamide ABPs and/or methylacrylamide ABPs, preferably diacrylamide ABPs and/or dimethyl diacrylamide ABPs with a further amine, and polymerizing the reaction product.
23. The method of claim 22, wherein the amine is a primary or secondary amine, preferably an amine selected from (1 ) to (94):
Figure imgf000069_0001
24. The method according to any one of claims 20 to 23, wherein said ABPs form linear polymers, 2d-sheets or 3d polymers.
25. The method according to any one of claims 20 to 24, wherein one or more ABPs may be chemically modified after polymerization.
26. The method according to any one of claims 20 to 25, wherein, proteins or natural lipids, in particular phospholipids, are admixed prior to polymerization.
27. Use of the ABPs of any one of claims 1 to 7 in the production of drug delivery vesicles, in particular nucleic acid transfection vesicles for medicine and research.
28. Use of the polymers of any one of claims 8 to 12 in the production of transfection vehicles and polymer surfaces according to claims 17 to 18.
29. Use of the ABPs of any one of claims 1 to 7 and the polymers of any one of claims 8 to 12 in vesicle based drug delivery.
30. Use of the polymer surfaces of claims 17 to 18 in clinical and medical use.
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WO2010128504A3 (en) * 2009-05-04 2011-01-13 Ben-Gurion University Of The Negev Research And Development Authority Nano-sized particles comprising multi-headed amphiphiles for targeted drug delivery
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