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WO2001005375A1 - Lipides d'ether vinyliques avec des groupes de tetes hydrophiles clivables - Google Patents

Lipides d'ether vinyliques avec des groupes de tetes hydrophiles clivables Download PDF

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Publication number
WO2001005375A1
WO2001005375A1 PCT/US2000/019430 US0019430W WO0105375A1 WO 2001005375 A1 WO2001005375 A1 WO 2001005375A1 US 0019430 W US0019430 W US 0019430W WO 0105375 A1 WO0105375 A1 WO 0105375A1
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Prior art keywords
lipid
vinyl ether
ethylene glycol
ether
poly
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PCT/US2000/019430
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English (en)
Inventor
David H. Thompson
Jeremy A. Boomer
Robert Haynes
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Purdue Research Foundation
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Publication date
Application filed by Purdue Research Foundation filed Critical Purdue Research Foundation
Priority to AU61053/00A priority Critical patent/AU6105300A/en
Priority to US10/031,033 priority patent/US6979460B1/en
Priority to EP00947445A priority patent/EP1202714A1/fr
Publication of WO2001005375A1 publication Critical patent/WO2001005375A1/fr

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    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers

Definitions

  • This invention relates to novel amphiphilic compounds with cleavable hydrophilic headgroups and their use in liposomes. More particularly, the invention relates to novel lipid compounds with hydrophilic headgroups linked to the molecule through a vinyl ether linkage, and their use in liposome vesicle formation and the triggered release of the liposomal contents or triggered permeabilization of, or fusion with, target lipid membranes.
  • the invention also relates to triggered cleavage of the headgroups of novel vinyl ether lipid compounds while incorporated in liposomes, to facilitate a phase transition of the liposome to effect the release of liposomal contents and/or the permeabilization of, and/or fusion with, cellular membranes by the liposomes.
  • Liposomes have been used as drug delivery vehicles with both passive and active-targeting schemes to attempt to site-specifically deliver the contents of the liposome to target tissues in vivo as well as in cell and tissue culture applications.
  • a significant drawback of previous methods of liposomal delivery systems has been constructing liposomes that have sufficient cell culture or in vivo stability to reach desired tissue sites and/or intra-cellular compartments, but will then efficiently release their contents once at the target site.
  • liposomal release mechanisms activated by light, heat, low pH, or enzymatic activity have been reported and reviewed.
  • the cleavage results in one or both of the hydrophobic tailgroups dissociating from the molecule, which causes local changes in the liposome structure leading to leaking of liposomal contents or to fusing of the liposome with adjacent membranes.
  • these lipids, with labile vinyl ether linkages joining the hydrophobic tail groups to the remainder of the molecule have limited sensitivity to desirable triggering conditions, as exhibited by slow liposomal content release rates and/or slow membrane fusion kinetics, to be optimal for many applications.
  • One theory for this is that the labile vinyl ether linkage may distribute in the hydrophobic region of lipid bilayers, where access to protons and oxidative agents is limited.
  • the cleavable hydrophilic headgroups of the vinyl ether lipids cleave from the remainder of the molecule under oxidative and/or acidic conditions and the dissociation of the hydrophilic headgroups causes a local phase transition in the lipid bilayer.
  • vinyl ether lipids with cleavable hydrophilic headgroups according to the present invention and their use in forming liposomes and the use of such liposomes to deliver desired therapeutic or diagnostic agents to desired tissues or cellular sites.
  • novel amphiphilic lipid compounds having a hydrophilic headgroup portion which is linked to the remainder of the molecule through a vinyl ether linkage, and a hydrophobic tailgroup portion, effective to anchor the compound in a lipid film or lipid bilayer membrane.
  • the hydrophilic headgroup is bonded to one double bonded carbon of the vinyl group and the ether oxygen is bonded to the other double bonded carbon in the vinyl group.
  • one or more hydrophobic tailgroups are bonded either directly to the ether oxygen, or bonded by an ether or ester linkage to a polyalcohol or other linking moiety, which is bonded directly to the ether oxygen.
  • each of said one or more hydrophobic tailgroups is independently selected from the group consisting of sterol, fatty acid ester, fatty alcohol, sphingosine, ceramide, phosphoglycerolipid, polyisoprenoid, and aryl ether.
  • R or R is a hydrophilic headgroup and the other is hydrogen, a second hydrophilic headgroup, or a crosslinker joining one or more other molecules of the vinyl ether lipid compound, each at the R 1 or R 2 position;
  • R 3 is an organic hydrophobic moiety; and
  • R is hydrogen or an electron donating group.
  • liposomes containing two or more species, or types, of lipids, at least one of which is a vinyl ether lipid as described above.
  • the liposome may optionally contain a therapeutic agent or diagnostic agent, which is desired to be transported to and released in a target tissue or across a target lipid bilayer membrane.
  • lipid compounds having a acid or oxidatively labile vinyl ether linked hydrophilic headgroup, said acid or oxidative conditions being effective for cleaving said hydrophilic headgroup from the compound.
  • Figure 1 is a flow chart for the synthesis of CVEP.
  • Figure 2 is a flow chart for the synthesis of DVEP.
  • Figure 3 is a comparison of calcein release from CVEP:DOPE liposomes under acidic conditions.
  • Figure 4 is a comparison of the release of calcein from DOPE liposomes containing varying concentrations of CVEP.
  • Figure 5 is a comparison of calcein release from CVEP:DOPE liposomes under acidic conditions in the presence of sink liposomes.
  • Figure 6 is a comparison of photooxidatively triggered calcein release from CVEP:DOPE liposomes.
  • Figure 7 is a flow chart of an alternative synthetic method for CVEP.
  • CVEP is l'-(4'-cholesteryloxy-3'-butenyl)- ⁇ -methoxy- polyethylene[112] glycolate
  • BVEP is (R)-l,2-di-O-(l'Z,9'Z-octadecadienyl)-glycerl-3-( ⁇ -methoxy- polyethylene [112] glycolate
  • DOPE is l,2-dioleoyl-s «-glycerophosphoethanolamine
  • DMAP is N,N-dimethyl-4-aminopyridine
  • EDCI is ethyldimethylaminopropyl carbodiimide
  • THF is tetrahydrofuran
  • DMF is dimethylformarnide
  • TBAF tetrabutylammonium fluoride
  • TOAB tetraoctylammonium bromide
  • TLC thin layer chromatography
  • amphiphilic describing a molecule, means having both a water-soluble polar head (hydrophilic) portion and a water-insoluble organic tail (hydrophobic) portion.
  • lipid is an inclusive term for fats and fat derived materials, including compounds which are or are related to glycerol esters and ethers, fatty acid esters, fatty alcohols, sterols, and waxes. They may be hydrophobic, or amphiphilic. When amphiphilic, the hydrophilic headgroup may be bonded directly to a hydrophobic tailgroup, such as a sterol, fatty acid or fatty alcohol, or the hydrophilic headgroup may be bonded to one or more hydrophobic tail groups through a linker group, such as, but not limited to a glycerol moiety.
  • vinyl ether means a moiety in a compound having two carbon atoms bonded to each other by a carbon-carbon double bond, and at least one ether oxygen bonded to one of said double-bonded carbons atoms.
  • acidic or oxidative conditions for triggering cleavage of the labile vinyl ether bond are to be understood as biologically suitable acidic or oxidative conditions; i.e. conditions compatible with biological systems.
  • the amphiphilic lipid compounds of the present invention comprise a hydrophobic tail portion effective for anchoring the compound in a lipid mono- or bilayer, a linking segment, which is a vinyl ether moiety bonded through the ether oxygen to the hydrophobic tail portion, and a hydrophilic headgroup bonded to the vinyl ether moiety either cis- or trans- to the vinyl ether oxygen.
  • the hydrophilic headgroup of a vinyl ether lipid compound of the present invention may be cleaved from the remainder of the compound by oxidation or acid hydrolysis of the ether bond.
  • the hydrophilic headgroups of the amphiphilic compounds are cis to the ether oxygen.
  • the cis isomers advantageously tend to be 3- 10 times more reactive than their corresponding trans-isomers.
  • Mixtures of cis- and trans-isomers may be used and may be blended to advantage to custom tailor the average rate of cleavage of the vinyl ether lipids in a population of liposomes to suit a given application.
  • hydrophobic and hydrophilic portions of amphiphilic lipids of the present invention may be selected to tailor the lipid to a given application.
  • Hydrophobic tailgroups may be bonded directly to the vinyl ether oxygen of the linking portion of the compound.
  • one or more hydrophobic groups may be bonded to a bridging moiety, which is bonded directly to the vinyl ether oxygen of the linking portion of the compound.
  • one or more hydrophobic tailgroups may be bonded through ether or ester linkages to a polyalcohol, as for example glycerol, butane- 1,4-diol, or a mono-, di-, or tri-saccharide moiety, which bridging moiety is the then bonded to the vinyl ether oxygen of the linking portion of the compound.
  • a polyalcohol as for example glycerol, butane- 1,4-diol, or a mono-, di-, or tri-saccharide moiety, which bridging moiety is the then bonded to the vinyl ether oxygen of the linking portion of the compound.
  • Preferred hydrophobic tailgroups include, but are not limited to, fatty acids and fatty alcohols, particularly C 5 -C 32 saturated and mono- or poly-unsaturated fatty acids and alcohols; sterols, particularly cholesterol and its derivatives, as for example, but without limitation, ergosterol, stigmasterol, sitosterol, lanosterol, pregnenolone, cortisol, estradiol, aldosterone, cholecalciferol, and cholic acid; sphingosine; ceramide, phosphoglycerolipids, polyisoprenoids; and aryl ethers, particularly phenolic ethers.
  • hydrophobic portion of lipids of the present invention may also be other amphiphilic lipids, particularly naturally occuring lipids, as for example, but without limitation, phosphlipids and sphingolipids, provided that the resulting amphiphilic compound is effective in inducing lamellar phase lipid bilayers, and that when the vinyl ether bond is cleaved, the dissociation of the hydrophilic headgroup effects a phase transition which destabilizes the lamellar phase, resulting in liposome leakage or permeablization of or fusion with a target membrane.
  • Oxidative conditions suitable for cleaving the hydrophilic headgroup include but are not limited to the generation of singlet oxygen by photoexcitation of oxidative sensitizer agents, as for example, but without limitation, bacteriochlorophyll a illuminated with near-infrared radiation at between about 670 nm and about 900 nm.
  • oxidative sensitizer agents include, without limitation, metallophthalocyanines, cyanines, metallopo ⁇ hyrins, phthalocyanines, porphyrins, phenathiazinequinones, pu ⁇ urins, chlorins, and other dyes which generate singlet oxygen, such as Rose Bengal, etc, each illuminated by radiation of a wavelength within their respective abso ⁇ tion bands.
  • Sensitizers can be introduced into the target tissues directly, or, in a preferred embodiment, by encapsulating the sensitizer in liposomes formed in part with the vinyl ether lipids of the present invention.
  • Several in vivo tissues and sub-cellular compartments also have oxidative environments able to cleave the hydrophilic headgroups of the lipid compounds of the present invention, as for example, but without limitation, phagosomes, activated macrophages, lymphocytes, neutrophiles, stratum cornium, epidermis tissue, dermis tissue, and subdermal tissue, and neurons undergoing demyelination.
  • Acidic conditions suitable for acid hydrolysis of the vinyl ether bond to dissociate the hydrophilic headgroups of the amphiphilic lipid compounds of the present invention include pH less than or equal to 6.5, preferably pH less than or equal to 5.5, and more preferably pH less than or equal to 4.5. Such conditions are typically found in cellular endosomes, ischemic tissues, skin tissues, and tissues in the gastrointestinal tract, among other tissues.
  • R 1 or R 2 is a hydrophilic headgroup and the other is hydrogen, a second hydrophilic headgroup, or a crosslinker joining at least one other molecule of tthhee vviinnyyll eetthheerr lliippiidd ccoommppoouunndd aatt tthhee RR 11 oorr RR 22 ppoossiittiioonn;
  • RR 33 is an organic hydrophobic moiety;
  • R 4 is hydrogen or an electron donating group.
  • Preferred vinyl ether lipid compounds of this aspect of the invention include compounds wherein R is selected from the group consisting of cholesterol, a cholesterol derivative, sphingosine, a sphingosine derivative, and a group of the formula
  • each R 5 is independently a hydrophobic group of the formula
  • n is an integer from 5 to 32 inclusive; y is an even integer from 2 to 12 inclusive, and wherein y is less than or equal to n.
  • hydrophobic tail groups are known in the art and are suitable for linkage through the ether oxygen of the lipid compounds of the present invention to provide a hydrophobic anchor for the compound in liposomes or other lipid mono- or bilayers.
  • R 4 may be either hydrogen or an electron donating group. Electron donating groups enhance the lability of the ether linkage to the hydrophobic tailgroup portion of the compound. Those skilled in the art may select electron donating groups to tailor the acid and/or oxidative lability of the vinyl ether lipid compound to suit a particular usage.
  • the skilled artisan may tailor the compound to cleave at a higher or lower pH and/or under greater or lesser oxidative conditions, and thereby better control the tissue or cellular location of cleavage and the rate of cleavage of the amphiphilic lipid compounds used in a given liposome population. This advantageously allows for the finer control of the rate of release of liposomal contents and/or the permeabilization of or membrane fusion with target membranes by liposomes containing the present vinyl ether lipid compounds to suit a particular application.
  • Suitable electron donating groups include, but are not limited to, C ⁇ -C 6 alkoxy, preferably CpC alkoxy, furan, thiophene, and mono-, di- or tri- C ⁇ -C 2 alkoxy substituted phenyl.
  • the hydrophilic headgroups of the vinyl ether lipid compounds of the present invention are hydrophilic moieties effective in producing an amphiphilic compound with the selected hydrophobic tail group(s) so as to induce formation of liposomes with other lipids which do not otherwise form stable liposomes under the desired target conditions.
  • the hydrophilic headgroups are likewise effective in stabilizing liposomes in the lamellar phase prior to the cleavage of the headgroups from the compounds. It is to be understood that when R 1 and R 2 are both hydrophilic headgroups, they are each independently selected from the same set of suitable hydrophilic headgroups.
  • hydrophilic headgroups include, but are not limited to, naturally occurring lipid hydrophilic headgroups, substituted and unsubstituted poly(ethylene glycol), water-soluble polymers with a molecular weight of about 10,000 or less, amino substituted carbamates, mono-, di-, tri-, and oligosaccharides.
  • Preferred hydrophilic headgroups include poly(ethylene glycol), C C 6 alkoxy poly(ethylene glycol), poly(ethylenimine), N,N-di(aminoethyl)carbamyloxyethyl-, choline, monosaccharide, disaccharide, ethanolamine, phosphatidylcholine, phosphatidylethanolamine, cardiolipin, phosphatidylmonosaccharides, such as phosphatidylinositol, and phosphatidyldisaccharides.
  • Particularly preferred hydrophilic headgroups include poly(ethylene glycol) and C]-C 6 alkoxy terminated poly(ethylene glycol) and N,N-di(aminoethyl)carbamyloxyethyl-.
  • the hydrophilic headgroup is a poly(ethylene glycol) chain containing an average of between 1 and about 300 glycol units, more preferably an average of between about 10 to about 150 glycol units. In another embodiment, the poly(ethylene glycol) chain contains an average of between about 40 to about 125 glycol units.
  • R 1 or R 2 is a crosslinker group to another molecule of the vinyl ether lipid compound
  • two or more lipid compounds according to the present invention may be crosslinked together to obtain a cascading amplification effect.
  • the vinyl ether bonds of all the crosslinked molecules would need to be cleaved before the hydrophilic headgroups would dissociate, but then the large number of headgroups dissociating at once induce a larger, potentially more instantaneous, effect on the local membrane structure.
  • crosslinker moiety bonds to the individual vinyl ether lipid units at either the R 1 or R" position, with the other position in the respective units being the hydrophilic headgroup moiety.
  • Several vinyl ether lipid units may be crosslinked in this fashion by selecting a crosslinker with a plurality of crosslinking functional moieties, as for example, a polymer with an appropriate funtionality.
  • Suitable crosslinkers include, but are not limited to triethylenediamine, ⁇ , ⁇ - polyethylene glycol, ⁇ , ⁇ -polyethylenimine, ⁇ , ⁇ -polygylcidol, ⁇ , ⁇ -polyacrylic acid, polylysine, polyarginine, spermine, and spermidine.
  • the vinyl ether lipid compounds of the present invention can be synthesized in a variety of methods.
  • One such method begins by protecting one hydroxyl group of 1 ,4-butanediol with a blocking reagent, such as t-butyldimethylsilylchloride, followed by oxidation of the second hydroxyl to provide a carboxyl group, as for example with potassium permanganate.
  • An ester bond is then formed by a condensation reaction with the carboxy group and a hydroxy group on the desired hydrophobic tailgroup portion, as for example by reaction with cholesterol or the dioleoyl ester of glycerol in DMAP and EDCI.
  • the ester bond is then converted to a vinyl ether bond through a phosphonyl intermediate, followed by reduction with a palladium/aluminum catalyst, as for example, by reacting the ester with n-butyllithium and diisopropylamine, followed by reaction with chlorodiethylphosphate to produce the diethylphosphonyl-1- butene, and then reacting the phosphonyl butene intermediate with tertrakistriphenylphosphate palladium and triethylaluminum in methylene chloride to produce the racemic vinyl ether lipid.
  • a palladium/aluminum catalyst as for example, by reacting the ester with n-butyllithium and diisopropylamine, followed by reaction with chlorodiethylphosphate to produce the diethylphosphonyl-1- butene, and then reacting the phosphonyl butene intermediate with tertrakistriphenylphosphate palladium and trie
  • the blocking group is removed to regenerate a hydroxyl group, which is then condensed with a free carboxy group on the hydrophilic headgroup to yield an amphiphilic lipid compound with a vinyl ether linked hydrophilic headgroup according to the present invention.
  • 2-Vinyl-l,3-dioxolane is reacted with an ⁇ -protected alkyllithium, such as 4'-t- butyldiphenylsilyl-2-(2'-butenyl)-l,2-dioxolane, to effect the vinyl addition of the alkyl group with the opening of the dioxolane ring, forming a vinyl ether group and a reactive oxide ion.
  • a weak acid such as water, or a sulfonyl chloride such as mesyl or tosyl chloride, is added to yield the corresponding alcohol or sulfonate.
  • liposomes comprising at least two different species, or types of lipid compounds, at least one of which is an amphiphilic lipid compound having a vinyl ether linked hydrophilic headgroup as described above.
  • Methods for forming lipid vesicles or liposomes, particularly those containing desirable agents such as therapeutic drugs and/or diagnostic indicators, are well known in the art. It is likewise known how to select lipids to provide a general targeting of the liposome for specific tissue or cell types.
  • the present invention provides for the modification for such liposomes or lipid vesicles to facilitate their interaction with biological membranes when the liposomes come in contact with the target tissues or membranes, primarily to allow the delivery of the liposome contents to the target tissue or across the target membrane.
  • liposomes are made to include at least one species of vinyl ether lipid compound of the present invention.
  • the one or more vinyl ether lipid compounds of the present invention constitute between about 0.1 % and about 20% of the molar lipid content of the liposomes. More preferably, the liposomes molar lipid content contains between about 1.0% and about 15% vinyl ether lipid compound. In one embodiment of the present invention the liposomes contain between about 3.0% and about 10% vinyl ether lipid on a molar basis of the lipid content. It is to be understood that the vinyl ether lipid concentrations may be the sum of one or more vinyl ether lipid compounds of the present invention as desired for a particular application.
  • liposomes By the controlling the selection of the specific vinyl ether lipid compound or compounds used in the liposome synthesis and their relative concentrations, as well as the selection of other lipids and targeting agents, etc., the skilled artisan can tailor construct liposomes of a given stability for circulation, and of a desired release rate profile or fusogenicity to suit a particular therapeutic or diagnostic indication.
  • the lipid does not form vesicles, but rather exists in aqueous media in a hexagonal tubular array.
  • the lipid mix can form liposomes.
  • About 3-5 mole percent CVEP:DOPE can be used to form liposomes of suitable stability for cell culture uses, whereas about 5-10 mole percent is preferred for in vivo applications.
  • the skilled chemist will be able to select optimum liposome compositions to suit a given application.
  • Liposomes comprising one or more vinyl ether lipid compounds of the present invention and containing a desired therapeutic or diagnostic agent encapsulated within the liposome may be used to deliver the agent to a desired target tissue or across a biological membrane, as for example delivering the agent to the interior of a living cell within a target tissue.
  • Target tissues or cells are contacted with liposomes encapsulating a desired therapeutic agent or diagnostic agent according to the present invention under acidic or oxidative conditions effective to cleave the vinyl ether bond of the vinyl ether lipids, thereby dissociating the hydrophilic headgroups from the hydrophobic tailgroup portions of the molecules.
  • the dissociation causes a destabilization of the liposome, as for example by a phase transition from the lamellar phase to a hexagonal phase, thereby causing leakage of the liposomal contents into the tissue or cellular site, or a permeabilization of or fusion with a target membrane resulting in releasing the liposomal contents into the cellular compartment across the membrane.
  • the liposomes encapsulating a therapeutic or diagnostic agent are designed to be endocytosed by the target cell population.
  • the endocytic vesicle Upon uptake, the endocytic vesicle is naturally acidified, which causes cleavage of the vinyl ether bond of the vinyl ether lipids, dissociating the hydrophilic headgroups therefrom. The dissociation destabilizes the liposome, inducing fusion of the liposomal membrane with the cellular endocytic vesicle membrane resulting in the release of the liposomal contents, including the therapeutic or diagnostic agent, into the cytoplasm of the cell.
  • liposomes encapsulating a therapeutic or diagnostic agent are designed to accumulate in a target tissue having an acidic interstitial environment, as for example certain tumor tissues or ischemic tissues.
  • the acidic conditions Upon reaching the target tissue, the acidic conditions cause cleavage of the vinyl ether bonds of the vinyl ether lipids, dissociating the hydrophilic headgroups therefrom.
  • the dissociation destabilizes the liposome, inducing the breakdown of the liposome to release the liposomal contents, including the therapeutic or diagnostic agent, into the interstitial fluid of the tissue.
  • the destabilization may induce fusion of the liposomal membrane with the cellular membrane resulting in the release of the liposomal contents into the cytoplasm of the cells in the target tissue.
  • the liposomes encapsulating a therapeutic or diagnostic agent also contain an oxidative sensitizer agent, as for example, bacteriochlorophyll a, or an agent that can be activated to induce acidification of the liposome (an acidification agent).
  • an oxidative sensitizer agent as for example, bacteriochlorophyll a
  • an agent that can be activated to induce acidification of the liposome an acidification agent.
  • the liposomes are designed to accumulate in a predetermined tissue type. When the liposomes reach the target tissue, the oxidative sensitizing agent is excited or the acidifying agent is activated, thereby causing the cleavage of the vinyl ether lipids, dissociating the hydrophilic headgroups therefrom.
  • the dissociation destabilizes the liposome, inducing the breakdown of the liposome to release the liposomal contents, including the therapeutic or diagnostic agent, into the interstitial fluid of the tissue.
  • the destabilization may induce fusion of the liposomal membrane with the cellular membrane resulting in the release of the liposomal contents into the cytoplasm of the cells in the target tissue.
  • reaction was run for 12 min before quenching with 4 M NaOH (100 ml) and product extraction with ether (3x 100ml). The ether layer was then dried over MgSO 4 , filtered, evaporated, and dried in vacuo. The crude reaction mixture was then purified via silica gel chromatography (60-200 mesh, 2 cm diameter x 5 cm height) using 1: 1 hexane:ether to elute the starting material (4.8 g), followed by a step gradient of ethyl acetate to elute the product (7.1 g, 61% yield; 97% yield based on converted starting material).
  • the crude product was purified via silica gel flash chromatography (230-400 mesh, 8: 1 hexane:ether, 2 cm diameter x 25 cm height) to give 1.05 g product (53% yield based on consumed starting material) and 751 mg starting material.
  • TLC 2: 1 hexane:ether, I 2 stain, 0.71 (dark, product), 0.51 (impurity), 0.41 (dark, starting material).
  • Tetrakistriphenyl-phosphine palladium 40 mg, 34.6 ⁇ mol
  • 13 250 mg, 269 ⁇ mol
  • Triethylaluminum (471 ⁇ l, 471 ⁇ mol, 1M hexane solution) was added dropwise via syringe.
  • the reaction was warmed to room temperature and stirred for 4h.
  • the product mixture was filtered through a small silica plug with anhydrous ether, the filtrate evaporated, and dried in vacuo.
  • the crude mixture was purified via silica gel chromatography (60-200 mesh, 8:1 hexane:ether, 2 cm diameter x 10 cm height).
  • the synthesis of these materials begins with the formation of the corresponding 1-X-propenone in good yields using a Mannich coupling and Hoffman elimination scheme. (See V. J. Gutzmann, P. Messinger, Arch. Pharm. 1995, vol. 328, pg. 523-525, or P. Messinger, Arch. Pharm. 1973, vol. 306, pg.
  • Example 4 Liposome Vesicle Preparation. DOPE and the labile PEG lipid were co-dissolved in chloroform that had been prefiltered through a 2.53 cm plug of anhydrous sodium carbonate to remove traces of acid and water from the solvent. This solution was evaporated under a gentle stream of N and further dried under vacuum ( ⁇ 200 ⁇ , 4 h). Vesicles were formed by hydrating the lipid film in the presence of 50 mM calcein using five LN 2 freeze-thaw- vortex cycles. This suspension was then extruded at 50°C through two lOOnm track- etch polycarbonate membrane filters [9].
  • Extraliposomal calcein was removed using a single pass through a 40 cm Sephadex G-50 gel column equilibrated with 150 mM NaCl. The fraction eluting at the void volume was collected and stored at 8°C until use.
  • Light-sensitive vesicles were prepared in the same manner, except that bacteriochlorophyll a (Bchl) was codissolved in the chloroform lipid solution. Other agents or mixtures of agents, such as therapeutic compounds or diagnostic agents, are substituted for calcein in the above protocol resulting in liposomal encapsulation of the substituted agent.
  • Example 5 Triggered Release Assay. Vesicle release of the liposomes made in example 4 above, was monitored at
  • Acid-trigged release was initiated by dilution of the vesicles into an acidic buffer solution as described by Gerasimov et al [10].
  • Light-triggered release was promoted by aerobic illumination (800 nm, 1 W) of a continuously stirred 1 cm quartz cuvette with a SDL 820 diode laser coupled to an optical fiber [11]. The fiber was mounted pe ⁇ endicular to the cuvette surface to produce a spot of -3 mm diameter. Both triggering methods were conducted either in the absence or presence of ten-fold excess of egg phosphatidylcholine (EPC) vesicles as a membrane "sink”. Results Acid-triggered Release.
  • EPC egg phosphatidylcholine
  • the release rate properties of 98:2 DOPE:DVEP vesicles ( Figure 5) demonstrates that DVEP shares some of the characteristics of DOPE:DVEP vesicles, namely the lack of leakage at pH 7.4, regardless of whether sink EPC is present or not.
  • DOPE:CVEP:Bchla (pH 7.4) under photooxidative conditions is shown in Figure 6. Irradiation of these vesicles lead to slow release in the absence of sink EPC, however, the observed release rate increased significantly when sink lipid was present. Background (dark) calcein leakage from the vesicles was not significant, even in the presence of sink EPC. Cytoplasmic Delivery. DOPE:CVEP/calcein vesicles using folate as a targeting ligand (DSPE-PEG3350-folate) initially revealed punctuated fluorescence, followed by diffuse cytoplasmic fluorescence.
  • DSPE-PEG3350-folate targeting ligand
  • Example 6 Comparative cleavage rates for CVEP and BVEP.
  • BVEP having two vinyl ether linkages, each bonding a hydrophobic tailgroup to a glycerol moiety, which in turn is bonded through an ester linkage to a polyethylene glycol hydrophilic headgroup is synthesized by known methods. See J.A. Boomer & D.H. Thompson, Chem. Phys. Lipids (1999) vol. 99, pg. 145-153. Rates of cleavage by acid hydrolysis and oxidation are measured in a comparative study for CVEP and BVEP as follows:

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Abstract

La présente invention concerne un composé lipidique amphiphilique avec un groupe de tête hydrophile relié à l'éther vinylique. L'invention porte également sur des loposomes contenant le composé de lipide et d'éther vinylique, qui peuvent être amorcés pour libérer leurs contenus et /ou perméabiliser ou se condenser avec des membranes lipidiques cibles. La liaison clivable d'éther vinylique permet au groupe de tête hydrophlique d'être dissociée du(des) groupe(s) de queue hydrophobes du composé lipidique afin de faciliter les transitions de phase dans la bicouche lipidique.
PCT/US2000/019430 1999-07-16 2000-07-17 Lipides d'ether vinyliques avec des groupes de tetes hydrophiles clivables WO2001005375A1 (fr)

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AU61053/00A AU6105300A (en) 1999-07-16 2000-07-17 Vinyl ether lipids with cleavable hydrophilic headgroups
US10/031,033 US6979460B1 (en) 1999-07-16 2000-07-17 Vinyl ether lipids with cleavable hydrophlic headgroups
EP00947445A EP1202714A1 (fr) 1999-07-16 2000-07-17 Lipides d'ether vinyliques avec des groupes de tetes hydrophiles clivables

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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003047499A2 (fr) 2001-12-04 2003-06-12 Ben Gurion University Of The Negev Research And Development Authority Composes amphiphiliques et vesicules/liposomes pour le ciblage de medicaments propre a un organe
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