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WO2012119781A2 - Novel lipids, phospholipids, phospholipid and lipid compositions and their use - Google Patents

Novel lipids, phospholipids, phospholipid and lipid compositions and their use Download PDF

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Publication number
WO2012119781A2
WO2012119781A2 PCT/EP2012/001053 EP2012001053W WO2012119781A2 WO 2012119781 A2 WO2012119781 A2 WO 2012119781A2 EP 2012001053 W EP2012001053 W EP 2012001053W WO 2012119781 A2 WO2012119781 A2 WO 2012119781A2
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group
cis
trans
double bonds
optionally substituted
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French (fr)
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WO2012119781A3 (en
Inventor
Till SAXER
Andreas Zumbuehl
Bert MULLER
Illya FEDOTENKO
Margaret HOLME
Pierre-Léonard ZAFFALON
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Universite de Geneve
Universitaet Basel
Hopitaux Universitaires De Geneve
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Universite de Geneve
Universitaet Basel
Hopitaux Universitaires De Geneve
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Publication of WO2012119781A3 publication Critical patent/WO2012119781A3/en
Anticipated expiration legal-status Critical
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    • 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
    • C07F9/10—Phosphatides, e.g. lecithin
    • C07F9/106—Adducts, complexes, salts of phosphatides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/688—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols both hydroxy compounds having nitrogen atoms, e.g. sphingomyelins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
    • 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
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
    • 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

Definitions

  • Phospholipids are composed of a hydrophilic "head” and a hydrophobic "tail".
  • the hydrophilic head contains the negatively charged phosphate group, and may contain other polar groups.
  • the hydrophobic tail usually consists of long fatty acid hydrocarbon chains. Their specific properties allow phospholipids to play an important role in phospholipid bilayers or cell membranes. In biological systems, the phospholipids often occur with other molecules (e.g. proteins, glycolipids and cholesterol) in a bilayer such as a cell membrane.
  • Nanoparticles are widely known and applied for delivery of active compounds in many applications like pharmaceuticals and cosmetics. They may be composed of various materials and often lipids, phospholipids or cholesterol are used to prepare them.
  • Nanoparticles have been described and they may be loaded with a so-called payload. Such loaded nanoparticles find many technical applications.
  • the release of the payload is caused by various mechanisms and means.
  • the nanoparticle may fuse with a bilayer membrane and thus release its content, be endocytosed by a cell, or mechanical force, e.g. shear stress, may be applied and result in the release of the nanoparticles' payload.
  • Shear stress denoted t
  • t can be defined as a force per area, which is applied parallel or tangential to a surface of a material, as opposed to a force applied perpendicularly.
  • a drug may be applied systematically or by way of a targeted delivery.
  • Various methods and approaches are known in the art.
  • Disease states of patients where a targeted delivery of a drug would be advantageous are e.g. vascular diseases and in particular ischemia.
  • Yet another object is to provide a means and methods of delivering a desired compound to a target site and a transport vehicle which may easily and controllable release the desired compound.
  • the invention relates to a composition
  • a composition comprising or consisting of 1,3- diamidophospholipids or/and 1 ,2-diamidophospholipids or/and 2,3- diamidophospholopids or/and another lipid and preferably at least a further compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant.
  • Another aspect of the invention relates to a method of making a 1,3- diamidophospholipid wherein a phosphoethanolamine is alkylated under appropriate conditions, preferably with the use of dimethyl sulfate.
  • the invention relates to a method of making a composition according to the invention and to a method of making nanoparticles.
  • the invention relates to a composition or nanoparticles according to the invention for use in a pharmaceutical or cosmetic application.
  • the invention relates to a composition or nanoparticles according to the invention for use in the prophylaxis or treatment of a vascular disorder or disease, or for use in the prophylaxis or treatment of a dermatological disorder or disease, or for use as cosmetic, or for use in a monitoring or diagnostic method.
  • the invention relates to a composition or nanoparticles according to the invention for use in the targeted delivery of a selected compound.
  • the selected compound is preferably released at a target site due to endogenous shear stress at the target site.
  • the invention relates to a method of a targeted release of an active or selected compound or mixture of compounds from nanoparticles at a release site in a tubular system, preferably a vascular vessel system, due to endogenous shear stress wherein the nanoparticles are being recycled at least 2 times in said vascular vessel system and the active or selected compounds or mixture of compounds are periodically released from the nanoparticles at a target site, preferably wherein the amount of compound(s) released is dependent on the shear stress within the vascular vessel system at the target site with an advantageously engineered release profile.
  • FIGS. 1 - 4 are depicting the characterization of preferred embodiments of the phospholipids according to the invention.
  • FIG. 5 is depicting vortex assisted release of carboxyiluorescein from vesicles formed from Pad-PC-Pad ( ), DPPC ( ) and SMI 6 ( ) as percentage release over time.
  • FIG. 6 is depicting free release (no vortex treatment) of carboxyfluorescein from vesicles formed from Pad-PC-Pad ( ), DPPC ( ) and SMI 6 (.. ) as percentage release over time.
  • FIG. 8 is depicting release of carboxyfluorescein from Egg-PC vesicles with varying percentage of Pad-PC-Pad incorporation, after one pass through the in vitro artery model as described in the examples.
  • FIG. 8B shows the difference in release between the healthy and stenosed artery models. Results are the averages of three experiments.
  • FIG. 9 is depicting release of carboxyfluorescein from 100% Pad-PC-Pad vesicles illustrating the fractional release under exposure to increasing shear stress during one pass through the in vitro artery model as described in the examples.
  • FIG. 12 depicts the shear stress dependent release from nanoparticles comprising Pad- PC-Pad, i.e. a lipid according to the invention, in conditions similar to physiologic conditions due to the addition of HSA (human serum albumin) and at physiological temperature of at 37 °C.
  • HSA human serum albumin
  • composition of the invention comprises or is consisting of 1,3-diamidolipids or/and 1 ,2-diamidolipids or/and 2,3-diamidolipids or/and 1 ,3-diurealipids or/and 1,2-diurealipids or/and 2,3-diurealipids or/and 1,3- dithiourealipids or/and 1 ,2-dithiourealipids or/and 2,3-dithiourealipids or/and 1 ,3- diacylurealipids or/and 1 ,2-diacylurealipids or/and 2,3-diacylurealipids or/and 1- amidolipids or/and 1 -urealipids or/and 1 -thiourealipids or/and 1 -acylurealipids or/and cyclic-amidolipids or/and cyclic urealipids or/and cyclic thiourealipids or/and cyclic
  • composition as described above wherein the 1,3-diamidophospholipid or 1, 3-diaminolipid has the following Formula la:
  • alkyl- such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
  • undecyl- preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
  • primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl- heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
  • acylureas such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl- heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
  • x preferably, decyl-, dodecyl-, tetradecyl, hexadecyl-amide
  • xi an optionally substituted C1-C8 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
  • xii an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
  • xiii an optionally substituted C16-C22 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
  • lipid may be fully or partially deuterated, or radioactively labeled;
  • a proton (H) a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide, and wherein "Ci" and "C 2 " may be H or a methyl.
  • 1, 3-diamidophospholipids of the invention of Formula la are defined as follows: i. "Bi” is equal or different from “B 2 ", and "Bi” and “B 2 " is selected from: ii. H;
  • undecyl- preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
  • lipid may be fully or partially deuterated, or radioactively labeled; wherein "A” is selected from:
  • a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide; and wherein "Ci" and "C 2 " is selected from H or methyl.
  • Ci 8 -C 24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds
  • primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
  • acylurea such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
  • x preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amide
  • xii an optionally substituted C9-C15 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
  • xiii an optionally substituted C16-C22 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
  • Di and D 2 can be the same or can be different and are either O (oxygen) or S (sulfur),
  • lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate
  • alkyl- such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
  • undecyl- preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
  • Ci-C 10 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds
  • Ci8-C 24 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds
  • x. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl- heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
  • xi preferably, decyl,- dodecyl-, tetradecyl, hexadecyl-amine
  • xii an optionally substituted C1-C8 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
  • xiii an optionally substituted C9-C15 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
  • acylureas such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
  • xix preferably, decyl,- dodecyl-, tetradecyl-, hexadecyl-amide
  • Di is either O (oxygen) or S (sulfur),
  • lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phospho inositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphat
  • alkyl- such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
  • undecyl- preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
  • Ci8-C 24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
  • primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
  • " and "B 2 " are the same or different and are selected from:
  • acylureay such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
  • x preferably, decyl,- dodecyl-, tetradecyl-, hexadecyl-amide
  • xii an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
  • xiii an optionally substituted C16-C22 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
  • Di and D 2 can be the same or can be different and are either O (oxygen) or S (sulfur),
  • lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate
  • alkyl is to be understood as any chain of C-atoms being linear or branched
  • optionally substituted is to be understood as having no substitutions or being substituted with residues being compatible with the remaining molecule without interfering with its structure and providing optional functionality in the context of the invention.
  • the non-natural phospholipids may be defined to have the form Xn-HG-Ym wherein HG (head group) may be selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA) and phosphatidylglycerol (PG) and X, Y are saturated or/and unsaturated aliphatic chains of 10, 12, 14, 16 and 18 carbons, wherein n, m may comprise an ester (es), amide (ad), amine (an), and ether (et) linkers.
  • HG head group
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • PA phosphatidic acid
  • PG phosphatidylglycerol
  • X, Y are saturated or/and unsaturated aliphatic chains of 10, 12, 14, 16 and 18 carbons
  • n, m may comprise an ester (es), amide (ad), amine (
  • HG for “head group” which may include “phosphoethanolamine” denoted as “PE”, “phosphocholine” as “PC”, “phosphatidic acid” as “PA”, “polyethylene glycol” as “PEG”, “phosphoglycerol” as “PG”, “EYPC” as “egg yolk phosphocholine”.
  • ester may be denoted “es”, “amide” as “ad”, “amine” as “an”, and “ether” as "et”.
  • acylurea is to be understood as generally known by the skilled person in the field of the invention and is an urea moiety containing an additional carbonyl group alpha to the amine.
  • Pad-PC-Pad of which the positive mechano-sensitive effect has been shown.
  • Pad-PC-Pad can e.g. be combined with any of the other compounds of the invention or described herein.
  • the surfactant applied in a preferred embodiment in the composition according to the invention may be any known and useful surfactant.
  • the surfactant is selected from an anionic, cationic or non-ionic surfactant. More preferred the surfactant is selected from the group consisting of a Brij, preferably Brij P4, Brij S4, Brij SlO or Brij P10.
  • the composition may contain any useful number of compounds as described above and also the ratios of single compounds and compound groups may vary. The ratios can be modified and adapted according to the particular application of the composition of the invention.
  • the first and second compounds are present in a ratio of from 99.9 : 0.1 to 1 : 99 mol-%, preferably from 90 : 10 to 40 : 60 mol-%, more preferably from 70 : 30 to 50 : 50 mol-%, even more preferably wherein the first compound is present in either 50 mol-%, 60 mol-%, 70 mol-%, 75 mol-%, 80 mol-%, 85 mol-%, 90 mol-%, 95 mol-%, or 98 mol-%
  • compositions according to the invention comprising one or more synthetic compounds according to the invention in an amount of 80 to 98 mol-% , preferably 90 to 98 mol-%, more preferably 95 to 98 mol-% in combination with any of the other compounds described above exhibit superior release features when applied in nanoparticles of the invention which are useful in the targeted delivery of active or selected compounds.
  • the advantageous release features can be demonstrated by way of the in vitro model described below and in the experimental section.
  • compositions of the invention are 1,3- diamidophospholipids while in various compositions of the invention also 1,2- diamidophospholipids and 2,3-diamidophospholipids will be particularly useful to achieve advantageous release features in nanoparticles according to the invention.
  • the inventors arrived at lowering the stability of nanoparticles by the addition of synthetic phospholipids and/or lipids according to the invention to natural phospholipids in an amount of at least 50 mol-%, preferably at least 60 mol-%, more preferably at least 70 mol-%, more preferably at least 75 mol-%, more preferably at least 80 mol-%, more preferably at least 85 mol-%, more preferably at least 90 mol- %, more preferably at least 95 mol-%, or more preferably at least 98 mol-% In these preferred compositions additional compounds as described above may be present.
  • a synthetic phospholipid or preferably lipid as described above will be mixed with egg yolk phosphatidylcholine (EYPC) and with the surfactant Brij S10 (decaethylene glycol octadecyl ether).
  • EYPC egg yolk phosphatidylcholine
  • Brij S10 decaethylene glycol octadecyl ether
  • compositions of the invention may contain any synthetic phospholipid (e.g. Pad-PC -Pad) or preferably lipids according to any of formula la, lb, Ic and Id with 0-10 mol% surfactants such as the Brij family (e.g. S10, P10 or/and P4), 0-50 mol% rigidifiers such as cholesterol, and 0-25 mol% natural phospholipids such as EYPC, DOPC, POPC, DPPC, sphingomyelin and miltefosine.
  • any synthetic phospholipid e.g. Pad-PC -Pad
  • lipids e.g. Pad-PC -Pad
  • lipids e.g. Pad-PC -Pad
  • lipids e.g. Pad-PC -Pad
  • surfactants such as the Brij family (e.g. S10, P10 or/and P4)
  • 0-50 mol% rigidifiers such as cholesterol
  • compositions of the invention according to any of formulae la, lb, Ic and Id may comprise natural phospholipids (e.g. EYPC, DOPC, POPC, DPPC, sphingomyelin) with cholesterol, miltefosine and octadecanol in various concentrations up to 50 mol%, preferably 10 - 40 mol%, 20 - 30 mol%, or 30 - 50 mol%.
  • natural phospholipids e.g. EYPC, DOPC, POPC, DPPC, sphingomyelin
  • cholesterol miltefosine
  • octadecanol in various concentrations up to 50 mol%, preferably 10 - 40 mol%, 20 - 30 mol%, or 30 - 50 mol%.
  • the achievement of the inventors is to a great extent that they found that the synthetic phospholipids, and in a preferred embodiment the lipids of the invention, preferably in combination with natural lipids and eventually additional compounds could be tailored to endogenous shear stress levels in diseases, disease states or disorders to achieve a targeted delivery of an active compound and overcome thus the disadvantages of the state of the art in treating such diseases, disease states or disorders.
  • compositions according to invention may further comprise an active compound or selected compound.
  • active compound is preferably used for compounds which are applied in a medical context and preferably relate to drugs or any compounds which are used for the purpose to achieve an effect in a system, e.g. an animal or human body.
  • selected compound preferably is used for any compound that is supplied or delivered by application of the invention to a system and may be used for monitoring or diagnostic purposes. In general the selected compound does not usually change the system wherein it is supplied to, however, it is predominantly used to monitor, measure or diagnose a certain change in a system.
  • This active or selected compound may be chosen from any known class of chemical compounds and have different properties. It may have pharmaceutical, diagnostic, biomarker or other properties as may be required for the particular application of the composition of the invention.
  • the active or selected compound may also be denoted as "payload". In the context of the invention this payload will be incorporated by known techniques with the composition of the invention and may be produced as vesicles. In particular applications of the invention, the payload will be transported in a medium or a system like the circulation of a patient to a particular target site where the payload is released. The amount and timing of the release can be engineered according to the circumstances and the particular mixture of chemical compounds in the composition to arrive at a desired release profile.
  • the active compound is selected from the group consisting of a fibrinolytic agent, an anti-coagulation agent, an anti-aggregation agent, an atherosclerotic plaque stabilizer (Statin), a vasodilatory agent, preferably a direct or indirect acting vasodilator (a NO-liberating agent, an alpha-adrenoreceptor antagonist, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a direct renin inhibitor, a calcium-channel blocker (CCB), an endothelin receptor antagonist, a phosphodisesterase inhibitor, a potassium-channel opener,), anti-arrhytmic drugs (a sodium-channel blocker, a beta-blocker, a potassium-channel blocker, a calcium-channel blocker), inotrop positive medication (a catecholamine, a non-catecholamine), heart muscle remodeling (ACE inhibitors or ARB), diastolic dysfonction treatment (antical
  • the active compound is selected from the group consisting of alteplasum (Actilyse®), heparin (Liquemine®), acetyl salicylic acid (Aspirin®), clopidogrelum (Plavix®), glycoprotein Hb/IIIa inhibitor such as ReoPro®, rosuvastatinum (Crestor®), NO-liberating agents such as nitroglycerin (Perlinganit®), nitroprussiate or molsidomine (Corvaton®), phentolamine (Regitine®), enalapril (Epril®), candesartanum (Atacand®), diltiazem (Dilzem®), bosentan (Tracleer®), milrinone (Corotrop®) or levosimendan (Simdax®), minoxidilum (Loniten®), aliskirenum (Rasilez®), quinidine ,metoprolol (Lo
  • composition according to the invention preferably may be provided in the form of nanoparticles wherein the nanoparticle is preferably a micelle, more preferably the nanoparticle is composed of a monolayer, a bilayer and/or a vesicle and/or a nanocontainer.
  • the nanoparticles according to the invention may be designed in any useful size and amount.
  • the nanoparticle has an average diameter of about 10 to 1000 nm, preferably of about 50 to 500 nm, more preferably of about 50 to 200 nm.
  • the invention related to a method of making a 1,3- diamidophospholipid wherein a phosphoethanolamine is alkylated under appropriate conditions, preferably with the use of dimethyl sulfoxide.
  • Other methods of alkylation with alternative alkylating agents may be used such as alkylation with the corresponding alkylhalide, preferably methyliodide (Lu, X.; Bittman, R. The Journal of Organic Chemistry 2005, 70, 4746-4750)
  • the inventors thus could provide for a simple and economically advantageous method of making the compounds according to the invention.
  • the invention relates to a method of making a composition according to the invention as described above.
  • Another aspect of the invention relates to a method of making the composition of the invention comprising mixing the first and second compound with appropriate means.
  • the invention in another aspect relates to a method of making nanoparticles comprising a composition and preferably a payload as described above.
  • Nanoparticles can be made according to techniques known in the art (e.g. "Preparation of Vesicles (Liposomes)" by Peter Walde in Encyclopedia of Nanoscience and Nanotechnology, Volume 9, pp. 43-79(37)).
  • the nanoparticles are produced by thin film hydration, and/or one or more freeze-thaw cycles, sonication or/and extrusion, or by a electroformation method or by hydrating spray-dried lipids or by sonication or by repetitive freezing and thawing or by dehydration and rehydration or by the extrusion technique or by the treatment of a multilamellar vesicle suspension with a microfluidizer, or the preparation of multilamellar novasomes or the preparation of multilamellar spherulites, or the preparation of multilamellar vesicles by the "bubble method” (1-1.
  • Another aspect of the invention relates to a pharmaceutical or cosmetic composition
  • a pharmaceutical or cosmetic composition comprising a composition according to the invention as described above or a nanoparticle as described above, and preferably further useful carriers or/and additives.
  • composition according to the invention and the nanoparticles according to the invention for use in a pharmaceutical or cosmetic application.
  • compositions of the invention will be prepared using known methods and useful auxiliary compounds as known in the art and applicable in the context of the invention.
  • compositions according to the invention or nanoparticles according to the invention for use in the prophylaxis or treatment of a vascular disorder or disease, or for use in the prophylaxis or treatment of a dermatological disorder or disease, or for use as cosmetic, or for use in a monitoring or diagnostic method.
  • the invention relates to a method for the prophylaxis or treating a patient in need thereof by administering nanoparticles according to the invention with an active compound as describe above to a patient in an effective dosage to a patient.
  • the treatment or prophylaxis may be for a vascular disorder or disease, or a dermatological disorder or disease.
  • the method may be as well a cosmetic method wherein preferably the cosmetic method comprises topical applications.
  • the invention may be applied in a method of treatment or prophylaxis or use for treatment or prophylaxis in any disease or disorder wherein a targeted delivery of an active compound is advantageous.
  • One advantage of the invention is that the dosage delivered to a patient may be reduced due to the use of the inventive compositions or nanoparticles.
  • Another or additional advantage that may be achieved by the invention is the reduction of undesired side effects due to the targeted delivery of the active compound.
  • the treatment of ischemia and heart attacks focuses on intravenous administration of vasodilators such as nitroglycerine to restore blood flow and prevent myocardial ischemia.
  • vasodilators such as nitroglycerine
  • systemic vasodilation is a common and serious side effect.
  • the invention advantageously overcomes this shortcoming by a targeted delivery to the diseased blood vessels of the myocardium.
  • the inventors could show that elevated shear stresses similar to those found in stenosed coronary arteries could be used as a localized physical trigger for the release of an active compound from the nanoparticles according to the invention.
  • the inventors have shown in controlled in vitro fluorescence release studies, that the inventive compositions could be used to make nanoparticles exhibiting a preferential release profile in diseased artery models.
  • the invention provides for phospholipids and compositions comprising different components as described above wherein their particular composition can be engineered in a manner to meet certain shear stress release profiles. These specially engineered compositions can be provided as nanoparticles and loaded with a desired active compound to serve as a delivery means to a particular site (target site) in a patient to treat a particular disease state or disorder.
  • Literature values are reported on the shear stress in healthy and diseased systems (Cheng, C, et al., Large variations in absolute wall shear stress levels within one species and between species. Atherosclerosis, 2007. 195 (2): p. 225-235.). Whereas in a healthy artery average stresses of around 1.5 Pa have been determined, this value rises to between 7 and 10 Pa in complex plaques. Furthermore, blood vessel constriction can lead to wall shear stress values well above 10 Pa.
  • nanoparticles have been developed that may specifically deliver nitroglycerin to the site of atherosclerotic constriction.
  • the positive effects of the invention could be shown by application of a fluorophore model of carboxyfluorescein encapsulated vesicles.
  • Different compositions according to the invention were applied in this model and their usefulness for a targeted delivery by shear stress-induced release could be shown. The inventors thus could show for the first time that endogenous shear stress could be applied as a physical trigger to locally release a payload carried to the target site by nanoparticles.
  • the inventors could show that a composition consisting of pure Pad-PC-Pad nanoparticles showed that an increase in stenosis of the artery model (i.e. increased atherosclerosis) led to an increase in release induced by endogenous shear stress as compared to known natural lipids.
  • the nanoparticles according to the invention will exhibit different stabilities with regard to shear stress.
  • the invention makes advantageously use thereof for the medical and non-medical applications and methods of the invention. Changing the size and ratios of hydrophobic and hydrophilic sites in the compounds applied in the invention will change, i.e. increase or reduce, the stabilization and destabilisation properties, respectively, of the novel molecules on the formulated nanoparticles according to the invention.
  • the use or method of treatment can be applied to a dermatological disease or disorder wherein the dermatological disease or disorder is preferably selected from the group consisting of acne, napkin dermatitis, atopic dermatitis, seborrhoeic dermatitis, psoriasis, warts, tinia pedis, seborrhoeic keratosis, hives, rosacea, dermatological viral infection and dermatological bacterial infection.
  • the dermatological disease or disorder is preferably selected from the group consisting of acne, napkin dermatitis, atopic dermatitis, seborrhoeic dermatitis, psoriasis, warts, tinia pedis, seborrhoeic keratosis, hives, rosacea, dermatological viral infection and dermatological bacterial infection.
  • the vascular disorder or disease is related to or is acute coronary syndrome (ACS), myocardial infarction, acute heart insufficiency, chronic heart insufficiency, cerebrovascular accident (CVA), stroke, atherosclerosis, vasospasm, tumor treatment, hemoptysis, pulmonary embolism, pulmonary arterial hypertension, intestinal ischemia, intestinal hemorrhage, renal infarction, renal hemorrhage, renal auto-regulation for hypertensive treatment, auto-immune glomerulonephritis or intersitial nephritis, treatment of fetal diseases, placental infarction, placental hemorrhage, retinal ischemia, retinal hemorrhage, or retinal neovascularization.
  • ACS acute coronary syndrome
  • CVA cerebrovascular accident
  • the invention in another aspect relates to a composition or nanoparticles according to the invention for use in the targeted delivery of a selected compound.
  • the selected compound is released at a target site due to endogenous shear stress at the target site.
  • endogenous shear stress refers to the shear stress that is present at a target site and which is preferably used to trigger the partial or complete release of an active or selected compound from the nanoparticles according to the invention.
  • non-endogenous shear stress is produced by way of an apparatus being applied in order to produce a shear stresss e.g. within a system or the body of an animal or human at a desired site therein.
  • a pharmaceutical or cosmetic formulation in the form of e.g., a lotion, cream or emulsion
  • endogenous shear stress is also understood as the shear stress produced by applying e.g. the lotion onto the skin or other parts of the human or animal body.
  • the invention in another aspect relates to a method of a targeted delivery of a selected compound or composition of compounds wherein i. in a first step the selected compound is loaded into a nanoparticle according to the invention, ii. the loaded nanoparticle is applied to a subject or object and the selected compound is released at the target site due to endogenous vascular shear stress at the target site.
  • composition or nanoparticles of invention are used in a monitoring method or a diagnostic method.
  • the selected compound is selected from the group consisting of a medium, a small molecule, a protein, peptide, nucleic acid, nucleotide or an antibody.
  • the selected compound is a marker, a contrast medium or a labeled compound.
  • the selected compound is selected from the group consisting of a iodine or gadolinium labeled antibody against glycoprotein- (GP)IIb/IIIa-(aIIb 3) receptors, iodine or gadolinium labeled abciximab (ReoPro®), an atherosclerosis associated marker such as CD 16a, CD 32, CD 36, CD 40, CD 44, CD 45RO, a general inflammatory marker like an interleukin, iodixanolum (Visipaque®), gadopentetate dimeglumine (Magnevist®), a coronary stenoses marker such as copper, or coagulation factor Vila (NovoSeven®).
  • a iodine or gadolinium labeled antibody against glycoprotein- (GP)IIb/IIIa-(aIIb 3) receptors iodine or gadolinium labeled abciximab (ReoPro®)
  • an atherosclerosis associated marker such
  • the use or methods according to the invention as described above may preferably be applied in coronary atherosclerosis, myocardial infarction, cerebrovascular accident (CVA), stroke, vasospasm, tumors, hemoptysis, pulmonary embolism, intestinal ischemia, digestive tract hemorrhage, renal infarction, renal hemorrhage, placental infarction, placental hemorrhage, retinal ischemia, retinal hemorrhage, diabetic retinopathy, or hypertensive retinopathy.
  • CVA cerebrovascular accident
  • compositions and nanoparticles of the invention may be designed to fit the needs of the desired applications and release profile.
  • the compositions and nanoparticles will be adapted to the chosen target sites wherein preferably the target site is characterized by an endogenous shear stress, preferably an endogenous vascular shear stress, of between 2 Pa and 20 Pa, preferably of between 2 Pa and 15 Pa, more preferably of between 2 Pa and 14 Pa, even more preferably of between 4 Pa and 14 Pa.
  • the selected compound is preferably released at a therapeutically effective amount.
  • terapéuticaally effective amount or "effective amount” of an active compound as understood in the context of the invention is meant as the amount released from the vesicles of the invention at the target site or in the vicinity of the target site and reaching the medical target producing the desired effect or response in the treatment or prophylaxis of the disease or disorder in question. It will be appreciated by the skilled person, that depending on the particular circumstances the amount loaded onto nanoparticles of the invention and the finally effective amount released to achieve a particular effect at the target site will vary significantly.
  • the "target site” as understood in the context of the invention is the area of the system or in the animal or human body whereto the active or selected compound is to be delivered. At the “target site” there can be under preferred circumstances a certain shear stress be present and preferably be determined by usual means (Cheng, C, et al., Atherosclerosis, 2007. 195 (2): p. 225-235.)
  • the structures according to the invention with regard to the desired release profile of an active or selected compound will be designed by combining a particular mixture of the different compounds as described above.
  • the amount of active or selected compound released can vary depending on the design of the vesicles according to the invention. Usually about the entire pay-load will be released at the target site. Preferably 80 to 95 %, 60 to 90 % or 50 to 90 % will be released. In other embodiments 30 to 40 %, even more preferably 40 to 50 %, even more preferably 40 to 60 % of the active or selected compound will be released
  • the invention relates to a method of a targeted release of an active or selected compound or mixture of compounds from loaded vesicles at a release site in a tubular system, preferably a vascular vessel system, due to endogenous shear stress wherein the vesicles are being recycled in said tubular system and the compounds or mixture of compounds are periodically released from the vesicles.
  • the release of the vesicles can be designed to meet particular characteristics and thus various release profiles depending on the needs can be engineered. It will be possible to design a continuous release, i.e. a similar amount of releases each time the vesicles pass through the release site. On the other hand an initial release of a great proportion of the loaded compound or compound mixture can be designed to achieve a fast effect of the compound or compound mixture at the release site with a lower level of release in the following to maintain the effect over a certain period of time.
  • the design of the releases profile will depend on the shear stress at the release site, the loaded compound or compound mixture, the composition of the vesicles and possibly the seize of the vesicles.
  • the released active or selected compound or compound mixture will be released during the first or several first passages through the release site at a high quantity. More preferably 30 to 40 %, even more preferably 40 to 50 %, even more preferably 40 to 60 % of the active or selected compound will be released during the first, preferably the first to third passage of the nanoparticles through the target site. The remainder of the active or selected compound will be released evenly during the fourth and following passages. In a preferred embodiment the release after the first burst or "bolus" release of the active compound will be in a fashion to maintain the appropriate effective level of the active compound at the target site or around the target side where the active compound is to act for treatment.
  • One example is a restricted or partially blocked artery of the circulation of a human or animal.
  • the anti-clotting compound or vasodilation compound in general the active compound
  • the anti-clotting compound or vasodilation compound will lead to opening the narrowed release site and thus the endogenous shear stress will be reduced.
  • less active compound will be released at the release site while circulating the body of the animal or human circulation.
  • the release site will narrow again and the shear stress will increase accordingly.
  • the recycling vesicles will release more of the active compound and thus there will be achieved a steady-state of an effective dosage of the active compound.
  • the invention achieves a continuous supply of active compound at the target site.
  • the invention achieves a sustained release of the active compound loaded onto the vesicles wherein preferably the effective dosage of active compound is maintained at an appropriate level or concentration to be effective for the particular application or disease or disorder.
  • the parameters will be chosen depending on the use and application as well as the combination of compounds constituting the vesicles and the active compound or compound mixture loaded onto them.
  • the inventors have developed thus novel phospholipids and mixtures comprising phospholipids useful in a targeted delivery of a selected or active compound.
  • the inventors found surprisingly that by providing particular mixtures of phospholipids, preferably compositions comprising or consisting of 1,3-diamidophospholipids or/and 1,2-diamidophospholipids or/and 2,3-diamidophospholipids and preferably at least a second compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant one could engineer nanoparticles for the targeted delivery of a selected or active compound or mixtures thereof.
  • the invention is also insofar advantageous as the release profile can preferably be modified by changing the amounts of lipids selected from the group consisting of 1,3- diamidophospholipids, 1,2-diamidophospholipids and 2,3-diamidophospholipids relative to natural lipids and preferably other components like cholesterol, cholesterol derivatives and surfactants in the nanoparticle forming composition.
  • the phospholipids with one CH tail and one fluorinated CH tail are phospholipids with one CH tail and one fluorinated CH tail:
  • Phospholipids with one fluorescent head group and 1,3-diamide are Phospholipids with one fluorescent head group and 1,3-diamide:
  • N ⁇ V-(2-hydroxypropane- 1 ,3-diyl)distearamide N ⁇ V-(2-(( 17-hydroxy-3,6,9, 12, 15-pentaoxaheptadecyl)oxy)propane- 1 ,3- diyl)distearamide
  • ESI-MS for the characterization of new compounds was performed on an ESI API 150EX and are reported as mass- per-charge ratio m/z.
  • IR spectra were recorded on a Perkin Elmer Spectrum One FT- IR spectrometer (ATR, Golden Gate). Melting point is uncorrected.
  • Palmitoyl chloride (5.56 mL, 17.8 mmol) and sodium azide (1.5 g, 23 mmol) were mixed in 40 mL of dry toluene and the solution was refluxed for 5 h under an N 2 atmosphere. The product was directly used without further purification.
  • the aqueous phase was washed 2 times with 50 mL of CH 2 C1 2 .
  • the organic phases were dried over MgS04.
  • the organic solvents were removed under reduced pressure.
  • silica gel column chromatographic purification (CH 2 Cl 2 -MeOH 95:5), a white solid was obtained (148 mg, 0.18 mmol, 38 %).
  • tert-butyl-(2-((amino(( 17,23-dioxo- 16, 18,22,24-tetraazanonatriacontan-20- yl)oxy)phosphoryl)oxy)ethyl)carbamate (140mg, 0.17 mmol) was dissolved in 9 mL dioxane and HC1 (3 mL, 12 mmol) in dioxane was added. After 4 h the reaction was stopped. Nitrogen was bubbled through the solution over 1 h and solvent was evaporated under reduced pressure. The crude material was the dissolved in 12 mL of methanol. Dimethyl sulfate (120 ⁇ , 1.27 mmol) was added and the solution was heated to 40 °C.
  • Tridecanoic acid (0.86 g, 4.0 mmol), triethylamine (1.0 mL, 7.2 mmol) and diphenylphosphoryl azide (0.95 mL, 4.4 mmoL) were dissolved in dry toluene (20 mL). The solution was refluxed over 3h. The solution was kept at 0 °C and ethanolamine (0.24 mL, 4.0 mmol) was added. The mixture was stirred at 20 °C overnight and mixed with 70 mL of CH 2 C1 2 to be extracted with 100 mL of water mixed with 10 mL of NH4OH (25%). The water phase was washed twice with 70 mL of CH2CI2.
  • Dodecyl isothiocyanate was first synthesized by variation of a procedure of Meijer. DCC (2.9 gl 7 mmol) and CS 2 (7.20 mL, 1 19 mmol) were dissolved in dry diethyl ether (40 mL). Dodecyl amine (3.2 g, 17 mmol) was added at 0°C to the mixture that was stirred overnight at room temperature. The precipated solid was filtered off and washed with 60 mL of dry diethyl ether. The solvent were removed by evaporation and the isothiocyanate was used without further purification.
  • Rf 0.28 (95 % CH 2 C1 2 , 5% MeOH).
  • Pentadecyl isocyanate was first synthesized by variation of a procedure of De Feyter and all. 2 Palmitoyl chloride (5.6 mL, 18 mmol) and sodium azide (1.5 g, 23 mmol) were mixed in dry toluene (40 mL). The solution was refluxed over 5h. The solution was directly used without further purification.
  • Pentadecyl isothiocyanate was first synthesized by variation of a procedure of Meijer. 1 DCC (3.1 g, 15 mmol) and CS 2 (6.3 mL, 104 mmol) were dissolved in dry diethyl ether (40 mL). Pentadecyl amine (3.4 g, 15 mmol) was added at 0°C to the mixture that was stirred overnight at room temperature. The precipated solid was filtered off and washed with 60 mL of dry diethyl ether. The solvent were removed by evaporation and the isothiocyanate was used without further purification.
  • Hexadecyl isothiocyanate was first synthesized by variation of a procedure of Meijer. 1 DCC (2.42 g, 14.5 mmol) and CS 2 (6.00 mL, 100 mmol) were dissolved in dry diethyl ether (40 mL). Hexadecyl amine (3.80 g, 14.2 mmol) was added at 0°C to the mixture that was stirred overnight at room temperature. The precipated solid was filtered off and washed with 60 mL of dry diethyl ether. The solvents were removed by evaporation and the isothiocyanate was used without further purification.
  • the crude product was purified on silica gel column (95 % CH 2 C1 2 , 5% MeOH) and then by recristallisation (dioxane/pentane, 4:1) to give the product as a white powder (756 mg, 2.19 mmol, 59%).
  • Rf 0.25 (95 % CH 2 C1 2 , 5% MeOH).
  • Free release was measured daily for 6 days for each sample. For every measurement a second sample of the liposomal suspension was taken and mixed with a solution of the detergent (Triton-X100). The latter values were used as a reference.
  • Liposome formulation is based on the techniques described in: Olson et. al., Biochimica et Biophysica Acta 1979, 557, 9-23.
  • a description of one of the methods for nanoparticle formulation is as follows: 30 ⁇ lipid (e.g. egg yolk phosphatidylcholine (EYPC, Avanti Polar Lipids)), Pad- PC -Pad or a combination of lipids) was weighed into a 25 mL round bottomed flask and dissolved in 1 mL chloroform. Any required surfactant (e.g. Brij S10) was added at this point from a freshly prepared 2.5 mg/mL methanol solution. After evaporation to dryness, the film was dried for 12 h under high vacuum.
  • EYPC egg yolk phosphatidylcholine
  • Pad- PC -Pad or a combination of lipids was weighed into a 25 mL round bottomed flask and dissolved in 1 mL chloroform. Any required surfactant (e.g. Brij S10) was added at this point from a freshly prepared 2.5 mg/mL methanol solution. After evaporation to dryness, the film was
  • a Medtronic extra-corporeal circulation pump (Medtronic Bio-Medicus 540 Bio Console (Model 5401) with tubing of approximately 10 mm diameter and a total volume of 180 mL was fitted with a Medtronic pressure gauge, flow reader and temperature bath. Pressure and flow rate were controlled by regulating pump speed.
  • the nanoparticle suspension was diluted to 30 nmol lipid concentration in external buffer and loaded into the in vitro Medtronic pump setup (described above). Either a healthy or stenosed artery model (Elastrat) was connected in series.
  • flow rates were typically 700 mL/min (healthy artery model) or 350 mL/min (unhealthy artery model), pressure was typically 60 mmHg (0.8kPa) and the reaction temperature 37 °C.
  • Samples of the circulating liposome solution were collected after 1 pass through the model artery and at 5, 10, 15 and 20 min circulation time. On each sample extraction, an equivalent amount of external buffer was reinjected into the system to counteract the fall in total liquid volume in the pump.
  • Fluorescence release from nanoparticles was analyzed by way of a fluorescence 96- well plate reader. From each collected sample, twelve 200 ⁇ , samples were prepared. To six of these, 4 ih 1.2% Triton X- 100 aqueous solution was added to facilitate liposome degradation and release of fluorescent contents. The samples were incubated for 30 min then fluorescence was measured (excitation 492 nm, emission 517 nm). The average fractional release for each sample was calculated as the mean of the sample fluorescence divided by the mean of the sample plus Triton X- 100 fluorescence. Errors were extrapolated appropriately.
  • a low viscocity rheometer will allow the dependence of dye release from the vesicle to be determined as a function of the applied shear stress.
  • the release patterns in (a) showed that there is a peak in specific shear induced release at around 0.6 mol% Brij S10. An example of release seen at 0.4 mol% is illustrated in fig. 8.
  • the release properties of 100% Pad-PC-Pad were vastly different to those of Pad-PC-Pad/EYPC mixtures and were unique in all release studies, with release of 40% of contents after one pass through the model artery. In all other formulations, release was in the region of ⁇ 5% after 1 pass.
  • Formulations are based on non-natural/synthetic phospholipids (e.g. Pad-PC-Pad) or other lipids according to one of formulae la, lb, Ic and Id with 0-10 mol% surfactants such as the Brij family (including, but not restricted to, SI 0, PI 0 and P4), 0-50 mol% rigidifiers such as cholesterol, and 0-25 mol% natural phospholipids such as EYPC, DOPC, POPC, DPPC, sphingomyelin and miltefosine.
  • non-natural/synthetic phospholipids e.g. Pad-PC-Pad
  • other lipids according to one of formulae la, lb, Ic and Id with 0-10 mol% surfactants such as the Brij family (including, but not restricted to, SI 0, PI 0 and P4), 0-50 mol% rigidifiers such as cholesterol, and 0-25 mol% natural phospholipids such as EYPC
  • formulations include synthetic phospholipids or lipids according to the invention (according to one of formulae la, lb, Ic and Id) and/or natural phospholipids (EYPC, DOPC, POPC, DPPC, sphingomyelin) with cholesterol, miltefosine and octadecanol in various concentrations up to 50 mol%.
  • DOPC l,2-dioleoyl-5 «-glycero-3-phosphocholine

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Abstract

The invention relates to a composition comprising or consisting of 1,3-diamidophospholipids or/and 1,2-diamidophospholipids or/and 2,3-diamidophospholopids or/and another lipid and preferably at least a further compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant, methods of making same, nanoparticles comprising same and their medical and non-medical use.

Description

NOVEL LIPIDS, PHOSPHOLIPIDS, PHOSPHOLIPID AND LIPID
COMPOSITIONS AND THEIR USE
FIELD OF THE INVENTION
The invention relates to new lipid and phospholipid compounds, mixtures of such compounds alone or in combination with additional compounds, nanoparticles comprising same, methods for making same and their diagnostic, non-medical as well as medical use.
BACKGROUND OF THE INVENTION
Phospholipids are a class of lipids and are a major component of all cell membranes as they can form lipid bilayers. Phospholipids may contain a diglyceride, a phosphate group, and a simple organic molecule such as choline. Sphingomyelin is another type of phospholipid, which is derived from sphingosine instead of glycerol. One of the first phospholipids identified as such in biological tissues was lecithin, or phosphatidylcholine, in the egg yolk.
Phospholipids are composed of a hydrophilic "head" and a hydrophobic "tail". The hydrophilic head contains the negatively charged phosphate group, and may contain other polar groups. The hydrophobic tail usually consists of long fatty acid hydrocarbon chains. Their specific properties allow phospholipids to play an important role in phospholipid bilayers or cell membranes. In biological systems, the phospholipids often occur with other molecules (e.g. proteins, glycolipids and cholesterol) in a bilayer such as a cell membrane.
Phospholipids may also play an important role as second messengers or in signal transduction. In the cellular context, phospholipids are synthesized adjacent to the endoplasmatic reticulum (ER). Lipids in contrast to phospholipids do not carry a phosphate group and are often defined as substances such as fat, oil or wax that dissolve in a non-polar solvent (e.g. alcohol) but not in a polar solvent (e.g. water). Cholesterol and triglycerides also belong to the class of lipids.
WO2009/056955 A2 describes amine bearing phospholipids and their use as a drug delivery system. Particular compositions, their synthesis and their use according to the present invention are not disclosed neither suggested in this patent application.
Fedotenko LA. et al., Tetrahedron Letters 51, 2010, 5382-5384 describe 1,3- diamidophospholipids and a method of making same. The particular compounds of the invention, their mixtures and use in nanoparticles as well as their use according to the invention are neither disclosed nor suggested therein.
Nanoparticles are widely known and applied for delivery of active compounds in many applications like pharmaceuticals and cosmetics. They may be composed of various materials and often lipids, phospholipids or cholesterol are used to prepare them.
Nanoparticles have been described and they may be loaded with a so-called payload. Such loaded nanoparticles find many technical applications. The release of the payload is caused by various mechanisms and means. The nanoparticle may fuse with a bilayer membrane and thus release its content, be endocytosed by a cell, or mechanical force, e.g. shear stress, may be applied and result in the release of the nanoparticles' payload.
Shear stress, denoted t, can be defined as a force per area, which is applied parallel or tangential to a surface of a material, as opposed to a force applied perpendicularly.
Shear stress has various technical applications. EP 1 056 473 Bl describes compositions and formulations for the controlled delivery of bioactive compounds to selected sites of a patient. The described vesicles are composed of phospholipids and ultrasound is applied for the targeted delivery and release of the bioactive compounds. The embodiments of the invention are not disclosed nor suggested. US 2003/01351 13 describes microspheres and their use in treating heart and other body tissues by injection of microsphere encapsulated macromolecule therapeutic agents. Upon degradation of the microspheres the active compound is released over time. The embodiments of the invention are not disclosed nor suggested.
In medical applications a drug may be applied systematically or by way of a targeted delivery. Various methods and approaches are known in the art.
In particular diseases or disorders, a targeted delivery of a drug would be preferred for a number of reasons. One being that a systemic application of many drugs involves significant side effects in non-target tissues.
Disease states of patients where a targeted delivery of a drug would be advantageous are e.g. vascular diseases and in particular ischemia.
However, the targeted delivery and release of a desired substance to a specific cell or tissue in a controlled manner is difficult to achieve.
Thus, it is one object of the present invention to provide new means for a targeted delivery and release of a compound to a target site in a system or a patient, or to improve the disadvantages of the state of the art.
Another object of the invention is to provide for methods of making compounds, compositions and means useful in the above object of providing solutions for a targeted delivery.
Yet another object of the invention is to provide tools for the use or methods in treating disorders, diseases or disease states wherein a directed or targeted and specific delivery of an active compound or a mixture of compounds is advantageous.
Yet another object is to provide a means and methods of delivering a desired compound to a target site and a transport vehicle which may easily and controllable release the desired compound. SUMMARY OF THE INVENTION
In one aspect the invention relates to a composition comprising or consisting of 1,3- diamidophospholipids or/and 1 ,2-diamidophospholipids or/and 2,3- diamidophospholopids or/and another lipid and preferably at least a further compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant.
In another aspect the invention is a composition wherein the composition is provided in the form of nanoparticles. The nanoparticles can be a micelle, or the nanoparticle is composed of a monolayer, a bilayer and/or a vesicle and/or a nanocontainer.
Another aspect of the invention relates to a method of making a 1,3- diamidophospholipid wherein a phosphoethanolamine is alkylated under appropriate conditions, preferably with the use of dimethyl sulfate.
In a further aspect the invention relates to a method of making a composition according to the invention and to a method of making nanoparticles.
In yet another aspect the invention relates to a pharmaceutical or cosmetic composition comprising a composition according to the invention or a nanoparticle according to the invention, and preferably comprising further useful carriers or/and additives.
In yet another aspect the invention relates to a composition or nanoparticles according to the invention for use in a pharmaceutical or cosmetic application.
In yet another aspect the invention relates to a composition or nanoparticles according to the invention for use in the prophylaxis or treatment of a vascular disorder or disease, or for use in the prophylaxis or treatment of a dermatological disorder or disease, or for use as cosmetic, or for use in a monitoring or diagnostic method. In yet another aspect the invention relates to a composition or nanoparticles according to the invention for use in the targeted delivery of a selected compound. The selected compound is preferably released at a target site due to endogenous shear stress at the target site.
In yet another aspect the invention relates to a method of a targeted delivery of a selected compound or composition of compounds wherein i. in a first step the selected compound is loaded into a nanoparticle according to the invention, ii. the loaded nanoparticle is applied to a subject or object and the selected compound is released at the target site due to endogenous, preferably vascular, shear stress at the target site.
In yet another aspect the invention relates to a method of a targeted release of an active or selected compound or mixture of compounds from nanoparticles at a release site in a tubular system, preferably a vascular vessel system, due to endogenous shear stress wherein the nanoparticles are being recycled at least 2 times in said vascular vessel system and the active or selected compounds or mixture of compounds are periodically released from the nanoparticles at a target site, preferably wherein the amount of compound(s) released is dependent on the shear stress within the vascular vessel system at the target site with an advantageously engineered release profile.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1 - 4 are depicting the characterization of preferred embodiments of the phospholipids according to the invention.
FIG. 5 is depicting vortex assisted release of carboxyiluorescein from vesicles formed from Pad-PC-Pad ( ), DPPC ( ) and SMI 6 ( ) as percentage release over time.
FIG. 6 is depicting free release (no vortex treatment) of carboxyfluorescein from vesicles formed from Pad-PC-Pad ( ), DPPC ( ) and SMI 6 (.. ) as percentage release over time. FIG. 7 is depicting release of carboxyfluorescein from Egg-PC vesicles with varying percentage of Pad-PC-Pad incorporation, following repeat passes through the in vitro artery model as described in the examples. (■■■ = 0% Pad-PC-Pad; = 10% Pad- PC-Pad; = 25% Pad-PC-Pad;—■ = 50% Pad-PC-Pad; = 75%
Pad-PC-Pad; = 100% Pad-PC-Pad; ·= stenosed model; o= healthy model)
FIG. 8 is depicting release of carboxyfluorescein from Egg-PC vesicles with varying percentage of Pad-PC-Pad incorporation, after one pass through the in vitro artery model as described in the examples. FIG. 8A depicts the absolute values. ( = untreated vesicles; = healthy artery model; HI = stenosed artery model)
FIG. 8B shows the difference in release between the healthy and stenosed artery models. Results are the averages of three experiments.
FIG. 9 is depicting release of carboxyfluorescein from 100% Pad-PC-Pad vesicles illustrating the fractional release under exposure to increasing shear stress during one pass through the in vitro artery model as described in the examples. ( = untreated vesicles; HtHi = syringe; = healthy artery model; S = stenosed arterymodel; = severely stenosed artery model).
FIG. 10 is depicting the dynamic light scattering (DLS) of samples of 100% Pad-PC- Pad vesicles collected from the same experiment as Fig. 8. After one pass through the artery model, an increase in mean size and polydispersity was observed. Area under each curve is constant. The number average depicts the number of particles of a defined size, whereas the volume average indicates the total encapsulated volume of particles of a defined size. (——— = untreated;■■■ = healthy artery model;— = stenosed artery model; = freshly prepared vesicles) .
FIG. 11 depicts (control/ 25 °C versus = healthy artery model; Q = stenosed artery model) the release of loaded vesicle composed of Pur-PC-Pur nanoparticles in an in vitro pump experiment setup wherein tubings representing arterial samples are tested for release under conditions resembling the in vivo situation of healthy versus stenosed vessels. The figure shows a significant difference in release induced by shear stress due to artery stenosis. This experiment supports the inventive concept of shear stress induced release from nanoparticles formed from lipids according to the invention.
FIG. 12 depicts the shear stress dependent release from nanoparticles comprising Pad- PC-Pad, i.e. a lipid according to the invention, in conditions similar to physiologic conditions due to the addition of HSA (human serum albumin) and at physiological temperature of at 37 °C. This figure supports the inventive concept that the lipids and lipid combinations according to the invention can be advantageously applied as delivery vehicles for medical and non-medical applications. In this use a directed and local release can be achieved by applying the lipids of the invention.
DETAILED DESCRIPTION
The invention provides novel phospholipids and lipids, compositions comprising phospholipids, preferably in combination with further phospholipids, lipids, cholesterol or/and a cholesterol derivative or/and a surfactant.
In particular, the invention provides for a composition comprising or consisting of 1,3-diamidophospholipids or/and 1 ,2-diamidophospholipids or/and 2,3- diamidophospholopids or/and preferably another lipid and preferably at least a second or further compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant.
In another preferred embodiment the composition of the invention comprises or is consisting of 1,3-diamidolipids or/and 1 ,2-diamidolipids or/and 2,3-diamidolipids or/and 1 ,3-diurealipids or/and 1,2-diurealipids or/and 2,3-diurealipids or/and 1,3- dithiourealipids or/and 1 ,2-dithiourealipids or/and 2,3-dithiourealipids or/and 1 ,3- diacylurealipids or/and 1 ,2-diacylurealipids or/and 2,3-diacylurealipids or/and 1- amidolipids or/and 1 -urealipids or/and 1 -thiourealipids or/and 1 -acylurealipids or/and cyclic-amidolipids or/and cyclic urealipids or/and cyclic thiourealipids or/and cyclic acylurealipids, and preferably at least a second compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant.
The inventors have developed novel phospholipids and lipids, and compositions comprising same useful in non-medical as well as medical applications. In particular the novel compositions are useful in medical, diagnostic and cosmetic applications, especially in a targeted delivery of a desired or active compound or composition.
The lipids of the invention are depicted in the following formulae la, lb, Ic and Id:
Formula la
Figure imgf000009_0002
Formula lb
Figure imgf000009_0003
Formula Ic
Figure imgf000010_0001
Formula Id wherein the residues "A", "B", "C" and "D" are as defined below in the preferred embodiments of the invention.
In a preferred embodiment a composition as described above is provided wherein the 1,3-diamidophospholipid or 1, 3-diaminolipid has the following Formula la:
Figure imgf000010_0002
Formula la and wherein
i. "Bi" is equal or different from "B2", and "B," and "B2" is selected from:
ii. H; alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
an optionally substituted Ci-Cio alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
an optionally substituted Cn-Ci7 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
an optionally substituted C 18-C24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
wherein 1 1, 13, 15, and 17 C-atoms are preferred;
a group listed in the figures below:
11
Figure imgf000012_0001
Figure imgf000012_0002
Figure imgf000012_0003
Figure imgf000012_0004
Figure imgf000012_0005
Figure imgf000013_0001
where m and n can be different or the same and
m = 0-7
preferably m=7
n=0-l l
preferably n=8, 10, or 1 1 or "Bi" and "B2" are the same or different and are selected from:
i. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl- heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
ii. preferably, decyl-, dodecyl-, tetradecyl, hexadecyl-amine
iii. an optionally substituted C1-C8 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
iv. an optionally substituted C9-C15 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
v. an optionally substituted C16-C22 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vi. wherein 9, 11, 13, and 15 C-atoms are preferred;
vii. a group listed in the figures below:
13
Figure imgf000014_0001
Figure imgf000014_0002
Figure imgf000014_0003
Figure imgf000015_0001
where m=0-6, and preferably m=6 n=0-l 1 , preferably n=8, 10, or 1 1
or
ix. primary amides (to give acylureas) such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl- heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
x. preferably, decyl-, dodecyl-, tetradecyl, hexadecyl-amide
xi. an optionally substituted C1-C8 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xii. an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C16-C22 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xiv. wherein 9, 1 1 , 13, and 15 C-atoms are preferred;
xv. a group listed in the figures below:
15
Figure imgf000016_0001
Figure imgf000017_0001
where m=0-5, and preferably m=5; n=0-l 1, preferably n=8, 10, or 1 1 ; wherein said lipid may be fully or partially deuterated, or radioactively labeled; and
wherein "A" is selected from:
a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide, and wherein "Ci" and "C2" may be H or a methyl.
In yet another preferred embodiment the 1, 3-diamidophospholipids of the invention of Formula la are defined as follows: i. "Bi" is equal or different from "B2", and "Bi" and "B2" is selected from: ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted CpCio alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds; vi. an optionally substituted Cn-C17 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds
vii. an optionally substituted Cig-Cw alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds
viiLwherein 113 13, 15, and 17 C-atoms are preferred;
ixa group listed in the figures below:
Figure imgf000018_0001
Figure imgf000018_0002
18
Figure imgf000019_0001
Figure imgf000019_0002
Figure imgf000019_0003
Figure imgf000019_0004
wherein said lipid may be fully or partially deuterated, or radioactively labeled; wherein "A" is selected from:
a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide; and wherein "Ci" and "C2" is selected from H or methyl.
In another preferred embodiment of the invention the lipids are defined according to formula lb:
Formula lb and wherein i. "Bi" is equal or different from "B2", and "B," and "B2" is selected from: ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted Ci-C10 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vi. an optionally substituted Cn-Ci7 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted Ci8-C24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 1 1 , 13, 15, and 17 C-atoms are preferred;
ix. a group listed in the figures below:
21
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000022_0003
Figure imgf000022_0004
Figure imgf000022_0005
or m = = Ή 'n
where m and n can be different or the same and
m = 0-7, preferably m=7
n=0-l 1 , preferably n=8, 10, or 1 1 or "Bi" and "B2" are the same or different and are selected from:
i. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
ii. preferably, decyl-, dodecyl-, tetradecyl, hexadecyl-amine
iii. an optionally substituted C1-C8 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
iv. an optionally substituted C9-C15 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
v. an optionally substituted C16-C22 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vi. wherein 9, 11, 13, and 15 C-atoms are preferred;
vii. a group listed in the figures below:
Figure imgf000024_0001
Figure imgf000024_0002
viii.
Figure imgf000024_0003
Figure imgf000025_0001
where m=0-6, and preferably m=6 n=0-l 1, preferably n=8, 10, or 11
or
ix. primary amides (to give acylurea) such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
x. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amide
xi. an optionally substituted C1-C8 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xii. an optionally substituted C9-C15 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C16-C22 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiv. wherein 9, 11, 13, and 15 C-atoms are preferred;
xv. a group listed in the figures below:
25
Figure imgf000026_0001
Figure imgf000027_0001
where m=0-5, and preferably m=5; n=0- 11 , preferably n=8, 10, or 11 ;
"Di" and "D2" can be the same or can be different and are either O (oxygen) or S (sulfur),
wherein said lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide; and wherein "CI" and "C2" may be H or a methyl.
In another preferred embodiment of the invention the lipids are defined according to formula Ic:
Figure imgf000027_0002
Formula Ic
and wherein i. "Bi" is selected from:
ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted Ci-C10 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vi. an optionally substituted Cn-Cn alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted Ci8-C24 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 1 1, 13, 15, and 17 C-atoms are preferred;
ix. a group listed in the figures below:
28
Figure imgf000029_0001
Figure imgf000029_0002
Figure imgf000029_0003
Figure imgf000029_0004
Figure imgf000029_0005
Figure imgf000030_0001
where m and n can be different or the same and
m = 0-7, preferably m=7;
n=0- 1 1 , preferably n=8, 10, or 1 1 ; or is selected from
x. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl- heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
xi. preferably, decyl,- dodecyl-, tetradecyl, hexadecyl-amine
xii. an optionally substituted C1-C8 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C9-C15 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xiv. an optionally substituted C16-C22 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xv. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
xvi. a group listed in the figures below:
χνϋ.
Figure imgf000031_0001
Figure imgf000032_0001
where m=0-6, and preferably m=6; n=0-l 1, and preferably n=8, 10, or 11 ;
or
xviii. primary amides (to give acylureas) such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
xix. preferably, decyl,- dodecyl-, tetradecyl-, hexadecyl-amide
xx. an optionally substituted C1-C8 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xxi. an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xxii. an optionally substituted C16-C22 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xxiii. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
xxiv. a group listed in the figures below:
32
Figure imgf000033_0001
Figure imgf000034_0001
where m=0-5, and preferably m=5; n=0-l 1, preferably n=8, 10, or 11; "Di" is either O (oxygen) or S (sulfur),
wherein said lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phospho inositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide; and wherein "CI" may be H or a methyl.
In another preferred embodiment of the invention the lipids are defined according to formula Id:
Figure imgf000034_0002
Formula Id wherein i. "B," is equal or different from "B2" or "E", and "B,", "B2" and "E" are selected from:
ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted CpCio alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vi. an optionally substituted Cn-Ci7 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted Ci8-C24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 11, 13, 15, and 17 C-atoms are preferred;
ix. a group listed in the figures below:
35
Figure imgf000036_0001
Figure imgf000036_0002
Figure imgf000036_0003
Figure imgf000036_0004
Figure imgf000036_0005
or
·Γ<Μ m = = ΤΜΓ n
where m and n can be different or the same and
m = 0-7, preferably m=7;
n=0- 11 ; preferably n=8, 10, or 11 ; or "Bi" and "B2" are equal or the same and are selected from:
i. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
ii. preferably, decyl,- dodecyl-, tetradecyl-, hexadecyl-amine
iii. an optionally substituted C1-C8 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
iv. an optionally substituted C9-C15 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
v. an optionally substituted C16-C22 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vi. wherein 9, 11, 13, and 15 C-atoms are preferred;
vii. a group listed in the figures below:
37
Figure imgf000038_0001
Figure imgf000039_0001
where m=0-6, and preferably m=6; n=0-l 1, preferably n=8, 10, or 1 1 ; or "B|" and "B2" are the same or different and are selected from:
ix. primary amides (to give acylureay) such as methyl-, ethyl-, propyl-, isopropyl-, butyl, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
x. preferably, decyl,- dodecyl-, tetradecyl-, hexadecyl-amide
xi. an optionally substituted C1-C8 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xii. an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C16-C22 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiv. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
xv. a group listed in the figures below:
39
Figure imgf000040_0001
Figure imgf000041_0001
where m=0-5, preferably m=5; n=0-l 1, preferably n=8, 10, or 11;
"Di" and "D2" can be the same or can be different and are either O (oxygen) or S (sulfur),
wherein said lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide.
In the context of the invention "alkyl" is to be understood as any chain of C-atoms being linear or branched, "optionally substituted" is to be understood as having no substitutions or being substituted with residues being compatible with the remaining molecule without interfering with its structure and providing optional functionality in the context of the invention.
In further preferred embodiments the non-natural phospholipids may be defined to have the form Xn-HG-Ym wherein HG (head group) may be selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA) and phosphatidylglycerol (PG) and X, Y are saturated or/and unsaturated aliphatic chains of 10, 12, 14, 16 and 18 carbons, wherein n, m may comprise an ester (es), amide (ad), amine (an), and ether (et) linkers. Throughout the description of the invention various chemical residues or compounds may be abbreviated. Examples of such abbreviation are "HG" for "head group" which may include "phosphoethanolamine" denoted as "PE", "phosphocholine" as "PC", "phosphatidic acid" as "PA", "polyethylene glycol" as "PEG", "phosphoglycerol" as "PG", "EYPC" as "egg yolk phosphocholine". Furthermore "ester" may be denoted "es", "amide" as "ad", "amine" as "an", and "ether" as "et".
The term acylurea is to be understood as generally known by the skilled person in the field of the invention and is an urea moiety containing an additional carbonyl group alpha to the amine.
Lipids according to the invention are not restricted to 1,3- or 1,2- or 2,3 -phospholipids and comprise non-phospholipids according to any of the formulae la, lb, Ic and Id as defined above. Lipids represented by either of these formulae can be used in individual form, as mixtures of lipids covered by either of these formulae or as a combination of one or several compounds originating from either of these formulae with one another.
One advantageous compound is Pad-PC-Pad of which the positive mechano-sensitive effect has been shown. Pad-PC-Pad can e.g. be combined with any of the other compounds of the invention or described herein.
The skilled person will adapt the particular compounds and compounds mixtures of the invention with regard to their combination, ratios, etc. depending on the particular need of the application. He thus will easily find the best suitable mixtures and the specific ratios, way of mixing etc. for the particular application and use by way of simple experimentation as described herein.
These lipids can be combined with each other in any useful manner including but not restricted with regard to the number of compounds, their ratios of mixtures etc. The lipids according to the invention can also be combined with any natural lipid as described below. The natural lipid may be any known natural lipid useful in the invention. Preferably the natural lipid chosen for the composition according to the invention is selected from the group consisting of egg yolk phosphatidylcholine (EYPC), a phosphatidylethanolamme (PE), a phosphatidylcholine (PC), a phosphatic acid (PA), a phosphatidylglycerol (PG), preferably l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l-palmitoyl-2-oleoyl-i?i-glycero-3-phosphocholine (POPC), 1,2- phosphatidylcholine, l,2-dipalmitoyl-j«-glycero-3-phosphocholine (DPPC), 1,2- dilauroyl-SH-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-i«-glycero-3- phosphocholine (DMPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), N- palmitoylsphingomyelin, miltefosine.
The composition of the invention may comprise a synthetic lipid selected from any synthetic lipid known in the art. It will be appreciated by the skilled person that preferably the synthetic lipid is chosen according to the particular requirements of the further application and use of the composition according to the invention. Particularly preferred are the synthetic lipids and the phospholipids described above. In particular the synthetic lipids and phospholipids according to any of formula la, lb, Ic and Id are preferred.
In a preferred embodiment the lipids may be modified and in particular double bonds may be replaced by methylation. The lipids may be bolaamphiphile lipids and they may contain iso and anteiso chains. Examples of such modified lipids can be found in archaebacteria. Examples of such lipids are described in Zhang Y.M. and Rock CO., Nature Vol. 6, March 2008, p. 222 - 233 which disclosure is herewith incorporated by reference.
The surfactant applied in a preferred embodiment in the composition according to the invention may be any known and useful surfactant. Preferably the surfactant is selected from an anionic, cationic or non-ionic surfactant. More preferred the surfactant is selected from the group consisting of a Brij, preferably Brij P4, Brij S4, Brij SlO or Brij P10.
The composition may contain any useful number of compounds as described above and also the ratios of single compounds and compound groups may vary. The ratios can be modified and adapted according to the particular application of the composition of the invention. In a preferred embodiment the first and second compounds are present in a ratio of from 99.9 : 0.1 to 1 : 99 mol-%, preferably from 90 : 10 to 40 : 60 mol-%, more preferably from 70 : 30 to 50 : 50 mol-%, even more preferably wherein the first compound is present in either 50 mol-%, 60 mol-%, 70 mol-%, 75 mol-%, 80 mol-%, 85 mol-%, 90 mol-%, 95 mol-%, or 98 mol-%
In particular, the compositions according to the invention comprising one or more synthetic compounds according to the invention in an amount of 80 to 98 mol-% , preferably 90 to 98 mol-%, more preferably 95 to 98 mol-% in combination with any of the other compounds described above exhibit superior release features when applied in nanoparticles of the invention which are useful in the targeted delivery of active or selected compounds. The advantageous release features can be demonstrated by way of the in vitro model described below and in the experimental section.
In particular, preferred in the compositions of the invention are 1,3- diamidophospholipids while in various compositions of the invention also 1,2- diamidophospholipids and 2,3-diamidophospholipids will be particularly useful to achieve advantageous release features in nanoparticles according to the invention.
The inventors arrived at lowering the stability of nanoparticles by the addition of synthetic phospholipids and/or lipids according to the invention to natural phospholipids in an amount of at least 50 mol-%, preferably at least 60 mol-%, more preferably at least 70 mol-%, more preferably at least 75 mol-%, more preferably at least 80 mol-%, more preferably at least 85 mol-%, more preferably at least 90 mol- %, more preferably at least 95 mol-%, or more preferably at least 98 mol-% In these preferred compositions additional compounds as described above may be present. In this manner the inventors arrived surprisingly to change the release features of nanoparticles consisting of natural lipids by addition of the synthetic phospholipids as described above and preferably other compounds. In this way the inventors arrived at providing vehicles for the delivery of active or selected compounds that may release a certain percentage or all of these compounds as described herein due to an endogenous shear stress at a desired target site. In a preferred embodiment a synthetic phospholipid or preferably lipid as described above will be mixed with egg yolk phosphatidylcholine (EYPC) and with the surfactant Brij S10 (decaethylene glycol octadecyl ether). The content of the surfactant, in particular of Brij S10, in the composition will be preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, 10, 15 and 20 mol%.
In another preferred embodiment EYPC is mixed with the synthetic phospholipid Pad-PC-Pad and preferably Pad-PC-Pad is contained in 10, 25, 50, 75 and 100 mol%. In yet another preferred embodiment the composition comprises or contains EYPC with cholesterol (preferably 1, 20, 25 and 50 mol%), octadecanol (1 and 5 mol%), miltefosine (5 mol%), octadecanol (1 and 5 mol%), Brij P10 (5 and 10 mol%). Another preferred embodiment contains synthetic phospholipid as described above and EYPC with the surfactant Brij P4 (0.2, 1 and 2 mol%). In yet another preferred embodiment Pad-PC-Pad may be replaced by a compound or compound mixture comprised by any of formula la, lb, Ic and Id according to the invention.
Further preferred compositions of the invention may contain any synthetic phospholipid (e.g. Pad-PC -Pad) or preferably lipids according to any of formula la, lb, Ic and Id with 0-10 mol% surfactants such as the Brij family (e.g. S10, P10 or/and P4), 0-50 mol% rigidifiers such as cholesterol, and 0-25 mol% natural phospholipids such as EYPC, DOPC, POPC, DPPC, sphingomyelin and miltefosine.
Other compositions of the invention according to any of formulae la, lb, Ic and Id may comprise natural phospholipids (e.g. EYPC, DOPC, POPC, DPPC, sphingomyelin) with cholesterol, miltefosine and octadecanol in various concentrations up to 50 mol%, preferably 10 - 40 mol%, 20 - 30 mol%, or 30 - 50 mol%.
It will be appreciated that each particular composition and the inclusion of the specific synthetic phospholipids or lipids of the invention will lead to a composition and consequently to nanoparticles which exhibit very particular release features which may be advantageously applied in the below described uses and methods. The skilled person will appreciate that various combinations of lipids according to any of the formulae la, lb, Ic or/and Id may be combined as described above or in an advantageous manner in order to adapt the release profile to the requirements of the desired medical or non-medical application and use.
The achievement of the inventors is to a great extent that they found that the synthetic phospholipids, and in a preferred embodiment the lipids of the invention, preferably in combination with natural lipids and eventually additional compounds could be tailored to endogenous shear stress levels in diseases, disease states or disorders to achieve a targeted delivery of an active compound and overcome thus the disadvantages of the state of the art in treating such diseases, disease states or disorders.
The compositions according to invention may further comprise an active compound or selected compound. In the context of the invention the term "active compound" is preferably used for compounds which are applied in a medical context and preferably relate to drugs or any compounds which are used for the purpose to achieve an effect in a system, e.g. an animal or human body. The term "selected compound" preferably is used for any compound that is supplied or delivered by application of the invention to a system and may be used for monitoring or diagnostic purposes. In general the selected compound does not usually change the system wherein it is supplied to, however, it is predominantly used to monitor, measure or diagnose a certain change in a system.
This active or selected compound may be chosen from any known class of chemical compounds and have different properties. It may have pharmaceutical, diagnostic, biomarker or other properties as may be required for the particular application of the composition of the invention. The active or selected compound may also be denoted as "payload". In the context of the invention this payload will be incorporated by known techniques with the composition of the invention and may be produced as vesicles. In particular applications of the invention, the payload will be transported in a medium or a system like the circulation of a patient to a particular target site where the payload is released. The amount and timing of the release can be engineered according to the circumstances and the particular mixture of chemical compounds in the composition to arrive at a desired release profile.
In a preferred embodiment the active compound is selected from the group consisting of a fibrinolytic agent, an anti-coagulation agent, an anti-aggregation agent, an atherosclerotic plaque stabilizer (Statin), a vasodilatory agent, preferably a direct or indirect acting vasodilator (a NO-liberating agent, an alpha-adrenoreceptor antagonist, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a direct renin inhibitor, a calcium-channel blocker (CCB), an endothelin receptor antagonist, a phosphodisesterase inhibitor, a potassium-channel opener,), anti-arrhytmic drugs (a sodium-channel blocker, a beta-blocker, a potassium-channel blocker, a calcium-channel blocker), inotrop positive medication (a catecholamine, a non-catecholamine), heart muscle remodeling (ACE inhibitors or ARB), diastolic dysfonction treatment (anticalcics, betablockers, ACE inhibitors or ARB), decongestion (Brain Natriuretic Peptide (BNP), nitro-release drugs), cardiomyocytes or stem cells (transplantation), radio contrast markers (for CT, MRI, Positron emission tomography (PET), scintigraphy, percutaneous coronary intervention), a chemotherapeutic agent, a coagulation factor agent, a heparin, and an antiinflammatory agent.
In a further preferred embodiment the active compound is selected from the group consisting of alteplasum (Actilyse®), heparin (Liquemine®), acetyl salicylic acid (Aspirin®), clopidogrelum (Plavix®), glycoprotein Hb/IIIa inhibitor such as ReoPro®, rosuvastatinum (Crestor®), NO-liberating agents such as nitroglycerin (Perlinganit®), nitroprussiate or molsidomine (Corvaton®), phentolamine (Regitine®), enalapril (Epril®), candesartanum (Atacand®), diltiazem (Dilzem®), bosentan (Tracleer®), milrinone (Corotrop®) or levosimendan (Simdax®), minoxidilum (Loniten®), aliskirenum (Rasilez®), quinidine ,metoprolol (Lopresor®; BelocZok®), amiodarone (Cordarone®), Verapamil (Isoptin®), epinephrin, norepinephrin, dopamine, dobutamine, isoprenalin, milrinone (Corotrop®), levosimendan (Simdax®), vasopressin, glypressin, Brain Natriuretic Petide (BNP), natural purified or recombinant forms such as nesiritidum (Noratak®), nitroglycerine, alteplasum (Actilyse®), Eptacogum alfa, NovoSeven®, Perlinganit®, nitroprussiate or molsidomine (Corvaton®), candesartanum (Atacand®), Iodine, Gadolinium containing contrast agents, positron-emitting radionuclide (tracer), cardiomyocytes or stem cells.
The composition according to the invention preferably may be provided in the form of nanoparticles wherein the nanoparticle is preferably a micelle, more preferably the nanoparticle is composed of a monolayer, a bilayer and/or a vesicle and/or a nanocontainer.
The nanoparticles according to the invention may be designed in any useful size and amount. Preferably the nanoparticle has an average diameter of about 10 to 1000 nm, preferably of about 50 to 500 nm, more preferably of about 50 to 200 nm.
In a further aspect the invention related to a method of making a 1,3- diamidophospholipid wherein a phosphoethanolamine is alkylated under appropriate conditions, preferably with the use of dimethyl sulfoxide. Other methods of alkylation with alternative alkylating agents may be used such as alkylation with the corresponding alkylhalide, preferably methyliodide (Lu, X.; Bittman, R. The Journal of Organic Chemistry 2005, 70, 4746-4750)
The inventors thus could provide for a simple and economically advantageous method of making the compounds according to the invention.
In a further aspect the invention relates to a method of making a composition according to the invention as described above.
Another aspect of the invention relates to a method of making the composition of the invention comprising mixing the first and second compound with appropriate means.
In another aspect the invention relates to a method of making nanoparticles comprising a composition and preferably a payload as described above.
Nanoparticles can be made according to techniques known in the art (e.g. "Preparation of Vesicles (Liposomes)" by Peter Walde in Encyclopedia of Nanoscience and Nanotechnology, Volume 9, pp. 43-79(37)). In preferred embodiments the nanoparticles are produced by thin film hydration, and/or one or more freeze-thaw cycles, sonication or/and extrusion, or by a electroformation method or by hydrating spray-dried lipids or by sonication or by repetitive freezing and thawing or by dehydration and rehydration or by the extrusion technique or by the treatment of a multilamellar vesicle suspension with a microfluidizer, or the preparation of multilamellar novasomes or the preparation of multilamellar spherulites, or the preparation of multilamellar vesicles by the "bubble method" (1-1. Talsma, M. J. van Steenbergen, J. C. H. Borchert and O. J. A. Crommelin, J. Phann. Sci. 83, 276 (1994)), or the preparation by the "Cochleate cylinder method" (S. Gould-Fogcrite and R. J. Mannino in Liposome Technology, Vol. 1, 2nd ed., edited by G. Gregoriadis, CRC Press, Boca Raton (1993), p. 67), or the preparation by the "Reversed-phase evaporation technique, or the preparation from water/oil and water/oil/water emulsions, or the preparation by the "solvent-spherule (w/o/w- emulsion) method" (S. Kim, R. E. Jacobs and S. H. White. Biochim. Biophys. Acta 812. 793 (1985)) or the "DepoFoam Technology" (S. Mantripragada, Progr. Lipid Res. 41, 392 (2002)), or the preparation from an organic aqueous two-phase system, or the preparation by the "ethanol injection method" (A. S. Domazou and P. L. Luisi,
I. Liposome Res. 12, 205 (2002)), or the preparation by the "pro-liposome method" (W. P. Williams. S. Perrett, M. Golding, J-P. Arnaud and M. Michez in Phospholipids: Characterization, Metabolism, and Novel Biological Application, edited by G. Cevc and F. Paltauf, AOCS Press, Champaign IL (1995). p. 181), or the preparation of multilamellar ethosomes, or the preparation by the "interdigitation- fusion method" (P. L. Ahl, L. Chen, W. R. Perkins, S. R. Minchey, L. T. Boni, T. F. Taraschi, A. S. Janoff. Biochim. Biophys. Acta 1195, 237 (1994)), or the preparation by the "coacervation technique" (F. Ishii, A. Takamura and Y. Ishigami, Langmuir
I I, 483 (1995)), or the preparation by the "supercritical liposome method" (L. Frederiksen. K. Anton. P. van Hoogevest. H. R. Keller and H. Leuenberger, J. Phann. Sci. 86, 921 (1997)), or the preparation from an initial oil/water emulsion, or the preparation by the "ether injection method" (D. W. Deamer, Ann. N. Y. Acad. Sci. 308, 250 (1978)), or the preparation by the "rapid solvent exchange method" (J. T. Buboltz and G. W. Feigenson, Biochim. Biophys. Acta 1417, 232 (1999)). or the preparation by the "Detergent-depletion method" (R. A. Parente and B. R. Lentz, Biochemistry 23, 2353 (1984); T. M. Allen. A. Y. Romans, H. Kercrel and 1. P. Segrest, Biochim. Biophys. Acta 601, 328 (1980)), or the preparation by mixing bilayer-forming and micelle-forming amphiphiles, or the preparation from lipids in chaotropidc ion solutions, or the preparation of vesicles prepared from a water/oil- emulsion with the help of a detergent.
Another aspect of the invention relates to a pharmaceutical or cosmetic composition comprising a composition according to the invention as described above or a nanoparticle as described above, and preferably further useful carriers or/and additives.
Yet another aspect of the invention is a composition according to the invention and the nanoparticles according to the invention for use in a pharmaceutical or cosmetic application.
The pharmaceutical and cosmetic compositions of the invention will be prepared using known methods and useful auxiliary compounds as known in the art and applicable in the context of the invention.
Another aspect of the invention relates to a composition according to the invention or nanoparticles according to the invention for use in the prophylaxis or treatment of a vascular disorder or disease, or for use in the prophylaxis or treatment of a dermatological disorder or disease, or for use as cosmetic, or for use in a monitoring or diagnostic method.
Alternatively, the invention relates to a method for the prophylaxis or treating a patient in need thereof by administering nanoparticles according to the invention with an active compound as describe above to a patient in an effective dosage to a patient.
The treatment or prophylaxis may be for a vascular disorder or disease, or a dermatological disorder or disease. The method may be as well a cosmetic method wherein preferably the cosmetic method comprises topical applications.
The invention may be applied in a method of treatment or prophylaxis or use for treatment or prophylaxis in any disease or disorder wherein a targeted delivery of an active compound is advantageous. One advantage of the invention is that the dosage delivered to a patient may be reduced due to the use of the inventive compositions or nanoparticles. Another or additional advantage that may be achieved by the invention is the reduction of undesired side effects due to the targeted delivery of the active compound.
In particular in acute and ambulatory situations the treatment of ischemia and heart attacks focuses on intravenous administration of vasodilators such as nitroglycerine to restore blood flow and prevent myocardial ischemia. Unfortunately, systemic vasodilation is a common and serious side effect. The invention advantageously overcomes this shortcoming by a targeted delivery to the diseased blood vessels of the myocardium. In an experimental model the inventors could show that elevated shear stresses similar to those found in stenosed coronary arteries could be used as a localized physical trigger for the release of an active compound from the nanoparticles according to the invention. The inventors have shown in controlled in vitro fluorescence release studies, that the inventive compositions could be used to make nanoparticles exhibiting a preferential release profile in diseased artery models.
Additionally, it was found that in a preferred embodiment of nanoparticles, in particular vesicles and/or nanocontainers, containing entirely the phospholipid Pad- PC-Pad according to the invention were considerably more sensitive to shear-induced release than mixtures containing solely natural phospholipids. The results show that the shear-induced release properties of vesicles can be tuned to allow preferential release in regions of higher shear stress. Accordingly, the invention provides for phospholipids and compositions comprising different components as described above wherein their particular composition can be engineered in a manner to meet certain shear stress release profiles. These specially engineered compositions can be provided as nanoparticles and loaded with a desired active compound to serve as a delivery means to a particular site (target site) in a patient to treat a particular disease state or disorder.
Literature values are reported on the shear stress in healthy and diseased systems (Cheng, C, et al., Large variations in absolute wall shear stress levels within one species and between species. Atherosclerosis, 2007. 195 (2): p. 225-235.). Whereas in a healthy artery average stresses of around 1.5 Pa have been determined, this value rises to between 7 and 10 Pa in complex plaques. Furthermore, blood vessel constriction can lead to wall shear stress values well above 10 Pa.
In a preferred embodiment, nanoparticles have been developed that may specifically deliver nitroglycerin to the site of atherosclerotic constriction. Thus, it is possible to overcome the problem of systemic vasodilation and allow the localized dilation of stenosed arteries without the negative side effects of systemic vasodilation. The positive effects of the invention could be shown by application of a fluorophore model of carboxyfluorescein encapsulated vesicles. Different compositions according to the invention were applied in this model and their usefulness for a targeted delivery by shear stress-induced release could be shown. The inventors thus could show for the first time that endogenous shear stress could be applied as a physical trigger to locally release a payload carried to the target site by nanoparticles.
In a preferred embodiment the inventors could show that a composition consisting of pure Pad-PC-Pad nanoparticles showed that an increase in stenosis of the artery model (i.e. increased atherosclerosis) led to an increase in release induced by endogenous shear stress as compared to known natural lipids.
The nanoparticles according to the invention will exhibit different stabilities with regard to shear stress. The invention makes advantageously use thereof for the medical and non-medical applications and methods of the invention. Changing the size and ratios of hydrophobic and hydrophilic sites in the compounds applied in the invention will change, i.e. increase or reduce, the stabilization and destabilisation properties, respectively, of the novel molecules on the formulated nanoparticles according to the invention.
For example the release properties of 100% Pad-PC-Pad are vastly different to those of Pad-PC-Pad/EYPC mixtures. It could be measured in the in vitro model as described in the examples section that these formulations showed a release of 40 % of its content after one pass through the model artery and of about < 5 %, respectively. In a preferred embodiment the use or method of treatment can be applied to a dermatological disease or disorder wherein the dermatological disease or disorder is preferably selected from the group consisting of acne, napkin dermatitis, atopic dermatitis, seborrhoeic dermatitis, psoriasis, warts, tinia pedis, seborrhoeic keratosis, hives, rosacea, dermatological viral infection and dermatological bacterial infection.
In a preferred embodiment the vascular disorder or disease is related to or is acute coronary syndrome (ACS), myocardial infarction, acute heart insufficiency, chronic heart insufficiency, cerebrovascular accident (CVA), stroke, atherosclerosis, vasospasm, tumor treatment, hemoptysis, pulmonary embolism, pulmonary arterial hypertension, intestinal ischemia, intestinal hemorrhage, renal infarction, renal hemorrhage, renal auto-regulation for hypertensive treatment, auto-immune glomerulonephritis or intersitial nephritis, treatment of fetal diseases, placental infarction, placental hemorrhage, retinal ischemia, retinal hemorrhage, or retinal neovascularization.
In another aspect the invention relates to a composition or nanoparticles according to the invention for use in the targeted delivery of a selected compound. The selected compound is released at a target site due to endogenous shear stress at the target site.
The term "endogenous shear stress" as understood in the context of the invention refers to the shear stress that is present at a target site and which is preferably used to trigger the partial or complete release of an active or selected compound from the nanoparticles according to the invention. In contrast thereto non-endogenous shear stress is produced by way of an apparatus being applied in order to produce a shear stresss e.g. within a system or the body of an animal or human at a desired site therein. In the context of application of a pharmaceutical or cosmetic formulation in the form of e.g., a lotion, cream or emulsion, endogenous shear stress is also understood as the shear stress produced by applying e.g. the lotion onto the skin or other parts of the human or animal body.
In another aspect the invention relates to a method of a targeted delivery of a selected compound or composition of compounds wherein i. in a first step the selected compound is loaded into a nanoparticle according to the invention, ii. the loaded nanoparticle is applied to a subject or object and the selected compound is released at the target site due to endogenous vascular shear stress at the target site.
In another aspect the composition or nanoparticles of invention are used in a monitoring method or a diagnostic method.
In a preferred embodiment the selected compound is selected from the group consisting of a medium, a small molecule, a protein, peptide, nucleic acid, nucleotide or an antibody. Preferably the selected compound is a marker, a contrast medium or a labeled compound.
In a further preferred embodiment the selected compound is selected from the group consisting of a iodine or gadolinium labeled antibody against glycoprotein- (GP)IIb/IIIa-(aIIb 3) receptors, iodine or gadolinium labeled abciximab (ReoPro®), an atherosclerosis associated marker such as CD 16a, CD 32, CD 36, CD 40, CD 44, CD 45RO, a general inflammatory marker like an interleukin, iodixanolum (Visipaque®), gadopentetate dimeglumine (Magnevist®), a coronary stenoses marker such as copper, or coagulation factor Vila (NovoSeven®).
The use or methods according to the invention as described above may preferably be applied in coronary atherosclerosis, myocardial infarction, cerebrovascular accident (CVA), stroke, vasospasm, tumors, hemoptysis, pulmonary embolism, intestinal ischemia, digestive tract hemorrhage, renal infarction, renal hemorrhage, placental infarction, placental hemorrhage, retinal ischemia, retinal hemorrhage, diabetic retinopathy, or hypertensive retinopathy.
It will be appreciated by the skilled person that for the use or methods according to the invention the compositions and nanoparticles of the invention may be designed to fit the needs of the desired applications and release profile. In particular the compositions and nanoparticles will be adapted to the chosen target sites wherein preferably the target site is characterized by an endogenous shear stress, preferably an endogenous vascular shear stress, of between 2 Pa and 20 Pa, preferably of between 2 Pa and 15 Pa, more preferably of between 2 Pa and 14 Pa, even more preferably of between 4 Pa and 14 Pa. In the use and methods according to the invention the selected compound is preferably released at a therapeutically effective amount.
The term "therapeutically effective amount" or "effective amount" of an active compound as understood in the context of the invention is meant as the amount released from the vesicles of the invention at the target site or in the vicinity of the target site and reaching the medical target producing the desired effect or response in the treatment or prophylaxis of the disease or disorder in question. It will be appreciated by the skilled person, that depending on the particular circumstances the amount loaded onto nanoparticles of the invention and the finally effective amount released to achieve a particular effect at the target site will vary significantly.
The "target site" as understood in the context of the invention is the area of the system or in the animal or human body whereto the active or selected compound is to be delivered. At the "target site" there can be under preferred circumstances a certain shear stress be present and preferably be determined by usual means (Cheng, C, et al., Atherosclerosis, 2007. 195 (2): p. 225-235.) The structures according to the invention with regard to the desired release profile of an active or selected compound will be designed by combining a particular mixture of the different compounds as described above.
The amount of active or selected compound released can vary depending on the design of the vesicles according to the invention. Usually about the entire pay-load will be released at the target site. Preferably 80 to 95 %, 60 to 90 % or 50 to 90 % will be released. In other embodiments 30 to 40 %, even more preferably 40 to 50 %, even more preferably 40 to 60 % of the active or selected compound will be released
In yet another aspect the invention relates to a method of a targeted release of an active or selected compound or mixture of compounds from loaded vesicles at a release site in a tubular system, preferably a vascular vessel system, due to endogenous shear stress wherein the vesicles are being recycled in said tubular system and the compounds or mixture of compounds are periodically released from the vesicles. The release of the vesicles can be designed to meet particular characteristics and thus various release profiles depending on the needs can be engineered. It will be possible to design a continuous release, i.e. a similar amount of releases each time the vesicles pass through the release site. On the other hand an initial release of a great proportion of the loaded compound or compound mixture can be designed to achieve a fast effect of the compound or compound mixture at the release site with a lower level of release in the following to maintain the effect over a certain period of time.
The design of the releases profile will depend on the shear stress at the release site, the loaded compound or compound mixture, the composition of the vesicles and possibly the seize of the vesicles.
In a preferred embodiment the released active or selected compound or compound mixture will be released during the first or several first passages through the release site at a high quantity. More preferably 30 to 40 %, even more preferably 40 to 50 %, even more preferably 40 to 60 % of the active or selected compound will be released during the first, preferably the first to third passage of the nanoparticles through the target site. The remainder of the active or selected compound will be released evenly during the fourth and following passages. In a preferred embodiment the release after the first burst or "bolus" release of the active compound will be in a fashion to maintain the appropriate effective level of the active compound at the target site or around the target side where the active compound is to act for treatment.
One example is a restricted or partially blocked artery of the circulation of a human or animal. The anti-clotting compound or vasodilation compound (in general the active compound) will lead to opening the narrowed release site and thus the endogenous shear stress will be reduced. In turn, i.e. during the following passage of the vesicles of the invention through the release site, less active compound will be released at the release site while circulating the body of the animal or human circulation. In case the effect of the active compound is reduced due to a lowered concentration, the release site will narrow again and the shear stress will increase accordingly. In turn the recycling vesicles will release more of the active compound and thus there will be achieved a steady-state of an effective dosage of the active compound. Thus the invention achieves a continuous supply of active compound at the target site. In this manner the invention achieves a sustained release of the active compound loaded onto the vesicles wherein preferably the effective dosage of active compound is maintained at an appropriate level or concentration to be effective for the particular application or disease or disorder.
The parameters will be chosen depending on the use and application as well as the combination of compounds constituting the vesicles and the active compound or compound mixture loaded onto them.
The inventors have developed thus novel phospholipids and mixtures comprising phospholipids useful in a targeted delivery of a selected or active compound. The inventors found surprisingly that by providing particular mixtures of phospholipids, preferably compositions comprising or consisting of 1,3-diamidophospholipids or/and 1,2-diamidophospholipids or/and 2,3-diamidophospholipids and preferably at least a second compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant one could engineer nanoparticles for the targeted delivery of a selected or active compound or mixtures thereof.
In particular by mixing natural and synthetic phospholipids as described above the inventors arrived surprisingly at nanoparticles with various desired release profiles adapted for the release of various selected or active compounds and wherein the release can be triggered by endogenous shear stress without the need of the application of additional means.
The invention is also insofar advantageous as the release profile can preferably be modified by changing the amounts of lipids selected from the group consisting of 1,3- diamidophospholipids, 1,2-diamidophospholipids and 2,3-diamidophospholipids relative to natural lipids and preferably other components like cholesterol, cholesterol derivatives and surfactants in the nanoparticle forming composition.
The following examples will further illustrate the invention without being understood as limiting or restricting the invention. The examples represent preferred embodiments of the invention or serve simply to explain and illustrate the mechanisms underlying the invention.
The following single compounds are disclaimed from the scope of the compounds as described above. These compounds are:
Mergen, F.; Lambert, D. M.; Poupaert, J. H.; Bidaine, A.; Dumont, P. Chem. Phys Lipids 1991, 59, 267-272.
Compounds of the invention as above defined relating to A=H; several amide linear tails and double bonded tails are herewith disclaimed.
Figure imgf000058_0001
ElKhihel, L.; Loiseau, P. M.; Bourass, J.; Gayral, P.; Letourneux, Y. Arzneim. Forsch/Drug. Res. 1994, 11, 1259-1264.
Compounds of the invention as above defined relating to A=H and CI and C2 =C15:
Figure imgf000059_0001
and in:
Morris, A. D.; Atassi, G.; Guilbaud, N.; Cordi, A. A. Eur. J. Med. Chem. 1997, 32,
Figure imgf000059_0002
Compounds of the invention as above defined with C1=C2=17:
Figure imgf000059_0003
and other groups in the patent
Figure imgf000060_0001
X31838 Upid-amide 1
Ugand Component =
fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUnTAfUrnAfCmA (VEGFHgand) SEQlDNO:6
Figure imgf000060_0002
NX31838 Upid-amide 2
Ugand Component =
fCmGmGrArAftfCmAmGfUmGrtiAmA^^
(VEGFHgand) SEQIDNO.7
Figure imgf000061_0001
X31838 20Km PEG
Ligand Component =
fCmGmGrArAfUfCmAmGfUmGjTiArnAfU
(VEGF Ifgand) SEQ IDNO:9
Figure imgf000061_0002
Ugand Component =
fCmGmGrArAfUfCmAmGfUmGmAmAfUmGtCfimJ
(VEGF Ugand) SEQ ID NO:8
Clary, L.; Santaella, C; Vierling, P. Tetrahedron Lett. 1995, 57, 13073-13088.
The phospholipids with one CH tail and one fluorinated CH tail:
Figure imgf000061_0003
Liu, H.; Kang, H.; Wu, Y.; Sefan, K.; Tan, W. Chem. Eur. J. 2010, 16, 3791-3797.
Phospholipids with one fluorescent head group and 1,3-diamide:
Figure imgf000062_0001
Compounds that are disclaimed from the scope of the application are also listed depicted below:
N,W-(2-hydroxypropane- 1 ,3-diyl)dihexanamide
N!N,-(2-hydroxypropane- 1 ,3-diyl)dioctanamide
NyV-(2-hydroxypropane- 1 ,3-diyl)bis(decanamide)
N V'-(2-hydroxypropane- 1 ,3-diyl)didodecanamide
N>N1-(2-hydroxypropane- 1 ,3-diyl)ditetradecanamide
N,W-(2-hydroxypropane- 1 ,3-diyl)dipalmitamide
N^V-(2-hydroxypropane- 1 ,3-diyl)distearamide
(E or Z)-NyA/'-(2-hydroxypropane-l,3-diyl)dioleamide
Ny/V-(2-hydroxypropane-l ,3-diyl)dibenzamide
N>N,-(2-hydroxypropane- 1 ,3-diyl)bis(3-phenylpropanamide)
(2E or Z,2'E or Z)-N^V-(2-hydroxypropane-l,3-diyl)bis(3-phenylacrylamide)
N> V-(2-hydroxypropane- 1 ,3-diyl)bis(3-cyclohexylpropanamide)
N;>N,-(2-hydroxypropane-l53-diyl)bis(2-isopropyl-3-methylbutanamide)
(3r,3V,5r,5V,7r,7V)-N^V-(2-hydroxypropane-l,3-diyl)bis(adamantane-l- carboxamide)
N^V-(2-hydroxypropane- 1 ,3-diyl)dipalmitamide
N, V-(2-hydroxypropane- 1 ,3-diyl)dipalmitamide
N^V-(2-hydroxypropane- 1 ,3-diyl)distearamide N^V-(2-(( 17-hydroxy-3,6,9, 12, 15-pentaoxaheptadecyl)oxy)propane- 1 ,3- diyl)distearamide
Figure imgf000063_0001
NX31838 Upid-amide 1
Ugand Component =
fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUn^fUn^fCmAfUf CmG-S'a'- ^
(VEGF ligand) SEQ IDNO:6
Figure imgf000063_0002
X31838 Upid-amide 2
Ugand Component =
fCmGmGrArAfUICfnAmGfUmGmArnAfUmGfCfUfUinAfUinA
(VEGF ligand) SEQ ID NO.7
Figure imgf000064_0001
NX3I838 20Km PEG
Ligand Component =
fCmGmGrArAfUfCmAmGfUmGmAmAfUn^fCfURI
(VEGF ligand) SEQ tt>NO:9
Figure imgf000064_0002
Ugand Component =
fCmGmGrArAfUfCrnAmGfUmGmAn^fUmGfCfUWmAWn^fCmAfUfCfCmG-a'a'-dT (VEGF ligand) SEQ ID NO:8
3-(hexadecylamino)-3-oxo-2-(12,12,13,13, 14,14,15,15,16,16,17,17,17- tridecafluoroheptadecanamido)propyl (2-(trimethylammonio)ethyl) phosphate
2- ( 12,12,13, 13, 14,14,15, 15,15-nonafluoropentadecanamido)-3 - ((12,12,13,13,14,14,15,15,15-nonafluoropentadecyl)amino)-3 -oxopropy 1 (2- (trimethylammonio)ethyl) phosphate
3- (( 12, 12, 13, 13, 14, 14, 15, 15, 15-nonafluoropentadecyl)amino)-3-oxo-2- (12,12,13,13,14, 14,15,15,16, 16,17,17,17-tridecafluoroheptadecanamido)propyl (2- (trimethylammonio)ethyl) phosphate
3-oxo-2-(12, 12, 13, 13, 14, 14, 15, 15, 16, 16, 17, 17, 17-tridecafluoroheptadecanamido)-3- ((12, 12, 13, 13, 14, 14, 15, 15, 16, 16, 17, 17, 17-tridecafluoroheptadecyl)amino)propyl (2- (trimethylammonio)ethyl) phosphate The above disclaimed compounds do however not relate to the gist of the invention in respect to their applications and uses. The inventors were the first to find the advantageous properties and useful applications of the compounds as described in this patent application. Accordingly, all described compounds, within the scope of the embodiments and the preferred embodiments are applicable in the used and methods as defined herein throughout the specification, examples and claims.
EXAMPLES
1. Synthesis of phospholipids and their characterization
Starting compounds and solvents were purchased from Sigma-Aldrich/Fluka or Acros and were used without further purification. Pad-PE-Pad and its homologues were synthesized using the procedures from Fedotenko et al.
Column chromatographic separation was carried out using 230-400 mesh silica gel. TLC plates were developed either with potassium permanganate mixture (1 g of KMn04, 2 g of Na2C03, 100 mL of H20) or ethanolic solution of phosphomolybdic acid. 1H,13C and 31P NMR spectra were recorded (as indicated) on either a Bruker 300 MHz or 400 MHz spectrometer and are reported as chemical shifts (δ) in ppm relative to TMS (δ = 0). Spin multiplicities are reported as a singlet (s) or triplet (t) with coupling constants (J) given in Hz, or multiplet (m). ESI-MS for the characterization of new compounds was performed on an ESI API 150EX and are reported as mass- per-charge ratio m/z. IR spectra were recorded on a Perkin Elmer Spectrum One FT- IR spectrometer (ATR, Golden Gate). Melting point is uncorrected. For the experiments with vortex was used a device IKA Vortex Genius 3.
1.1 Synthesis of compounds of the invention
Synthesis of l,3-dipalmitamidopropan-2-yl 2-(trimethylammonio)ethyl phosphate (Pad-PC-Pad).
Figure imgf000065_0001
l g (1.45 mmol) of Pad-PE-Pad (non-methylated phosphoethanolamine) was solubilized in 100 mL of methanol. 1 mL (1.33 g, 10.5 mmol) of dimethylsulfate was added, the mixture was warmed up to 40 °C and then a solution of 1.45 g (10.5 mmol) of potassium carbonate in 20 mL water was added in one minute, while the mixture was being strongly stirred for 30 min and then cooled down to 20 °C. The solvent was removed under reduced pressure. Then the solid was purified on a silica gel column (75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%)). Obtained were 0.56 g of the product (0.81 mmol, 53%) .
Compounds with the aliphatic chains containing 12, 14 and 18 carbon atoms were synthesized in the same way starting from the corresponding phosphoethanolamine. R = 0.32 (75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%)).
Ή NMR (400 MHz, CDC13) δ 7.58 (s, 2H), 4.39 (s, 2H), 4.12 (s, 1H), 3.89 (s, 2H), 3.48 (s, 2H), 3.36 (s, 9H), 3.25 - 3.02 (m, 2H), 2.18 (t, J = 7.3 Hz, 4H), 1.56 (s, 4H), 1.24 (s, 48H), 0.87 (t, J = 6.7 Hz, 6H).
,3C NMR (101 MHz, CDC13) 6 174.7 (C=0), 72.2 (CH), 66.7, 59.6, 54.7(CH3s at the headgroup), 41.0, 36.9, 32.1 , 29.89, 29.84, 29.72, 29.70, 29.59, 26.2, 22.9, 14.3 (CH3s of the tails).
3,P NMR (121 MHz, CDC13) 6 2.97.
LRMS (ESI+) m/z calcd for C40H83N3O6P [M+H]+ 732.6, found 732.7.
FTIR (cm"1): 3285, 2917, 2850, 1651, 1545, 1468, 1239, 1088, 1058, 967, 720.
mp 203°C.
Synthesis of l,3-lauramidopropan-2-yl 2-(trimethylammonio)ethyl phosphate (Lad-PC-Lad, 1)
0.80 g (1.38 mmol) of Lad-PE-Lad (non-methylated phosphoethanolamine) was mixed with 100 mL of methanol. 1.30 mL (1.74 g, 18.5 mmol) of dimethylsulfate was added shortly after that. The mixture was warmed up to 40 °C and then a solution of 1.91 g (18.5 mmol) of potassium carbonate in 20 mL water was added in one minute, while the mixture was strongly stirring. It was then stirred further 30 min at 40 °C and then cooled down to 20 °C. Methanol-water was removed under reduced pressure. To the solid residue was added methanol and the solvent was again removed under reduced pressure. Then the solid was mixed with 10 mL of a solution containing by volume 75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%) and purified on a silica gel column using the mobile phase of the same composition. Obtained was 0.26 g of the product (0.42 mmol, 30%).
R/= 0.47 (75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%))
Ή NMR (400 MHz, CDC13) δ 7.44 (s, 2H), 4.40 (s, 2H), 4.14 (s, 1H), 3.88 (s, 2H), 3.37 (s, 9H), 3.12 (s, 2H), 2.17 (t, J = 7.7 Hz, 4H), 1.57 (s, 4H), 1.24 (s, 31.3H), 0.87 (t, J= 6.8 Hz, 6H).
13C NMR (126 MHz, CDC13) δ 174.62, 72.10, 66.37, 59.47, 54.36, 40.53, 36.64,
31.93, 29.72, 29.67, 29.62, 29.51, 29.49, 29.38, 25.92, 22.69, 14.12.
3,P NMR (121 MHz, CDC13) δ -0.6.
HRMS (ESI+) m/z calcd [M+H]+ 620.4762, obs. 620.4775
FTIR (cm-1): 3280, 2919, 2851, 1648, 1551, 1468, 1219, 1084, 1058, 966, 798.
mp=145-150°C.
Synthesis of l,3-dimyristamidopropan-2-yI 2-(trimethylammonio)ethyl phosphate (Mad-PC-Mad, 2)
0.82 g (1.29 mmol) of Mad-PE-Mad (non-methylated phosphoethanolamine) was mixed with 100 mL of methanol. 2 mL (2.66 g, 21.1 mmol) of dimethylsulfate was added shortly after that. The mixture was warmed up to 40 °C and then a solution of 2.92 g (21.1 mmol) of potassium carbonate in 20 mL water was added in one minute, while the mixture was strongly stirring. It was then stirred further 30 min at 40 °C and then cooled down to 20 °C. Methanol-water was removed under reduced pressure. To the solid residue was added methanol and the solvent was again removed under reduced pressure. Then the solid was partially mixed with 10 mL of a solution containing by volume 75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%) and purified over a silica gel column using the mobile phase of the same composition. Obtained was 0.56 g of the product (0.83 mmol, 63%).
R/ ).51 (75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%)).
Ή NMR (400 MHz, CDCI3) 5 7.52 (s, 2H), 4.37 (s, 2H), 4.19 - 4.05 (m, 1H), 3.85 (s, 2H), 3.55 (s, 2H), 3.35 (s, 9H), 3.20 - 2.98 (m, 2H), 2.17 (t, J = 7.3 Hz, 4H), 1.57 (s, 4H), 1.24 (s, 39H), 0.87 (t, J= 6.8 Hz, 6H).
I3C NMR (126 MHz, CDC13) δ 174.61, 72.10, 66.36, 66.31, 59.47, 54.34, 40.52, 36.64, 31.94, 31.80, 29.74, 29.69, 29.64, 29.53, 29.50, 29.39, 25.92, 22.83, 22.70, 22.55, 14.12. 3,P NMR (121 MHz, CDC13) δ -2.9.
HRMS (ESI+) m/z calc. [M+H]+ 676.5388, obs. 676.5385.
FTIR (cm-1): 3287, 2918, 2851, 1651, 1547, 1468, 1235, 1058, 969, 788.
mp=189-193°C
Improved synthesis of l,3-distearamidopropan-2-yl 2-(trimethylammonio)ethyl phosphate (Pad-PC-Pad 3)
1 g (1.45 mmol) of Pad-PE-Pad (non-methylated phosphoethanolamine) was mixed with 100 mL of methanol. 1 mL (1.33 g, 10.5 mmol) of dimethylsulfate was added shortly after that. The mixture was warmed up to 40 °C and then a solution of 1.45 g (10.5 mmol) of potassium carbonate in 20 mL water was added in one minute, while the mixture was strongly stirring. It was then stirred further 30 min at 40 °C and then cooled down to 20 °C. Methanol-water was removed under reduced pressure. To the solid residue was added methanol and the solvent was again removed under reduced pressure. Then the solid was partially mixed with 10 mL of a solution containing by volume 75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%) and purified on an alumina column using the mobile phase of the same composition. The second time it was purified over a silica gel column. Obtained were 0.56 g of the product (0.81 mmol, 53%).
R/= 0.56 (75% CH2C12, 22% MeOH, 3% aq. ΝΗ,ΟΗ (25%)).
Ή NMR (400 MHz, CDC13) δ 7.58 (s, 2H), 4.39 (s, 2H), 4.12 (s, 1H), 3.89 (s, 2H), 3.36 (s, 9H), 3.12 (s, 2H), 3.25 - 3.02 (m, 2H), 2.18 (t, J= 7.3 Hz, 4H), 1.56 (s, 4H), 1.24 (s, 48H), 0.87 (t, J= 6.7 Hz, 6H).
13C NMR (101 MHz, CDC13) δ 174.7 (C=0), 72.2 (CH), 66.7, 59.6, 54.7(CH3s at the headgroup), 41.0, 36.9, 32.1, 29.89, 29.84, 29.72, 29.70, 29.59, 26.2, 22.9, 14.3 (CH3s of the tails).
3,P NMR (121 MHz, CDC13) δ 2.97.
HRMS (ESI+) m/z calcd for C40H83N3O6P [M+H]+ 732.6, found 732.7.
FTIR (cm'1): 3285, 2917, 2850, 1651, 1545, 1468, 1239, 1088, 1058, 967, 720.
mp 202-204°C.
Synthesis of l,3-distearamidopropan-2-yl 2-(trimethylammonio)ethyl phosphate (Sad-PC-Sad, 4)
1 16 mg (0.155 mmol) of Sad-PE-Sad (non-methylated phosphoethanolamine) was mixed with 15 mL of methanol. 0.1 mL (133 mg , 1.05 mmol) of dimethylsulfate was added shortly after that. The mixture was warmed up to 40 °C and then a solution of 145 mg (1.05 mmol) of potassium carbonate in 2 mL water was added in one minute, while the mixture was strongly stirring. It was then stirred further 30 min at 40 °C and then cooled down to 20°C. Methanol-water was removed under reduced pressure. To the solid residue was added methanol and the solvent was again removed under reduced pressure. Then the solid was partially mixed with 2 mL of a solution containing by volume 75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%) and run over an alumina and a silica gel column using the mobile phase of the same composition. Obtained were 60 mg of the product (0.08 mmol, 49%).
R = 0.64 (75% CH2C12, 22% MeOH, 3% aq. NH4OH (25%)).
Ή NMR (400 MHz, CDC13) δ 7.47 (s, 2H), 4.39 (s, 2H), 4.13 (s, 1H), 3.88 (s, 2H), 3.66 - 3.43 (m, 2H), 3.36 (s, 9H), 3.26 - 2.99 (m, 2H), 2.17 (t, J = 7.3 Hz, 4H), 1.57 (s, 4H), 1.24 (s, 55H), 0.87 (t, J= 6.8 Hz, 6H).
13C NMR (101 MHz, CDC13) 6 174.71, 66.36, 59.43, 54.41, 40.37, 36.61, 31.97,
29.80, 29.72, 29.69, 29.57, 29.55, 29.41, 25.97, 22.73, 14.15.
3,P NMR (121 MHz, CDC13) 6 3.0.
HRMS (ESI+) m/z calcd [M+H]+ 788.6616, obs. 788.6641
FTIR (cm"1): 3297, 2917, 2850, 1649, 1549, 1469, 1226, 1087, 1058, 970, 720.
mp=222-225°C
Figure imgf000069_0001
Palmitoyl chloride (5.56 mL, 17.8 mmol) and sodium azide (1.5 g, 23 mmol) were mixed in 40 mL of dry toluene and the solution was refluxed for 5 h under an N2 atmosphere. The product was directly used without further purification.
Ή NMR (300 MHz, CDC13) δ 3.29 (t, J = 6.7 Hz, 2H), 1.69 - 1.52 (m, 2H), 1.26 (s, 24H), 0.88 (t, J = 6.6 Hz, 3H). Tert-butyl (2-((amino((17,23-dioxo-16,18,22,24-tetraazanonatriacontan-20- yl)oxy)phosphoryl)oxy)ethyl)carbaraate
Figure imgf000070_0001
Tert-butyl-(2-((amino((l,3-diaminopropan-2-yl)oxy)phosphoiyl)oxy)ethyl)carbamate (210 mg, 0.672 mmol) and 5 mL of the solution of 1 -isocyanatopentadecane (51 1 mg, 2.02 mmol) and triethylamine (470 μΐ,, 3.36 mmol) were mixed with 6 mL of dry acetonitrile. The solution was heated up to 40 °C for 50 min. 100 mL of CH2C12 was added and the solution was extracted with saturated NaHC03 (100 mL). The aqueous phase was washed 2 times with 50 mL of CH2C12. The organic phases were dried over MgS04. The organic solvents were removed under reduced pressure. After silica gel column chromatographic purification (CH2Cl2-MeOH 95:5), a white solid was obtained (148 mg, 0.18 mmol, 38 %).
Rf: 0.1 1 (CH2Cl2-MeOH 95:5).
Pur-PEBoc-Pur
Ή NMR (500 MHz, CDC13) δ 6.17 (br, 1H), 6.09 (br, 1H), 5.77 (br, 1H), 5.43 (br, 1H), 5.21 (br, 1H), 4.32 (s, 1H), 4.01 (d, J = 34.2 Hz, 4H), 3.37 (s, 6H), 3.1 1 (q, J = 6.4 Hz, 4H), 1.44 (s, 13H), 1.25 (s, 48H), 0.88 (t, J= 7.0 Hz, 6H).
,3C NMR (126 MHz, CDC13) δ 159.28, 156.42, 79.49, 76.16, 65.85, 41.05, 40.42, 31.93, 30.38, 30.33, 29.73, 29.47, 29.38, 28.43, 27.02, 22.70, 14.12.
31P NMR (122 MHz, CDC13) δ 10.68.
FTIR (cm"1) 3360, 31 10, 2921, 2852, 1694, 1629, 1570, 1466, 1366, 1245, 1 173, 992, 721.
HRMS (ESI+) m/z calcd for C42H88N607P [M+H]+ 819.6446 found 819.6448. 17^23- 10X0-16, 18,22,24-tetraazanonatriacontan-20-yl-(2- (trimethylammonio)ethyl) phosphate (Pur-PC-Pur)
Figure imgf000071_0001
tert-butyl-(2-((amino(( 17,23-dioxo- 16, 18,22,24-tetraazanonatriacontan-20- yl)oxy)phosphoryl)oxy)ethyl)carbamate (140mg, 0.17 mmol) was dissolved in 9 mL dioxane and HC1 (3 mL, 12 mmol) in dioxane was added. After 4 h the reaction was stopped. Nitrogen was bubbled through the solution over 1 h and solvent was evaporated under reduced pressure. The crude material was the dissolved in 12 mL of methanol. Dimethyl sulfate (120 μί, 1.27 mmol) was added and the solution was heated to 40 °C. 3.5 mL of a solution of potassium carbonate (210 mg, 1.5 mmol) was added and stirring was continued for 1 h at 40 °C. Then the solvents were evaporated under reduced pressure and the product was purified by silica gel column chromatography (CH2Cl2-MeOH-NH40H 25% 75:22:3) to get a white solid (40 mg, 0.053 mmol, 30%).
1H NMR (400 MHz, CDC13) δ 7.81 (br, 2H), 6.49 (s, 1H), 5.86 (br, 1H), 4.45 - 4.30 (m, 2H), 4.10 (d, J - 13.8 Hz, 1H), 3.85 (d, J = 10.0 Hz, 2H), 3.54 - 2.93 (m, 18H), 1.55 - 1.34 (m, 4H), 1.25 (s, 44H), 0.88 (t, J= 7.8 Hz, 6H).
13C NMR (126 MHz, CDC13) δ 159.45, 74.59, 66.20, 59.60, 54.28, 41.48, 40.49, 31.96, 30.59, 29.83, 27.19, 22.72, 14.13.
3,P NMR (122 MHz, CDC13) δ 2.76.
FTIR (cm"1) 3286, 3042, 2919, 2851, 1641 , 1569, 1469, 1231, 1060, 971.
HRMS (ESI+) m/z calcd for C4oH85N506P [M+H]+ 762.6232 found 762.6228.
R/H).5 (CH2Cl2-MeOH-NH4OH 25% 75:22:3)
Synthesis of l-dodecyI-3-(2-hydroxyethyl)urea (1). 0
OH
9 H H
1
Tridecanoic acid (0.86 g, 4.0 mmol), triethylamine (1.0 mL, 7.2 mmol) and diphenylphosphoryl azide (0.95 mL, 4.4 mmoL) were dissolved in dry toluene (20 mL). The solution was refluxed over 3h. The solution was kept at 0 °C and ethanolamine (0.24 mL, 4.0 mmol) was added. The mixture was stirred at 20 °C overnight and mixed with 70 mL of CH2C12 to be extracted with 100 mL of water mixed with 10 mL of NH4OH (25%). The water phase was washed twice with 70 mL of CH2CI2. After drying over magnesium sulfate, the solvent were evaporated and the crude product was purified by silica gel column (95 % CH2C12, 5% MeOH). A white powder (440 mg, 1.62 mmol, 40.4%) was obtained.
Rf = 0.17 (95 % CH2C12, 5% MeOH).
Ή NMR (500 MHz, Methanol-d4) δ 3.67 (t, J = 5.3 Hz, 2H), 3.58 (s, 2H), 3.46 (s, 2H), 1.65 - 1.47 (m, 2H), 1.30 (s, 18H), 0.90 (t, J= 7.0 Hz, 3H).
13C NMR (126 MHz, MeOD) δ 161.47 (s), 62.66 (s), 43.46 (s), 41.06 (s), 33.09 (s), 32.15 - 29.47 (m), 27.98 (s), 14.44 (s).
IR (cm-'): 3340, 3317, 3030, 2955, 2921, 2849, 1619, 1591, 1462, 1268, 1057, 620. HRMS (ESI+) m/z calcd for Ci5H33N202 [M+H]+ 273.2536 found 273.2532.
Synthesis of l-dodecyl-3-(2-hydroxyethyl)thiourea (2).
Figure imgf000072_0001
Dodecyl isothiocyanate was first synthesized by variation of a procedure of Meijer. DCC (2.9 gl 7 mmol) and CS2 (7.20 mL, 1 19 mmol) were dissolved in dry diethyl ether (40 mL). Dodecyl amine (3.2 g, 17 mmol) was added at 0°C to the mixture that was stirred overnight at room temperature. The precipated solid was filtered off and washed with 60 mL of dry diethyl ether. The solvent were removed by evaporation and the isothiocyanate was used without further purification.
Ethanolamine (0.23 mL, 3.8 mmol) and triethylamine (0.61 mL, 4.37 mmol) were dissolved in dry THF (40 mL). At 0°C was added dropwise dodecyl isothiocyanate (1.0 mL, 3.8 mmol). After stirring at 20°C for 3h30, the mixture was added to CH2C12 (70 mL) to be extracted with 100 mL of water mixed with 10 mL of NH4OH (25%). The water phase was washed twice with 70 mL of CH2C12. After drying over magnesium sulfate, the solvent were evaporated and the crude product was purified by silica gel column (95 % CH2C12, 5% MeOH). The product was recristallised from dioxane/pentane (1 :1) at 8°C overnight. A white powder (850 mg, 2.95 mmol, 778%) was obtained.
Rf = 0.28 (95 % CH2C12, 5% MeOH).
Ή NMR (500 MHz, Methanol-d4) δ 3.67 (t, J = 5.3 Hz, 2H), 3.58 (s, 2H), 3.46 (s, 2H), 1.65 - 1.47 (m, 2H), 1.30 (s, 18H), 0.90 (t, J = 7.0 Hz, 3H).
,3C NMR (126 MHz, MeOD) 6 183.84, 61.82, 47.43, 45.39, 33.09, 30.79, 30.79, 30.49, 30.20, 27.99, 23.75, 14.45.
IR (cm-1): 3293, 3233, 3071, 2918, 2849, 1565, 1471 , 1461 , 1294, 1276, 1255, 1210, 1163, 1059, 1034, 728, 650.
HRMS (ESI+) mfz calcd for C,5H33 2OS [M+H]+ 289.2308 found 289.2305.
Synthesis of l-(2-hydroxyethyl)-3-pentadecylurea (3).
Figure imgf000073_0001
Pentadecyl isocyanate was first synthesized by variation of a procedure of De Feyter and all.2 Palmitoyl chloride (5.6 mL, 18 mmol) and sodium azide (1.5 g, 23 mmol) were mixed in dry toluene (40 mL). The solution was refluxed over 5h. The solution was directly used without further purification.
Ethanolamine (0.22 mL, 3.7 mmol) and dry Et3N (1.04 mL, 7.42 mmol) were dissolved in dry THF (25 mL). 4.9 mL of the toluene solution of 1- isocyanatopentadecane (0.94 g, 3.7 mmol) in dry THF (10 mL) was added dropwise (30 min) to the solution under stirring at 0 °C and still stirred 3 h at room temperature. Then the solution was treated with 10 mL NH4OH 25% in 100 mL water, and extracted with CH2C12 (4 x 50 mL). The solvents from the combined organic phases were dried with MgS04 and removed under reduced pressure. The crude product was purified on silica gel column (95 % CH2C12, 5% MeOH) to give a white powder (555 mg, 1.76 mmol, 48%).
0.20 (95 % CH2C12, 5% MeOH). Ή NMR (500 MHz, CDCl3/Methanol-d4 1 :1) δ 3.56 (t, J = 5.4 Hz, 2H), 3.21 (t, J = 5.3 Hz, 2H), 3.07 (t, J = 7.1 Hz, 2H), 1.52 - 1.35 (m, 2H), 1.22 (s, 24H), 0.84 (t, J = 7.0 Hz, 3H).
,3C NMR (126 MHz, CDCl3/Methanol-d4 1 :1) δ 159.66, 61.40, 41.98, 39.68, 31.40,
29.56, 29.15, 29.13, 29.11, 29.09, 28.87, 28.82, 26.37, 22.13, 13.29.
IR (cm-1): 3309, 3214, 3079, 2919, 2850, 1572, 1471, 1348, 1271, 1037, 718, 650.
Synthesis of l-(2-hydroxyethyl)-3-pentadecylthiourea (5).
Figure imgf000074_0001
Pentadecyl isothiocyanate was first synthesized by variation of a procedure of Meijer.1 DCC (3.1 g, 15 mmol) and CS2 (6.3 mL, 104 mmol) were dissolved in dry diethyl ether (40 mL). Pentadecyl amine (3.4 g, 15 mmol) was added at 0°C to the mixture that was stirred overnight at room temperature. The precipated solid was filtered off and washed with 60 mL of dry diethyl ether. The solvent were removed by evaporation and the isothiocyanate was used without further purification.
Ethanolamine (0.23 mL, 3.8 mmol) and triethylamine (1.0 mL, 7.4 mmol) were dissolved in dry THF (40 mL). At 0°C was added dropwise pentadecyl isothiocyanate (1.0 mL, 3.7 mmol) in dry THF (20 mL) over lh30. After stirring at 20°C for 4h30, the mixture was added to CH2C12 (70 mL) to be extracted with 100 mL of water mixed with 10 mL of NH4OH (25%). The water phase was washed twice with 70 mL of CH2C12. After drying over magnesium sulfate, the solvent were evaporated and the crude product was purified by silica gel column (95 % CH2C12, 5% MeOH). The product was recristallised from dioxane/pentane (1 :4) at 8°C overnight. A white powder (700 mg, 2.12 mmol, 57%) was obtained.
Rf = 0.36 (95 % CH2C12, 5% MeOH).
1H NMR (300 MHz, CDC13) δ 3.92 - 3.76 (m, 2H), 3.68 (s, 2H), 3.37 (s, 2H), 1.69 - 1.49 (m, 2H), 1.25 (s, 24H), 0.88 (t, J= 6.5 Hz, 3H).
,3C NMR (126 MHz, MeOD) δ 183.71, 61.82, 47.43, 45.39, 33.08, 30.78, 30.48, 30.20, 27.98, 23.74, 14.44.
IR (cm-1): 3228, 3073, 2915, 2848, 1567, 1470, 1365, 1293, 1276, 1216, 1049, 738, 720, 665. HRMS (ESI+) m/z calcd for C18H39N2OS [M+H]+ 331.2777 found 331.2774.
Synthesis of l-(2-hydroxyethyl)-3-hexadecylthiourea (6).
Figure imgf000075_0001
Hexadecyl isothiocyanate was first synthesized by variation of a procedure of Meijer.1 DCC (2.42 g, 14.5 mmol) and CS2 (6.00 mL, 100 mmol) were dissolved in dry diethyl ether (40 mL). Hexadecyl amine (3.80 g, 14.2 mmol) was added at 0°C to the mixture that was stirred overnight at room temperature. The precipated solid was filtered off and washed with 60 mL of dry diethyl ether. The solvents were removed by evaporation and the isothiocyanate was used without further purification.
Aminoethanol (0.22 mL, 3.7 mmol) and dry Et3N (1.0 mL, 7.4 mmol) were dissolved in dry THF (25 mL). 1 -isothiocyanatohexadecane (1.0 mL, 3.7 mmol) in dry THF (15 mL) was added dropwise (lhl5) to the solution under stirring at 0 °C and still stirred 3h30 at room temperature. Then the solution was treated with 10 mL NH4OH 25% in 100 mL water, and extracted with CH2C12 (4 x 50 mL). The solvents from the combined organic phases were dried with MgS04 anhyd and removed under reduced pressure. The crude product was purified on silica gel column (95 % CH2C12, 5% MeOH) and then by recristallisation (dioxane/pentane, 4:1) to give the product as a white powder (756 mg, 2.19 mmol, 59%).
Rf = 0.25 (95 % CH2C12, 5% MeOH).
Ή NMR (500 MHz, Methanol-d4) δ 3.66 (t, J = 5.4 Hz, 2H), 3.57 (s, 2H), 3.43 (s, 2H), 1.64 - 1.47 (m, 2H), 1.29 (s, 26H), 0.90 (t, J= 7.0 Hz, 3H).
13C NMR (126 MHz, MeOD) δ 183.01, 61.82, 47.45, 45.57, 33.08, 30.79, 30.73, 30.71, 30.48, 30.20, 27.98, 23.74, 14.44.
IR (cm"'): 3298, 3231, 3073, 2917, 2849, 1565, 1461, 1366, 1286, 1272, 1211, 1059, 1036, 728, 651.
Synthesis of l,l'-(5-hydroxydihydropyrimidine-l,3(2H,4H)-diyl)bis(hexadecan-l- one) (Cyclo[6]Pad-OH-Pad) Method A. 100 mg (0.98 mmol) of 5- hydroxy-l,3-diaziridine and 0.349 mL (253 mg, 2.5 mmol) of Et3N were mixed with 10
Figure imgf000076_0001
mL of dry CH2C12. 0.61 1 mL (550 mg, 1.96 mmol) of palmitoyl chloride were added. The mixture was left stirring for 10 h. CH2C12 was removed under reduced pressure. The product was purified over a silica gel column, to give 142 mg of a white solid (0.24 mmol, 25%).
Method B. 5-hydroxy-l,3-diaziridine (402 mg, 3.94 mmol) was dissolved in a mixture of CH2C12 (18 mL), toluene (7 mL) and NaOH (693 mg, 17.3 mmol) in H20 (13 mL). At 20 °C, palmitoyl chloride (2.4 ml, 7.87 mmol) was added in one step. After stirring for 3 h, the solution was extracted with 50 mL of NaHC03 saturated and washed 2 times with 50 mL of CH2C12. (If an emulsion appears because of palmitic acid, the solution must be filtered and re-extracted) The organic phases were dried over MgS04 and the solvents were evaporated.
Purification was done with a silica gel column, eluted with CH2C12 then CH2C12/Ethyl acetate 1 : 1. Obtained 935 mg of a white solid (1.62 mmol, 41%)
R = 0.5 (CH2C12 Ethyl acetate 1 : 1)
'H NMR (500 MHz, CDC13) 5 5.69 (d, J= 13.1 Hz, 1H), 4.54 (d, J= 13.1 Hz, 1H), 4.16 (dd, J= 13.6, 3.4 Hz, 1H), 3.95 - 3.85 (m, 1H), 3.73 (dd, J = 13.8, 4.1 Hz, 1H), 3.54 (d, J= 13.6 Hz, 1H), 3.46 (d, J= 13.5 Hz, 1H), 2.67 - 2.50 (m, 2H), 2.35 (ddt, J = 30.7, 15.4, 7.9 Hz, 2H), 1.66 (d, J= 46.1 Hz, 9H), 1.26 (s, 49H), 0.89 (t, J= 6.8 Hz, 6H).
,3C NMR (126 MHz, CDC13) δ 174.35, 172.91, 64.20, 55.91, 51.01, 47.83, 33.16, 32.91 , 31.94, 29.71 , 29.38, 25.29, 24.95, 22.71 , 14.21.
HRMS (ESI+) m/z calcd for C36H71N203 [M+H]+ 579.5459 obs. 579.5466
FTIR (cm'1): 3414, 2918, 2850, 1658, 1619, 1468, 1255, 1 145, 885, 722
Mp= 84-86 °C ilmitoylhexahydropyrimidin-5-yl (2-(trimethylammonio)ethyl) phosphate
(Cyclo[6]Pad-PC-Pad)
To a solution of 150 mg (0.259 mmol) of the cyclo[6]Pad-OH-Pad and 0.253 mL (184 mg, 1.81
Figure imgf000077_0001
mmol) of NEt3 in 15 mL of CH2C12 was added ethylene chlorophosphite (0.099 mL, 142 mg, 1.09 mmol) at 0 °C. After that the mixture was stirred for 24 h. Then 1 M bromine solution (1.74 mL, 1.74 mmol) was added at 0 °C. After 10 min, the solvent was removed under reduced pressure. The residue was dissolved in 9 mL of CH3CN//-PrOH/CHCl3 (1.5:1.5: 1), and 5 mL of 45% aqueous trimethylamine was added at rt. After 24 h, the solvents were removed. The product was purified first - by passing through a column chromatography on silica gel (elution with CH2C12-CH3-0H-H20 65:25:4). Then the small portions (30- 50 mg) were purified on a SEPHADEX LH-20 column (elution with CH2C12-CH3- OH-H20 65:25:4). Purity was controlled by 1H- NMR for each portion. If the purity was insufficient, the separation was repeated. Yield 140 mg (0.189 mmol, 73%)
Ή NMR (400 MHz, CDC13) δ 5.38 (d, J = 14.4 Hz, 1H), 4.79 (d, J = 13.0 Hz, 1H), 4.49 - 4.25 (m, 3H), 4.08 - 3.96 (m, 1H), 3.87 (s, 2H), 3.82 - 3.71 (m, 1H), 3.68 - 3.58 (m, 1H), 3.54 - 3.44 (m, 1H), 3.37 (s, 7H), 2.69 - 2.33 (m, 4H), 1.71 - 1.48 (m, 4H), 1.46 - 1.09 (m, 48H), 0.92 (t, J= 6.8 Hz, 6H).
13C NMR (126 MHz, CDC13) δ 173.30, 172.65, 66.78, 66.33, 59.45, 55.64, 54.44,
50.48, 46.46, 33.38, 32.85, 31.94, 29.75, 29.69, 29.62, 29.55, 29.46, 29.38, 25.63,
24.99, 22.70, 14.13.
31P NMR (121 MHz, CDC13) δ 2.63.
Mp=214-218°C
HRMS (ESI+) m/z calcd for C4iH82N306P [M+H]+ 744.6014 obs. 744.6003
FTIR (cm-1): 3341, 2921, 2853, 1642, 1466, 1430, 1087, 1054, 970, 920, 875, 778, 722 (1) Boas, U.; Karlsson, A. J.; de Waal, B. F. M.; Meijer, E. W. J. Org. Chem. 2001, (5(5, 2136-2145.
(2) De Feyter, S.; Larsson, M.; Schuurmans, N.; Verkuijl, B.; Zoriniants, G.; Gesquiere, A.; Abdel Mottaleb, M. M.; van Esch, J.; Feringa, B. L.; van Stam, J.; De Schryver, F. Chemistry - A European Journal 2003, 9, 1198-1206.
1.2. Studies on the release of the liposomal content
Free release and vortex. Large unilamellar vesicles of average diameter of 100 nm were formulated using the procedure described below (2 mg of Pad-PC-Pad, DPPC (AvantiLipids) or N-palmitoylsphingomyelin (AvantiLipids) in 0.5 mL of carboxyfluoresceine buffer (10 mM HEPES, NaOH pH=7.4, 10 mM NaCl, 50 mM carboxyfluoresceine). The liposomal suspension was diluted to 100 mL with a second buffer (10 mM HEPES, NaOH pH 7.4, 10 mM NaCl). Five aliquots (3 mL each) were filled into 20 mL vials with polystyrene caps and vortexed for a discrete amount of time times (0, 5, 10, 20, 60 sec) at 2500 rpm. Release of the content was measured by a plate reader at the wavelengths of 492 (excitation) and 517 nm (emission).
Free release was measured daily for 6 days for each sample. For every measurement a second sample of the liposomal suspension was taken and mixed with a solution of the detergent (Triton-X100). The latter values were used as a reference.
1.3. Influence of vesicle concentration on the release by vortex.
Large unilamellar vesicles of a diameter of 100 nm were formulated using the procedure described below (2 mg of Pad-PC-Pad in 0.5 mL of carboxyfluoresceine buffer (10 mM HEPES, NaOH pH=7.4, 10 mM NaCl, 50 mM carboxyfluoresceine). The suspension was diluted to 50 ml with a second buffer (10 mM HEPES, NaOH pH
7.4. 10 mM NaCl). The suspension was diluted (1 :1, 1 :0.5; 1 :0.25). Five aliquots (3 mL each) were filled into 20 mL vials with polystyrene caps and vortexed for a discrete amount of time times (0, 5, 10, 20, 60 sec) at 2500 rpm. Release of the content was measured by a plate reader at the wavelengths of 492 (excitation) and 517 nm (emission).
2. Production of nanoparticles and loading with active compounds 2.1. Preparation of nanoparticles
All compounds are purchased from Sigma-Aldrich or Avanti Polar Lipids and used without further purification unless otherwise stated. Liposome formulation is based on the techniques described in: Olson et. al., Biochimica et Biophysica Acta 1979, 557, 9-23.
A description of one of the methods for nanoparticle formulation is as follows: 30 μπιοΐ lipid (e.g. egg yolk phosphatidylcholine (EYPC, Avanti Polar Lipids)), Pad- PC -Pad or a combination of lipids) was weighed into a 25 mL round bottomed flask and dissolved in 1 mL chloroform. Any required surfactant (e.g. Brij S10) was added at this point from a freshly prepared 2.5 mg/mL methanol solution. After evaporation to dryness, the film was dried for 12 h under high vacuum. 1 mL internal buffer (50 mM 5 (6)-Carboxy fluorescein, 10 mM HEPES buffer (AppliChem), 10 mM NaCl dissolved in pure water, pH=7.4 (NaOH)) was added to the flask to cover the film, and was allowed to hydrate the film for 30 min. The suspension was sequentially frozen (liquid nitrogen bath) and melted (water bath at 40 °C) five times, then extruded 1 1 times using a mini-extruder (Avanti Polar Lipids) and 100 nm filter (Whatman). Purification by size exclusion chromatography (20 g Sephadex G-50 column) in external buffer (107 mM NaCl, 10 mM HEPES dissolved in ultra pure water, pH=7.4 (NaOH)) afforded the pure 5(6)-carboxyfluorescein loaded vesicles. Vesicles were stored at 5 °C in the dark until use. All experiments were conducted within 24 h of vesicles formulation.
3. Characterization of release features of various compositions useful in making nanoparticles
3.1. Shear-Stress Induced Release Studies with a Cardiovascular Pump
A Medtronic extra-corporeal circulation pump (Medtronic Bio-Medicus 540 Bio Console (Model 5401) with tubing of approximately 10 mm diameter and a total volume of 180 mL was fitted with a Medtronic pressure gauge, flow reader and temperature bath. Pressure and flow rate were controlled by regulating pump speed. The nanoparticle suspension was diluted to 30 nmol lipid concentration in external buffer and loaded into the in vitro Medtronic pump setup (described above). Either a healthy or stenosed artery model (Elastrat) was connected in series. During experiments, flow rates were typically 700 mL/min (healthy artery model) or 350 mL/min (unhealthy artery model), pressure was typically 60 mmHg (0.8kPa) and the reaction temperature 37 °C. Samples of the circulating liposome solution were collected after 1 pass through the model artery and at 5, 10, 15 and 20 min circulation time. On each sample extraction, an equivalent amount of external buffer was reinjected into the system to counteract the fall in total liquid volume in the pump.
Fluorescence release from nanoparticles was analyzed by way of a fluorescence 96- well plate reader. From each collected sample, twelve 200 μΐ, samples were prepared. To six of these, 4 ih 1.2% Triton X- 100 aqueous solution was added to facilitate liposome degradation and release of fluorescent contents. The samples were incubated for 30 min then fluorescence was measured (excitation 492 nm, emission 517 nm). The average fractional release for each sample was calculated as the mean of the sample fluorescence divided by the mean of the sample plus Triton X- 100 fluorescence. Errors were extrapolated appropriately.
3.2. Further Analysis
Further analytical methods can be applied to define the parameters of the vesicles of the invention. For example, an online direct nitroglycerine detection method using electrochemical methods, specifically using a silver working electrode and differential pulse amperometric technique can be used.
Furthermore a low viscocity rheometer will allow the dependence of dye release from the vesicle to be determined as a function of the applied shear stress.
Further formulations based on different artificial diamidophospholipids according to the inventon can be studied under elevated shear stress.
3.3. Shear-Stress Induced Release Studies with a Cardiovascular Pump in healthy versus stenosed arteries model with Pur-PC-Pur nanoparticles This experiment was also performed in the artery model system as described herein. The experiment was performed with and describes the fractional release of entrapped 5 (6)-carboxy fluorescein from vesicles made from Pur-PC-Pur at 37 °C when pumped once through a stenosed and healthy artery model system, respectively. The results are depicted in FIG. 11.
3.4. Shear dependent release from vesicles of the invention in similar to physiological conditions including HSA (human serum albumin) with Pad-PC- Pad nanoparticles at 37 °C.
In this experiment the release conditions are as described herein with the addition of HSA (human serum albumin) and thus this experimentation resembles better the physiological situation in vivo. The results are depicted in FIG. 12.
4. Investigated further formulations
4.1. Formulations
a. EYPC with the synthetic phospholipid Pad-PC-Pad in 0, 10, 25, 50, 75 and 100 mol%.
b. synthetic phospholipids according to the invention and/or EYPC (each in equal amounts or in varying proportions as advantageous) with cholesterol (1, 20, 25 and 50 mol%), octadecanol (1 and 5 mol%), miltefosine (5 mol%), octadecanol (1 and 5 mol%), Brij P10 (5 and 10 mol%) and mixtures thereof.
The release patterns in (a) showed that there is a peak in specific shear induced release at around 0.6 mol% Brij S10. An example of release seen at 0.4 mol% is illustrated in fig. 8. The release properties of 100% Pad-PC-Pad were vastly different to those of Pad-PC-Pad/EYPC mixtures and were unique in all release studies, with release of 40% of contents after one pass through the model artery. In all other formulations, release was in the region of <5% after 1 pass.
4.2. Additional Formulations
Formulations are based on non-natural/synthetic phospholipids (e.g. Pad-PC-Pad) or other lipids according to one of formulae la, lb, Ic and Id with 0-10 mol% surfactants such as the Brij family (including, but not restricted to, SI 0, PI 0 and P4), 0-50 mol% rigidifiers such as cholesterol, and 0-25 mol% natural phospholipids such as EYPC, DOPC, POPC, DPPC, sphingomyelin and miltefosine.
Other formulations include synthetic phospholipids or lipids according to the invention (according to one of formulae la, lb, Ic and Id) and/or natural phospholipids (EYPC, DOPC, POPC, DPPC, sphingomyelin) with cholesterol, miltefosine and octadecanol in various concentrations up to 50 mol%.
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Abbreviations
EYPC Egg yolk phosphatidylcholine
DOPC l,2-dioleoyl-5«-glycero-3-phosphocholine
POPC 1 -palmitoy 1-2-oleoy l-s/z-glycero-3 -phosphocholine
DPPC 1 ,2-dipalmitoy l-i«-glycero-3 -phosphocholine
PEG Polyethyleneglycol
Brij S10 decaethylene glycol octadecyl ether
Brij P4 tetraethylene glycol hexadecyl ether
Brij P10 decaethylene glycol hexadecyl ether
Pa, Pa Pascal, kilopascal

Claims

Claims
1. Composition comprising or consisting of 1,3-diamidophospholipids or/and 1,2- diamidophospholipids or/and 2,3-diamidophospholipids or/and another lipid and preferably at least a second compound which compound is i. at least one natural lipid, ii. at least one synthetic lipid, iii. cholesterol or a cholesterol derivative or/and a surfactant.
2. Composition according to claim 1 wherein the lipid has one of the following formulae:
Figure imgf000086_0001
Formula la
Figure imgf000086_0002
Formula lb
Figure imgf000087_0001
Formula Ic
Figure imgf000087_0002
Formula Id wherein Formula la is characterized in that i. "B," is equal or different from "B2", and "B," and "B2" is selected from:
ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl- , heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted Ci-Ci0 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vi. an optionally substituted Cn-Ci7 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted Ci8-C24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 1 1 , 13, 15, and 17 C-atoms are preferred;
ix. a group listed in the figures below: 87
Figure imgf000088_0001
Figure imgf000088_0002
Figure imgf000088_0003
Figure imgf000088_0004
Figure imgf000088_0005
Figure imgf000088_0006
Figure imgf000089_0001
where m and n can be different or the same and
m = 0-7
preferably m=7
n=0-l l
preferably n=8, 10, or 1 1 or "Bi" and "B2" are the same or different and are selected from:
i. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine ii. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amine
iii. an optionally substituted C1-C8 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
iv. an optionally substituted C9-C15 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
v. an optionally substituted C16-C22 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vi. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
vii. a group listed in the figures below:
Figure imgf000090_0001
Figure imgf000090_0002
Figure imgf000091_0001
where m=0*6, and preferably m=6 n=(Ml, preferably ό=8, 10, or 11
or
ix. primary amides (to give acyhjrcas) such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyK deeyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
x. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amide xi. an optionally substituted C1-C8 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xii; an Optionally substituted C9-C15 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C16-C22 amidoalkyl group with 1 , 2, 3, 4, or
5 cis- or trans- double bonds;
xiv; wherein 9, 11 , 13, and 15 C-atoms are preferred;
xv. a grou listed in the figures below:
91
Figure imgf000092_0001
Figure imgf000093_0001
where m=0-5, and preferably m=5; n=0-l 1, preferably n=8, 10, or 1 1 ; wherein said lipid may be fully or partially deuterated, or radioactively labeled; and
wherein "A" is selected from:
a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide, and wherein "Ci" and "C2" may be H or a methyl; wherein Formula lb is characterized in that i. "Bi" is equal or different from "B2", and "Bt" and "B2" is selected from: ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted Ci-Cio alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds; vi. an optionally substituted Cu-Ci7 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted C|8-C24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 1 1, 13, 15, and 17 C-atoms are preferred;
ix. a group listed in the figures below:
94
Figure imgf000095_0001
Figure imgf000095_0002
Figure imgf000095_0003
Figure imgf000095_0004
Figure imgf000096_0001
where m and n can be different or the same and
m = 0-7, preferably m=7
n=0-l 1 , preferably n=8, 10, or 1 1 or "Bi" and "B2" are the same or different and are selected from:
i. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine ii. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amine
iii. an optionally substituted C1-C8 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
iv. an optionally substituted C9-C15 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
v. an optionally substituted C16-C22 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vi. wherein 9, 1 1 , 13, and 15 C-atoms are preferred;
vii. a group listed in the figures below:
96
Figure imgf000097_0001
Figure imgf000097_0002
Figure imgf000097_0003
Figure imgf000097_0004
Figure imgf000098_0001
where m=0-6, and preferably m=6 n=0-l 1, preferably n=8, 10, or 1 1
or
ix. primary amides (to give acylurea) such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
x. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amide
xi. an optionally substituted C1-C8 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xii. an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C16-C22 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiv. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
xv. a group listed in the figures below:
98
Figure imgf000099_0001
Figure imgf000100_0001
where m=0-5, and preferably m=5; n=0- 11 , preferably n=8, 10, or 11 ;
"Di" and "D2" can be the same or can be different and are either O (oxygen) or S (sulfur),
wherein said lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide; and wherein "CI" and "C2" may be H or a methyl; wherein Formula Ic is characterized in that i. "Bi" is selected from:
ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-; v. an optionally substituted Cj-Cio alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vi. an optionally substituted Cn-Cn alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted Ci8-C24 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 1 1 , 13, 15, and 17 C-atoms are preferred;
ix. a group listed in the figures below:
101
Figure imgf000102_0001
Figure imgf000102_0002
Figure imgf000102_0003
Figure imgf000102_0004
Figure imgf000102_0005
Figure imgf000103_0001
where m and n can be different or the same and
m = 0-7, preferably m=7;
n=0-l 1 , preferably n=8, 10, or 1 1 ; or is selected from
x. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl- , pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine
xi. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amine
xii. an optionally substituted C1-C8 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C9-C15 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiv. an optionally substituted C16-C22 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xv. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
xvi. a group listed in the figures below:
103
Figure imgf000104_0001
Figure imgf000104_0002
Figure imgf000104_0003
where m=0-6, and preferably m=6; n=0-l 1, and preferably n=8, 10, or 1 1 ;
or
xviii. primary amides (to give acylureas) such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
xix. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amide
xx. an optionally substituted C1-C8 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xxi. an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xxii. an optionally substituted C16-C22 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xxiii. wherein 9, 11, 13, and 15 C-atoms are preferred;
xxiv. a group listed in the figures below:
105
Figure imgf000106_0001
Figure imgf000107_0001
where m=0-5, and preferably m=5; n=0-l 1, preferably n=8, 10, or 1 1 ; "Di" is either O (oxygen) or S (sulfur),
wherein said lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide; and wherein "CI" may be H or a methyl; wherein Formula Id is characterized in that i. "B," is equal or different from "B2" or "E", and "B,", "B2" and "E" are selected from:
ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl- , heneicosyl-, docosyl-, tricosyl-, or tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted C|-Ci0 alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds; vi. an optionally substituted Cn-Cn alkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted C| 8-C24 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 1 1, 13, 15, and 17 C-atoms are preferred;
ix. a group listed in the figures below:
Figure imgf000109_0001
sf* Sf ^( jj( jj; j|{
Figure imgf000109_0002
Figure imgf000109_0003
Figure imgf000109_0004
Figure imgf000109_0005
Figure imgf000110_0001
where m and n can be different or the same and
m = 0-7, preferably m=7;
n=0-l 1 ; preferably n=8, 10, or 1 1 ; or "Bi" and "B2" are equal or the same and are selected from:
i. primary amines such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amine ii. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amine
iii. an optionally substituted C1-C8 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
iv. an optionally substituted C9-C15 aminoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
v. an optionally substituted C16-C22 aminoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
vi. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
vii. a group listed in the figures below:
110
Figure imgf000111_0001
Figure imgf000111_0002
Figure imgf000112_0001
where m=0-6, and preferably m=6; n=0-l 1, preferably n=8, 10, or 1 1 ; or "Bi" and "B2" are the same or different and are selected from:
ix. primary amides (to give acylureay) such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl-, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or tetracosyl-amide
x. preferably, decyl-, dodecyl-, tetradecyl-, hexadecyl-amide
xi. an optionally substituted C1-C8 amidoalkyl group with 1 , 2, 3, 4, or 5 cis- or trans- double bonds;
xii. an optionally substituted C9-C15 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiii. an optionally substituted C16-C22 amidoalkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
xiv. wherein 9, 1 1, 13, and 15 C-atoms are preferred;
xv. a group listed in the figures below:
112
Figure imgf000113_0001
Figure imgf000114_0001
where m=0-5, preferably m=5; n=0-l 1, preferably n=8, 10, or 1 1 ;
"Di" and "D2" can be the same or can be different and are either O (oxygen) or S (sulfur),
wherein said lipid preferably may be fully or partially deuterated, or radioactively labeled; and wherein "A" is selected from: a proton (H), a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide.
3. Composition according to claim 1 wherein the 1 ,3-diamidophospholipid has the following Formula la:
Figure imgf000114_0002
Formula la wherein
i. "Br" is equal or different from "B2", and "Bi" and is selected from:
ii. H;
iii. alkyl-, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl-, pentadecyl-, hexadecyl-, heptadecylr, nonadecyl-, eicosyl-, heneicosyl-, docosyl-, tricosyl-, or- tetracosyl-;
iv. preferably, undecyl-, tridecyl-, pentadecyl-, heptadecyl-;
v. an optionally substituted Cio alkyl group with 1, 2, 3, 4, or 5 cis- or transr double bonds;
vi. an optionally substituted C11-C17 alkyl group with 1, 2, 3, 4, or 5 cis- or trans- double bonds;
vii. an optionally substituted Cig-C2 alkyl group With 1, 2, 3, 4, or 5 cis- or trans- double bonds;
viii. wherein 11, 13, 15, and 17 C-atoms are preferred;
ixa group listed in the figures below:
* = rans
Figure imgf000115_0001
115
Figure imgf000116_0001
Figure imgf000116_0002
Figure imgf000116_0003
Figure imgf000116_0004
Figure imgf000116_0005
Figure imgf000117_0001
wherein said lipid may be fully or partially deuterated, or radioactively labeled; wherein "A" is selected from:
a phosphatic acid or a phosphate ester substituted group such as phosphocholine, phosphoethanolamine, phosphoglycerol, phosphoserine, phosphoinositol, phosphoinositol 4,5-bisphosphate, a phosphate ester substituted sugar, a phosphate ester substituted polyethyleneglycol, a 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 and a phosphate substituted azide; and wherein "Cj" and "C2" is selected from H or methyl.
4. Composition according to claim 1 wherein the natural lipid is selected from the group consisting of egg yolk phosphatidylcholine (EYPC), a phosphatidylethanolamine (PE), a phosphatidylcholine (PC), a phosphatic acid (PA), a phosphatidylglycerol (PG), preferably l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-phosphatidylcholine, 1 ,2-dipalmitoyl-jn-glycero-3- phosphocholine (DPPC), l,2-diIauroyl-s«-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-SH-glycero-3-phosphocholine (DMPC), 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), N-palmitoylsphingomyelin, miltefosine.
5. Composition according to claim 1 wherein the synthetic lipid is selected from the group consisting of any lipid according to claim 2.
6. Composition according to claim 1 wherein the surfactant is selected from an anionic, cationic or non-ionic surfactant.
7. Composition according to claim 6, wherein the surfactant is selected from the group consisting of a Brij, preferably Brij P4, Brij S4, Brij S10 or Brij P10.
8. Composition according to any of the preceding claims wherein the first and second compound are present in a ratio of from 99.9 : 0.1 to 1 : 99 mol-%, preferably from 90 : 10 to 40 : 60 mol-%, more preferably from 70 : 30 to 50 : 50 mol-%, even more preferably wherein the first compound is present in either 50 mol-%, 60 mol-%, 70 mol-%, 75 mol-%, 80 mol-%, 85 mol-%, 90 mol-%, 95 mol-%, or 98 mol-%.
9. Composition according to any of the preceding claims wherein it further comprises an active compound.
10. Composition according to claim 9 wherein the active compound is selected from the group consisting of a fibrinolytic agent, an anti-coagulation agent, an anti-aggregation agent, an atherosclerotic plaque stabilizer (Statin), a vasodilatory agent, preferably a direct or indirect acting vasodilator (a NO- liberating agent, an alpha-adrenoreceptor antagonist, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a direct renin inhibitor, a calcium-channel blocker (CCB), an endothelin receptor antagonist, a phosphodisesterase inhibitor, a potassium-channel opener,), anti-arrhytmic drugs (a sodium-channel blocker, a beta-blocker, a potassium-channel blocker, a calcium-channel blocker), inotrop positive medication (a catecholamine, a non-catecholamine), heart muscle remodeling (ACE inhibitors or ARB), diastolic dysfonction treatment (anticalcics, betablockers, ACE inhibitors or ARB), decongestion (Brain Natriuretic Peptide (BNP), nitro-release drugs), cardiomyocytes or stem cells (transplantation), radio contrast markers (for CT, MRI, Positron emission tomography (PET), scintigraphy, percutaneous coronary intervention), a chemotherapeutic agent, a coagulation factor agent, a heparin, and an antiinflammatory agent.
1 1. Composition according to claim 10 wherein the active compound is selected from the group consisting of alteplasum (Actilyse®), heparin (Liquemine®), acetyl salicylic acid (Aspirin®), clopidogrelum (Plavix®), glycoprotein Ilb/IIIa inhibitor such as ReoPro®, rosuvastatinum (Crestor®), NO-liberating agents such as nitroglycerin (Perlinganit®), nitroprussiate or molsidomine (Corvaton®), phentolamine (Regitine®), enalapril (Epril®), candesartanum (Atacand®), diltiazem (Dilzem®), bosentan (Tracleer®), milrinone (Corotrop®) or levosimendan (Simdax®), minoxidilum (Loniten®), aliskirenum (Rasilez®), quinidine ,metoprolol (Lopresor®; BelocZok®), amiodarone (Cordarone®), Verapamil (Isoptin®), epinephrin, norepinephrin, dopamine, dobutamine, isoprenalin, milrinone (Corotrop®), levosimendan (Simdax®), vasopressin, glypressin, Brain Natriuretic Petide (BNP), natural purified or recombinant forms such as nesiritidum (Noratak®), nitroglycerine, alteplasum (Actilyse®), Eptacogum alfa, NovoSeven®, Perlinganit®, nitroprussiate or molsidomine (Corvaton®), candesartanum (Atacand®), Iodine, Gadolinium containing contrast agents, positron-emitting radionuclide (tracer), cardiomyocytes or stem cells.
12. Composition according to any of the preceding claims wherein the composition is provided in the form of nanoparticles.
13. Nanoparticles according to claim 12 wherein the nanoparticle is a micelle, the nanoparticle is composed of a monolayer, a bilayer, multilayer, and/or a vesicle.
14. Nanoparticles according to claim 12 or 13 wherein the nanoparticle has an average diameter of about 10 to 1000 nm, preferably of about 50 to 500 ran, more preferably of about 50 to 200 nm.
15. Method of making a 1,3-diamidophospholipid wherein a phosphoethanolamine is alkylated under appropriate conditions, preferably with the use of dimethyl sulfoxide.
16. Method of making a composition according to any of claims 1 to 12.
17. Method according to claim 16 comprising mixing the first and second compound with appropriate means.
18. Method of making nanoparticles according to any of claims 13 - 14.
19. Method according to claim 18 by thin film hydration, and/or one or more freeze-thaw cycles, sonication or/and extrusion, or by an electroformation method or by hydrating spray-dried lipids or by sonication or by repetitive freezing and thawing or by dehydration and rehydration or by the extrusion technique or by the treatment of a multilamellar vesicle suspension with a microfluidizer, or the preparation of multilamellar novasomes or the preparation of multilamellar spherulites, or the preparation of multilamellar vesicles by the "bubble method", or the preparation by the "Cochleate cylinder method", or the preparation by the "Reversed-phase evaporation technique, or the preparation from water/oil and water/oil/water emulsions, or the preparation by the "solvent-spherule (W/O/W-emulsion) method" or the "DepoFoam Technology", or the preparation from an organic aqueous two- phase system, or the preparation by the "ethanol injection method, or the preparation by the "pro-liposome method", or the preparation of multilamellar ethosomes, or the preparation by the "interdigitation-fusion method", or the preparation by the "coacervation technique", or the preparation by the "supercritical liposome method", or the preparation from an initial oil/water emulsion, or the preparation by the "Detergent-depletion method", or the preparation by mixing bilayer-forming and micelle-forming amphiphiles, or the preparation from lipids in chaotropidc ion solutions, or the preparation of vesicles prepared from a water/oil-emulsion with the help of a detergent, or a vesicle prepared by the hydration of a multilayer of lipids on a hydrogel, or a vesicle prepared by using a microfluidics device.
20. Pharmaceutical or cosmetic composition comprising a composition according to any of claims 1 - 12 or a nanoparticle according to any of claims 13 - 14, and preferably further useful carriers or/and additives.
21. Composition according to any of claims 1 - 12 or nanoparticles according to any of claims 13 - 14 for use in a pharmaceutical or cosmetic application.
22. Composition according to any of claims 1 - 12 or nanoparticles according to any of claims 13 - 14 for use in the prophylaxis or treatment of a vascular disorder or disease, or for use in the prophylaxis or treatment of a dermatological disorder or disease, or for use as cosmetic, or for use in a monitoring or diagnostic method.
23. Use according to claim 22 wherein the dermatological disease or disorder is selected from the group consisting of acne, napkin dermatitis, atopic dermatitis, seborrhoeic dermatitis, psoriasis, warts, tinia pedis, seborrhoeic keratosis, hives, rosacea, dermatological viral infection and dermatological bacterial infection.
24. Use according to claim 22 wherein the use as cosmetic comprises topical applications.
25. Use according to claim 22 wherein the vascular disorder or disease is related to or is acute coronary syndrome (ACS), myocardial infarction, acute heart insufficiency, chronic heart insufficiency, cerebrovascular accident (CVA), stroke, atherosclerosis, vasospasm, tumor treatment, hemoptysis, pulmonary embolism, pulmonary arterial hypertension, intestinal ischemia, intestinal hemorrhage, renal infarction, renal hemorrhage, renal auto-regulation for hypertensive treatment, auto-immune glomerulonephritis or intersitial nephritis, treatment of fetal diseases, placental infarction, placental hemorrhage, retinal ischemia, retinal hemorrhage, or retinal neovascularization.
26. Composition according to any of claims 1 - 12 or nanoparticles according to any of claims 13 - 14 for use in the targeted delivery of a selected compound.
27. Use according to claim 26 wherein the selected compound is released at a target site due to endogenous shear stress at the target site.
28. Method of a targeted delivery of a selected compound or composition of compounds wherein i. in a first step the selected compound is loaded into a nanoparticle according to any of claims 13 - 14, ii. the loaded nanoparticle is applied to a subject or object and the selected compound is released at the target site due to endogenous vascular shear stress at the target site.
29. Use according to claim 22, 26 or 27 wherein the use is a monitoring method or a diagnostic method.
30. Use or method according to claim 26-29 wherein the selected compound is selected from the group consisting of a medium, a small molecule, a protein, peptide, nucleic acid, nucleotide or an antibody.
31. Use or method according to claim 30 wherein the selected compound is a marker, a contrast medium or a labeled compound.
32. Use or method according to claim 26-29 wherein the selected compound is selected from the group consisting of a iodine or gadolinium labeled antibody against glycoprotein-(GP)IIb IIIa-(aIIbp3) receptors, iodine or gadolinium labeled abciximab (ReoPro®), an atherosclerosis associated marker such as CD 16a, CD 32, CD 36, CD 40, CD 44, CD 45RO, a general inflammatory marker like an interleukin, iodixanolum (Visipaque®), gadopentetate dimeglumine (Magnevist®), a coronary stenoses marker such as copper, or coagulation factor Vila (NovoSeven®).
33. Use or method according to claim 25-28 wherein the method is applied in coronary atherosclerosis, myocardial infarction, cerebrovascular accident (CVA), stroke, vasospasm, tumors, hemoptysis, pulmonary embolism, intestinal ischemia, digestive tract hemorrhage, renal infarction, renal hemorrhage, placental infarction, placental hemorrhage, retinal ischemia, retinal hemorrhage, diabetic retinopathy, or hypertensive retinopathy.
34. Use or method according to claim 26-29 wherein the target site is characterized by an endogenous shear stress, preferably an endogenous vascular shear stress, of between 2 Pa and 20 Pa, preferably of between 2 Pa and 15 Pa, more preferably of between 2 Pa and 14 Pa, even more preferably of between 4 Pa and 14 Pa.
35. Use or method according to claim 28-29 wherein the selected compound is released at a therapeutically effective amount.
36. Method of a targeted release of an active or selected compound or mixture of compounds from nanoparticles at a release site in a tubular system, preferably a vascular vessel system, due to endogenous shear stress wherein the nanoparticles are being recycled at least 2 times in said tubular system, preferably vascular vessel system, and the active or selected compounds or mixture of compounds are periodically released from the nanoparticles at a target site, preferably wherein the amount of the active or selected compound or mixture of compounds released is dependent on the shear stress within the tubular system, preferably vascular vessel system, at the target site.
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