WO2014081299A1 - Liposome pouvant être actif - Google Patents

Liposome pouvant être actif Download PDF

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Publication number
WO2014081299A1
WO2014081299A1 PCT/NL2013/050846 NL2013050846W WO2014081299A1 WO 2014081299 A1 WO2014081299 A1 WO 2014081299A1 NL 2013050846 W NL2013050846 W NL 2013050846W WO 2014081299 A1 WO2014081299 A1 WO 2014081299A1
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alkyl
group
independently
aryl
tco
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PCT/NL2013/050846
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English (en)
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Marc Stefan Robillard
Sander Martinus Johannes VAN DUIJNHOVEN
Maarten Jozef Pouderoijen
Ronny Mathieu Versteegen
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Tagworks Pharmaceuticals B.V.
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Publication of WO2014081299A1 publication Critical patent/WO2014081299A1/fr

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    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention pertains to reactive (activatable) liposomes, and particularly to liposomal compositions designed for improved target delivery of an entrapped agent.
  • the goal of drug delivery systems is to increase the efficacy and safety of both new and existing drugs.
  • a number of drug compounds cannot be delivered safely and/or effectively by conventional routes or dosage forms such as oral tablets or injection.
  • Alternative delivery methods can increase safety by sequestering drugs in carriers that reduce systemic exposure and decrease dose-limiting toxicity and side effects, or by providing sustained delivery so that therapeutic levels can be achieved with fewer and smaller doses.
  • New delivery systems can also increase efficacy by several strategies, including: increasing stability of the drug; increasing the ability of the drug to reach its therapeutic target by prolonging the circulating half -life; and targeting delivery to the therapeutic site and effecting drug release in order to reduce the total circulating dose without diminishing efficacy.
  • the most prominent delivery systems used in the clinic are based on liposomes (e.g.
  • Doxil which are liposomes filled with doxorubicin.
  • the mechanism of liposome accumulation may be a combination of the leakiness of the newly forming or damaged capillaries and enhanced vascular permeation by the coated liposomal particles themselves.
  • Specific targeting involves the use of antibodies or ligands to tag liposomes so that they bind specifically to cells that express the appropriate cell-surface antigens or ligand receptors, respectively.
  • liposomes can be targeted to any cell surface structure that can be recognized by a fragment of a specific antibody, or to any receptor for which a small and specific ligand can be produced.
  • liposomes can be directed to specific classes of T and B lymphocytes or to tumor cells, preferentially expressing high levels of specific cell surface proteins.
  • the goals of ligand targeting of liposomes are to concentrate them selectively at the therapeutic site, decrease the required dose by reducing non-specific losses, and reduce systemic exposure to drugs with toxic side effects.
  • liposome release With respect to liposome release, this can be governed by multiple processes and variables. Localization of passive or active targeted liposomes is usually followed by a relatively lengthy process, which can involve an internalization pathway followed by intracellular processing and drug release or expulsion back to the extracellular domain.
  • liposomal delivery systems capable of release of their content under the influence of e.g. pH, thiols, and light have been developed.
  • pH-sensitive liposomes liposomes that destabilize under mildly acidic conditions
  • pH-sensitive liposomes have been described as an approach to intracellular deliver an entrapped agent (Slepushkin et al., J. Biol. Chem., 272(4):2382 (1997); Wang et al., Proc. Natl. Acad. Sci.,
  • liposomes are primarily composed of a lipid, such as
  • DOPE dioleoylphosphatidylethanolamine
  • Fusogenic liposomes typically include a hydrophobic polymer extending from the liposomes' outer surfaces for penetration into a target cell membrane.
  • the hydrophobic polymers are initially shielded by a hydrophilic polymer coating, and then exposed for fusion with the target membrane when the hydrophilic polymer coating is released by reaction with thiols.
  • MscL Mechanism-sensitive channel of large conductance
  • E. coli E. coli
  • MscL a well- studied bacterial channel protein
  • the channel creates a large non-selective pore of 3-4 nm in diameter in the membrane and allows the passage of ions, small molecules, peptides and smaller proteins (up to 7 kDa).
  • MscL opens in response to the tension in the membrane. It has been shown that the hydrophilicity of the 22nd amino acid position of MscL affects the mechanosensitivity of the channel up to a point where it starts to open even in the absence of tension (Yoshimura et al. (1999) Biophys. J. 77, 1960-1972). Hydrophilic substitutions in this narrow pore constriction area of the channel cause hydration of the pore and
  • MscL protein was re-engineered to site-selectively- incorporate (masked) amine-functionalized molecules.
  • a series of small modulators were designed, synthesized and specifically attached to an engineered Cysteine at position 22 in MscL.
  • the working principle is that the protein-attached modulators would be charged only in response to a pre-defined stimulation (pH, light, etc) leading to hydration of the hydrophobic constriction zone of the pore and channel opening in the absence of the natural stimulus.
  • the masked reagents possess a
  • the liposome membrane contains a channel protein modified with a chemical Trigger, and this channel protein-Trigger conjugate does not allow efflux of the liposome contents and is not activated endogeneously by e.g. thiols or a specific pH. Instead it is activated by a controlled administration of the Activator, i.e.
  • a cysteine in the channel protein was modified with moiety comprising an amine, which was masked by an azide Trigger.
  • the Trigger is removed, unveiling the amine moiety, which due to its pKa is
  • Staudinger approach for this concept has turned out not to work well, and its area of applicability is limited in view of the specific nature of the release mechanism imposed by the Staudinger reaction.
  • Other drawbacks for use of Staudinger reactions are their limited reaction rates, and the oxidative instability of the phosphine components of these reactions. Therefore, it is desired to provide reactants for an abiotic, bio- orthogonal reaction that are stable in physiological conditions, that are more reactive towards each other, and that are capable of activating liposomes and inducing release of a entrapped drugs by means of a variety of mechanisms, thus offering a greatly versatile activated drug release method.
  • liposomes selectively and predictably at the target site without being dependent on homogenous penetration and targeting, and on endogenous parameters which may vary en route to and within the target, and from indication to indication and from patient to patient.
  • endogenous activation mechanisms eg pH, thiols
  • Selective activation of liposomes when and where required allows control over many processes within the body, including cancer. Therapies may thus be made more specific and effective, providing an increased therapeutic contrast between normal cells and tumour to reduce unwanted side effects.
  • a reactive liposome comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a linkage to an eight-membered non-aromatic cyclic alkenylene group, preferably a cyclooctene group, and more preferably a trans -cyclooctene group.
  • the invention provides a kit for the administration and activation of an activatable liposome, the kit comprising a liposome linked, directly or indirectly, to a Trigger, and an Activator for the Trigger, wherein the Trigger comprises an eight- membered non-aromatic cyclic alkenylene group as a dienophile, preferably a cyclooctene group, and more preferably a trans -cyclooctene group, and the Activator comprises a diene.
  • the invention is a liposomal composition, comprising:
  • a cyclooctene group preferably a cyclooctene group, and more preferably a trans- cyclooctene group, linked to both the Construct and the Masking Moiety.
  • the invention resides in the use of an eight- membered non-aromatic cyclic alkenylene group as a dienophile, preferably a cyclooctene group, and more preferably a ircms-cyclooctene group, as a Trigger on an activatable liposome, wherein the reaction of the dienophile with a diene results in activation of the liposome.
  • the invention in a broad sense, is based on a judicious use of the so-called inverse electron-demand Diels-Alder reaction (also referred to as the reiroDiels-Alder or rDA reaction), as a chemical tool in liposome activation.
  • Diels-Alder reaction also referred to as the reiroDiels-Alder or rDA reaction
  • the present use of the rDA reaction is irrespective of the precise chemical components further present.
  • liposomes are known, as is their use as a carrier for therapeutic agents.
  • activatable liposomes are known.
  • the invention expressly is directed to providing a breakable linkage in an activatable liposome assembly. Breaking said linkage through the judiciously chosen rDA reaction, results in the liposome becoming
  • the Retro Diels-Alder coupling chemistry generally involves a pair of reactants that couple to form an unstable intermediate, which intermediate ehminates a small molecule (depending on the starting compounds this may be e.g. N2, CO2 , RCN), as the sole by-product through a retro Diels-Alder reaction to form the retro Diels-Alder adduct.
  • the paired reactants comprise, as one reactant (i.e. one Bio-orthogonal
  • a suitable diene such as a derivative of tetrazine, e.g. an electron -deficient tetrazine and, as the other reactant (i.e. the other Bio- orthogonal Reactive Group), a suitable dienophile, such as a strained trans -cyclooctene (TCO).
  • TCO strained trans -cyclooctene
  • the inititally formed 4,5- dihydropyridazine product may tautomerize to a 1,4-dihydropyridazine product.
  • the two reactive species are abiotic and do not undergo fast metabolism or side reactions in vivo. They are bio-orthogonal, e.g. they selectively react with each other in physiologic media.
  • the compounds and the method of the invention can be used in a living organism.
  • the reactive groups are relatively small and can be introduced in biological samples or living organisms without significantly altering the size of biomolecules therein. References on the Inverse electron demand Diels Alder reaction, and the behavior of the pair of reactive species include: Thalhammer, F; Wallwise, U; Sauer, J,
  • the aforementioned retro Diels-Alder coupling and subsequent liposome activation chemistry can be applied to basically any pair of molecules, groups, or moieties that are capable of being used in liposomal drug delivery.
  • one of such a pair will comprise a Construct linked to a dienophile (the Trigger).
  • the other one will be a complementary diene for use in reaction with said dienophile.
  • the Trigger T R dienophile is an eight-membered non- aromatic cyclic alkenylene group, preferably a cyclooctene group, and more preferably a ircms-cyclooctene group. These eight-membered groups are herein collectively abbreviated as TCO.
  • the ircms-cyclooctene (TCO) moiety comprises at least two exocyclic bonds fixed in substantially the same plane, and/or it optionally comprises at least one substituent in the axial position, and not the equatorial position.
  • the person skilled in organic chemistry will understand that the term "fixed in substantially the same plane” refers to bonding theory according to which bonds are normally considered to be fixed in the same plane. Typical examples of such fixations in the same plane include double bonds and strained fused rings.
  • the at least two exocyclic bonds can also be single bonds on two adjacent carbon atoms, provided that these bonds together are part of a fused ring (i.e. fused to the TCO ring) that assumes a substantially flat structure, therewith fixing said two single bonds in substantially one and the same plane.
  • a fused ring i.e. fused to the TCO ring
  • Examples of the latter include strained rings such as cyclopropyl and cyclobutyl.
  • the TCO satisfies the following formula
  • fused rings are present that result in two exocyclic bonds being fixed in substantially the same plane. These are selected from fused 3-membered rings, fused 4-membered rings, fused bicyclic 7-membered rings, fused aromatic 5-membered rings, fused aromatic 6-membered rings, and fused planar conjugated 7-membered rings as defined below:
  • Fused 3-membered rings are:
  • E, G are part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are CR a or CX D , and such that CX D can only be present in A and P.
  • E-G is CR a -CR a or CR a -CX D
  • Fused 4-membered rings are:
  • E-G is part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are C,
  • E is C
  • G is CR a , CX D or N
  • Fused bicyclic 7-membered rings are:
  • E-G is part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are C, CR a or CX D , and such that CX D can only be present in A and P;
  • E,G are C, CR a , CX D ; K is N and L is CR a ; D,M form a
  • E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ.
  • E and G are C; one of the groups L, K, or M are O, NR b , S and the remaining two groups are independently from each other CR a or N; or E is C and G is N; L, K, M are independently from each other CR a or N.
  • Fused aroma ic 6-membered rings are: E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ.
  • E,G is C; L, K, D , M are independently from each other CR a or N.
  • E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ
  • E,G is C; L, K, D, M are CR a ; J is S, O, CR3 ⁇ 4, NR b .
  • D D is either a masking moiety M M or a Construct C c (possibly two or more Constructs C c linked via self-immolative linkers), preferably linked via S, N, NH, or O, wherein these atoms are part of M M or C c .
  • T each independently denotes H, or a substituent selected from the group consisting of alkyl, F, CI, Br, or I.
  • the inventors believe that in the foregoing embodiments, the rDA reaction results in a cascade-mediated release or elimination (i.e. cascade mechanism) of the Construct (or for that matter, the Masking Moiety).
  • said release or elimination is believed to be mediated by a strain release mechanism.
  • one of the bonds PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY consists of -CR a X D -CR a Y D -, the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR3 ⁇ 4, S, O, SiR c 2, such that P and A are CR a 2, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR3 ⁇ 4 or O.
  • X D is 0-C(0)-(LD)n-(DD), S-C(O)-(LD)n-(E>D), O-C(S)-(LD)n-(E>D),
  • X D is NR d -C(O)-(LD) N -(D D ), and Y D is NHR d .
  • the X D and Y D groups may be positioned cis or trans relative to each other, where depending on the positions on the TCO, cis or trans are preferred: if PQ, QP, AY or YA is - CR a X D -CR a Y D -, then X D and Y D are preferably positioned trans relative to each other; if ZX or XZ is -CR a X D -CR a Y D -, then X D and Y D are preferably positioned cis relative to each other.
  • A is CR a X D and Z is CR a Y D
  • Z is CR a X D and A is CR a Y D
  • P is CR a X D and X is CR a Y D
  • X is CR a X D and P is CR a Y D , such that X D and Y D are positioned in a trans conformation with respect to one another; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR3 ⁇ 4, S, O, SiR c 2, such that P and A are CR a 2, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR3 ⁇ 4 or O;
  • X D is O-C(O)-(L D ) n -(D D ), S-C(O)-(L D ) n
  • A is CR a Y° and one of P, Q, X, Z is CR a X D , or P is CR a Y D and one of A, Y, Z, X is CR a X D , or Y is CR a Y D and X or P is CR a X D , or Q is CR a Y D and Z or A is CR a X D , or either Z or X is CR a Y D and A or P is CR a X D , such that X D and Y 13 are positioned in a trans conformation with respect to one another; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR a 2 , S, O, SiR c 2 , such that P and A are CR3 ⁇ 4, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si
  • X D is (O-C(O))p-(LD) n -(DD), S-C(O)-(LD) n -(DD), 0-C(S)-(LD) n - (D D ), S-C(S)-(L D ) n -(D D );
  • P is CR a Y D and Y is CR a X D
  • A is CR a Y D and Q is CR a X D
  • Q is CR a Y D and A is CR a X D
  • Y is CR a Y D and P is CR a X D , such that X D and Y° are positioned in a trans conformation with respect to one another; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR a 2 , S, O, SiR c 2 , such that P and A are CR a 2 , and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR a 2 or O.
  • Y is Y D and P is CR a X D , or Q is Y 15 and A is CR a X D ; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR a 2 , S, O, SiR c 2 , such that P and A are CR a 2 , and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR a 2 or O.
  • X D is (0-C(0))p-(L D ) n -(DD), S-C(0)-(L D ) n -(DD), 0-C(S)-(L D ) n -
  • Y is Y D and P or Q is X D , or Q is Y 0 and A or Y is X D ; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR3 ⁇ 4, S, O, SiR3 ⁇ 4 such that P and A are CR a 2, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR3 ⁇ 4 or O.
  • X D is N-C(O)-(L D ) n -(DD), N-C(S)-(L D ) n -(DD); Y ⁇ is NH;
  • X D is N-C(O)-(L D ) n -(D D )
  • D D is either a masking moiety M M or a Construct C c , preferably linked via S, N, NH, or O, wherein these atoms are part of M M or C c .
  • T, F each independently denotes H, or a substituent selected from the group consisting of alkyl, F, CI, Br, or I.
  • this NH is a primary amine (-NH2) residue from D D
  • this N is a secondary amine (-NH-) residue from D D
  • said O or S are, respectively, a hydroxy! (-OH) residue or a sulfhydryl (-SH) residue from D D .
  • S, N, NH, or O moieties comprised in D D are bound to an aliphatic or aromatic carbon of D D .
  • this NH is a primary amine (-NH2) residue from L D
  • this N is a secondary amine (-NH-) residue from L D
  • O or S are, respectively, a hydroxyl (-OH) residue or a sulfhydryl (-SH) residue from L D .
  • S, N, NH, or O moieties comprised in L D are bound to an aliphatic or aromatic carbon of L D .
  • linker L D this can be self-immolative or not, or a combination thereof, and which may consist of multiple self-immolative units. It will be understood that if L D is not self-immolative, the linker equals a spacer S p .
  • the position and ways of attachment of linkers L D and moieties D D are known to the skilled person (see for example Papot et al, Anti- Cancer Agents in Medicinal Chemistry, 2008, 8, 618-637).
  • self-immolative linkers L D are benzyl-derivatives, such as those drawn below.
  • an example of a self-immolative linker with multiple units is shown; this linker will degrade not only into CO2 and one unit of 4-aminobenzyl alcohol, but also into one l,3-dimethylimidazolidin-2-one unit.
  • the TCO of formula (la) is an all-carbon ring. In another preferred embodiment, the TCO of formula (la) is a
  • R c as above indicated is independently selected from the group consisting of H, alkyl, aryl, O-alkyl, O-aryl, OH;
  • R d as above indicated is independently selected from H, Ci-6 alkyl and Ci-6 aryl;
  • one of A, P, Q, Y, X, and Z, or the substituents or fused rings of which they are part, or the self- immolative linker L D is bound, optionally via a spacer or spacers S p , to the species Y M .
  • Y M is either a masking moiety M M or a Construct C c , such that when D D is C c , Y M is M M , and such that when D D is M M , Y M is C c .
  • TCO's as described above is well available to the skilled person. This expressly also holds for TCO's having one or more heteroatoms in the strained cycloalkene rings. References in this regard include Cere et al. Journal of Organic Chemistry 1980, 45, 261 and Prevost et al. Journal of the American Chemical Society 2009, 131, 14182.
  • the ircms-cyclooctene moiety satisfies formula (lb):
  • the at least two exocyclic bonds fixed in the same plane are selected from the group consisting of (a) the single bonds of a fused cyclobutyl ring, (b) the hybridized bonds of a fused aromatic ring, (c) an exocyclic double bond to an oxygen, and (d) an exocyclic double bond to a carbon.
  • the TCO containing one or two X D moieties, may consist of multiple isomers, also comprising the equatorial vs. axial positioning of substituents, such as X D , on the TCO.
  • substituents such as X D
  • C Whitham et al. J. Chem. Soc. (C), 1971, 883-896, describing the synthesis and characterization of the equatorial and axial isomers of ircms-cyclo-oct- 2-en-ol, identified as (IRS, 2RS) and (1SR, 2RS), respectively.
  • the OH substituent is either in the equatorial or axial position.
  • Preferred dienophiles which are optimally selected for D D release believed to proceed via a cascade elimination mechanism, are selected from the following structures:
  • Preferred dienophiles which are optimally selected for D D release beheved to proceed via a strain release mechanism, are selected from the following structures:
  • the dienophile is a compound selected
  • the dienophile is a compound selected from
  • the dienophile of formula (la) and the diene are capable of reacting in an inverse electron-demand Diels-Alder reaction. Activation of the Liposome by the retro Diels-Alder reaction of the Trigger with the Activator leads to release of the Drug.
  • the invention is based on the recognition that a species D D can be released from ircms-cyclooctene derivatives satisfying formula (la) upon cyclooaddition with compatible dienes, such as tetrazine derivatives.
  • compatible dienes such as tetrazine derivatives.
  • the dienophiles of formula (la) have the advantage that they react (and effectuate D D release) with substantially any diene.
  • the inventors believe that the molecular structure of the retro Diels-Alder adduct is such that a spontaneous elimination reaction within this rDA adduct releases D D .
  • the inventors believe that appropriately modified rDA components lead to rDA adducts wherein the bond to D D on the dienophile is destabilized by the presence of a lone electron pair on the diene.
  • the inventors believe that the molecular structure of the retro Diels-Alder adduct is such that a spontaneous elimination or cyclization reaction within this rDA adduct releases D D .
  • the inventors believe that appropriately modified rDA components, i.e. according to the present invention, lead to rDA adducts wherein the bond to D D on the part originating from the dienophile is broken by the reaction with a
  • nucleophile on the part originating from the dienophile while such an intramolecular reaction within the part originating from the dienophile is precluded prior to rDA reaction with the diene.
  • Scheme 1 general scheme of activation of a masked liposome according to this invention.
  • Construct is a chemical assembly of a liposome and a lipid that is part of a lipid bilayer of the liposome, particularly present in an outer bilayer thereof.
  • D D and Y M stand for either of the Construct C c and the Masking moiety M M , such that when D D is C c , Y M is M M , and such that when D D is M M , Y M is 0°.
  • TCO stands for ircms-cyclooctene.
  • trans -cyclooctene is used here as possibly including one or more hetero- atoms, and particularly refers to a structure satisfying formula (la).
  • the inventors have found that - other than the attempts made on the basis of the Staudinger reaction - the selection of a TCO as the trigger moiety for a masked liposome Construct, provides a versatile tool to render unstable drug containing liposomes into stable drug containing liposomes, wherein the drug release occurs through a powerful, abiotic, bio-orthogonal reaction of the dienophile (Trigger) with the diene (Activator), viz the aforementioned retro Diels-Alder reaction, and wherein the masked liposome Construct is a Construct-dienophile conjugate.
  • Trigger dienophile
  • Activator diene
  • a requirement for the successful application of an abiotic bio- orthogonal chemical reaction is that the two participating functional groups have finely tuned reactivity so that interference with coexisting functionality is avoided.
  • the reactive partners would be abiotic, reactive under physiological conditions, and reactive only with each other while ignoring their cellular/physiological surroundings (bio-orthogonal). The demands on selectivity imposed by a biological environment preclude the use of most conventional reactions.
  • the inverse electron demand Diels Alder reaction has proven utility in animals at low concentrations and semi-equimolar conditions (R. Rossin et al, Angewandte Chemie Int Ed 2010, 49, 3375- 3378).
  • the reaction partners subject to this invention are strained trans- cyclooctene (TCO) derivatives and suitable dienes, such as tetrazine derivatives.
  • TCO strained trans- cyclooctene
  • suitable dienes such as tetrazine derivatives.
  • the cycloaddition reaction between a TCO and a tetrazine affords an intermediate, which then rearranges by expulsion of dinitrogen in a retro-Diels-Alder cycloaddition to form a dihydropyridazine conjugate.
  • This and its tautomers is the retro Diels-Alder adduct.
  • the invention provides, in one aspect, the use of a tetrazine as an activator for the release, in a physiological environment, of a species D D (i.e. as defined above) linked to a ircms-cyclooctene.
  • the invention also pertains to a tetrazine for use as an activator for the release, in a physiological environment, of a substance linked to a ircms-cyclooctene, and to a method for activating, in a physiological environment, the release of a substance linked to a ircms-cyclooctene, wherein a tetrazine is used as an activator.
  • the present inventors have come to the non-obvious insight, that the structure of the TCO of formula (la), par excellence, is suitable to provoke the release of a species D D linked to it, as a result of the reaction involving the double bond available in the TCO dienophile, and a diene.
  • the features believed to enable this are (a) the nature of the rDA reaction, which involves a re-arrangement of double bonds, which can be put to use in provoking an elimination cascade; (b) the nature of the rDA adduct that bears a dihydro pyridazine group that is non-aromatic (or another non- aromatic group) and that can rearrange by an elimination reaction to form conjugated double bonds or to form an (e.g.
  • the feature believed to enable this is the change in nature of the eight membered ring of the TCO in the dienophile reactant as compared to that of the eight membered ring in the rDA adduct.
  • the eight membered ring in the rDA adduct has significantly more conformational freedom and has a significantly different conformation as compared to the eight membered ring in the highly strained TCO prior to rDA reaction.
  • nucleophilic site is properly positioned within the rDA adduct and will react intramolecularly, thereby releasing D D .
  • D D release is mediated by strain-release of the TCO-dienophile after and due to the rDA reaction with the diene Activator.
  • the invention puts to use the recognition that the rDA reaction, using a dienophile of formula (la), as well as the rDA adduct embody a versatile platform for enabling provoked release of a D D ( in the context of a liposome) in a bioorthogonal reaction.
  • the invention is thus of a scope well beyond specific chemical structures.
  • the invention puts to use the recognition that the rDA reaction using a dienophile of formula (la) as well as the rDA adduct embody a versatile platform for enabling provoked D D release in a bioorthogonal reaction.
  • the invention also presents the use of the inverse electron-demand Diels-Alder reaction between a ircms-cyclooctene and a tetrazine as a chemical tool for the release, in a physiological environment, of a bound substance.
  • the reaction is bio-orthogonal, and that many structural options exist for the reaction pairs, will be clear to the skilled person.
  • the rDA reaction is known in the art of pre-targeted medicine. Reference is made to, e.g., WO 2010/119382, WO 2010/119389, and WO 2010/051530. Whilst the invention presents an entirely different use of the reaction, it will be understood that the various structural possibilities available for the rDA reaction pairs as used in pre-targeting, are also available in the field of the present invention.
  • the dienophile trigger moiety used in the present invention comprises a trans -cyclooctene ring, the ring optionally including one or more hetero-atoms.
  • this eight-membered ring moiety will be defined as a ircms-cyclooctene moiety, for the sake of legibility, or abbreviated as "TCO" moiety.
  • TCO ircms-cyclooctene moiety
  • the invention is not limited to strictly D D - substituted ircms-cyclooctene.
  • the person skilled in organic chemistry will be aware that other eight-membered ring-based dienophiles exist, which comprise the same endocyclic double bond as the ircms-cyclooctene, but which may have one or more heteroatoms elsewhere in the ring.
  • the invention generally pertains to eight-membered non-aromatic cyclic alkenylene moieties, preferably a cyclooctene moiety, and more preferably a ircms-cyclooctene moiety, comprising a conjugated D D .
  • the present invention first and foremost requires the right chemical reactivity combined with an appropriate design of the D D - conjugate.
  • the possible structures extend to those of which the skilled person is familiar with that these are reactive as dienophiles.
  • the TCO dienophile may also be denoted E -cyclooctene.
  • E the conventional nomenclature
  • any substituted variants of the invention whether or not formally "E” or “Z,” or “cis” or “trans” isomers, will be considered derivatives of unsubstituted ircms-cyclooctene, or unsubstituted E- cyclooctene.
  • trans -cyclooctene TCO
  • E-cyclooctene E-cyclooctene
  • alkyl and "aryl.”
  • alkyl each independently, indicates an aliphatic, straight, branched, saturated, unsaturated and/or or cyclic hydrocarbyl group of up to ten carbon atoms, possibly including 1- 10 heteroatoms such as O, N, or S
  • aryl each independently, indicates an aromatic or heteroaromatic group of up to twenty carbon atoms, that possibly is substituted, and that possibly includes 1-10 heteroatoms such as O, N, P or S.
  • Aryl also include “alkylaryl” or “arylalkyl” groups (simple example: benzyl groups).
  • Ci-io alkyl means that said alkyl may contain from 1 to 10 carbon atoms.
  • Certain compounds of the invention possess chiral centers and/or tautomers, and all enantiomers, diasteriomers and tautomers, as well as mixtures thereof are within the scope of the invention.
  • groups or substituents are indicated with reference to letters such as "A”, “B”, “X”, “ ⁇ ', and various (numbered) "R” groups. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.
  • alkyl is preferably lower alkyl (C 1.4 alkyl), and each aryl preferably is phenyl.
  • the TCO is preferably an all-carbon TCO.
  • the Activator comprises a Bio-orthogonal Reactive Group, wherein this Bio-orthogonal Reactive Group of the Activator is a diene. This diene reacts with the other Bio-orthogonal Reactive Group, the Trigger, and that is a dienophile (vide supra).
  • the diene of the Activator is selected so as to be capable of reacting with the dienophile of the Trigger by undergoing a Diels-Alder cycloaddition followed by a retro Diels-Alder reaction, giving the Retro Diels-Alder adduct. This intermediate adduct then releases the D D or several D D s, where this D D release can be caused by various circumstances or conditions that relate to the specific molecular structure of the retro Diels-Alder adduct.
  • the Activator in one embodiment, is selected such as to provoke D D release via an elimination or cascade elimination (via an intramolecular elimination reaction within the Retro Diels-Alder adduct).
  • elimination reaction can be a simple one step reaction, or it can be a multiple step reaction that involves one or more intermediate structures. These intermediates may be stable for some time or may immediately degrade to the thermodynamic end product or to the next intermediate structure. When several steps are involved, one can speak of a cascade reaction. In any case, whether it be a simple or a cascade process, the result of the elimination reaction is that the D D gets released from the retro Diels-Alder adduct. Without wishing to be bound by theory, the design of both components (i.e.
  • the diene Activator and the dienophile Trigger is such that the distribution of electrons within the retro Diels- Alder adduct is unfavorable, so that a rearrangement of these electrons must occur.
  • This situation initiates the intramolecular (cascade) elimination reaction to take place, and it therefore induces the release of the D D or D D s.
  • Occurrence of the elimination reaction in and Trigger release from the D D is not efficient or cannot take place prior to the Retro Diels-Alder reaction, as the Trigger-D D itself is relatively stable as such. Elimination can only take place after the Activator and the Trigger-D D have reacted and have been assembled in the retro Diels-Alder adduct.
  • the above two examples illustrate how the unfavorable distribution of electrons within the retro Diels-Alder adduct can be relieved by an elimination reaction, thereby releasing the D D .
  • the elimination process produces end product A, where this product has a conjugation of double bonds that was not present in the retro Diels-Alder adduct yet.
  • Species A may tautomerize to end product B, or may rearrange to form end product C.
  • the non-aromatic dihydro pyridazine ring in the retro Diels-Alder adduct has been converted to the aromatic pyridazine ring in the end product C.
  • the distribution of electrons in the retro Diels-Alder adduct is generally unfavorable relative to the distribution of the electrons in the end products, either species A or B or C.
  • the formation of a species more stable than the retro Diels- Alder adduct is the driving force for the (cascade) elimination reaction.
  • the D D here the amine D D -NH2
  • the D D is effectively expelled from the retro Diels-Alder adduct, while it does not get expelled from the Trigger-D D alone.
  • the below scheme depicts a possible alternative release mechanism for the cascade elimination, in addition to the two discussed above.
  • the below examples illustrates how the unfavorable distribution of electrons within the retro Diels-Alder adduct may be relieved by an elimination reaction, thereby releasing the D D .
  • This process may evolve via various tauromerisations that are all equilibria.
  • the rDA reaction produces tautomers A and B, which can interchange into one and other.
  • Tautomer B can lead to the elimination into product C and thereafter into D.
  • the releasing effect of the rDA reaction is, in one embodiment, caused by an intramolecular cyclization/elimination reaction within the part of the Retro Diels-Alder adduct that originates from the TCO dienophile.
  • a nucleophilic site present on the TCO i.e. the dienophile, particularly from the Y° group in this Trigger, vide supra
  • the part of the rDA adduct that originates from the TCO i.e. the eight membered ring of the rDA adduct, has a different conformation and has an increased
  • cyclization/elimination reaction takes place, as the nucleophilic site and the electrophilic site have been brought together in close proximity within the Retro Diels-Alder adduct, and produce a favorable structure with a low strain. Additionally, the formation of the cyclic structure may also be a driving force for the intramolecular reaction to take place, and thus may also contribute to an effective release of the leaving group, i.e. release of the Construct or the Masking Moiety. Reaction between the nucleophilic site and the electrophilic site does not take place or is relatively inefficient prior to the Retro Diels-Alder reaction, as both sites are positioned unfavorably for such a reaction, due to the relatively rigid,
  • the Liposomal composition itself is relatively stable as such and elimination is favored only after the
  • Activator and the Liposomal composition have reacted and have been assembled in a retro Diels-Alder adduct that is subject to intramolecular reaction.
  • the TCO ring is in the crown conformation.
  • the above example illustrates how the intramolecular cyclization/ehmination reaction within the retro Diels-Alder adduct can result in release of a Construct or Masking Moiety.
  • the rDA reaction produces A, which may tautomerize to product B and C. Structures B and C may also tautomerize to one another (not shown). rDA products A, B, and C may intramolecularly cyclize, releasing the bound moiety, and affording structures D, E, and F, which optionally may oxidise to form product G.
  • the tautomerization of A into B and C in water is very fast (in the order of seconds) it is the inventors' belief, that release occurs predominantly from structures B and C.
  • nucleophilic site assists in expelling the D D species by a nucleophilic attack on the electrophilic site with subsequent release, but without actually forming a (stable) cyclic structure.
  • no ring structure is formed and the nucleophilic site remains intact, for example because the ring structure is shortlived and unstable and breaks down with
  • Preferred nucleophiles are amine, thiol or alcohol groups, as these are generally most nucleophilic in nature and therefore most effective.
  • amine functional D D species these can be e.g. primary or secondary amine, aniline, imidazole or pyrrole type of moieties, so that the D D is varying in leaving group character.
  • D D with other functionalities may also be possible (e.g. thiol functionnalized D D ), in case corresponding hydrolytically stable TCO-D D conjugates are applied.
  • the drawn fused ring products may or may not tautomerize to other more favorable tautomers.
  • TCO- D D conjugates and tetrazine Activators illustrate the possibilities for cascade elimination induced model D D release from the retro Diels-Alder adduct.
  • the D D is preferably attached to a carbon atom that is adjacent to the double bond in the TCO ring.
  • urethane (or carbamate) substituted TCOs gives release of an amine functional D D from the adduct.
  • the tetrazine Activator is symmetric and electron deficient.
  • urethane (or carbamate) substituted TCOs gives release of an amine functional D D from the adduct.
  • the tetrazine Activator is asymmetric and electron deficient. Note that use of an asymmetric tetrazine leads to formation of retro Diels-Alder adduct regiomers, apart from the stereo-isomers that are already formed when symmetric tetrazine are employed.
  • L D self-immolatoive linker comprising Y M
  • urethane (or carbamate) TCOs gives release of an amine functional D D from the adduct.
  • the tetrazine Activator is symmetric and electron sufficient.
  • L D self-immolatoive linker comprising Activator
  • the Activator is a diene.
  • the person skilled in the art is aware of the wealth of dienes that are reactive in the Retro Diels-Alder reaction.
  • the diene comprised in the Activator can be part of a ring structure that comprises a third double bond, such as a tetrazine (which is a preferred Activator according to the invention).
  • the Activator is a molecule comprising a
  • heterocyclic moiety comprising at least 2 conjugated double bonds.
  • B is O or S;
  • A is selected from the group consisting of N, C-alkyl, C-aryl, and N + 0-;
  • B is N;
  • particularly useful dienes are 1,2- diazine, 1,2,4-triazine and 1,2,4,5-tetrazine derivatives, as given in formulas (5), (6) and (7), respectively.
  • R and R" each independently being H, aryl or alkyl and R" independently being aryl or alkyl
  • X-Y may be a single or a double bond
  • X and Y may be connected in a second ring structure apart from the 6-membered diazine.
  • Electron -deficient 1,2-diazines (5), 1,2,4-triazines (6) or 1,2,4,5-tetrazines (7) are especially interesting as such dienes are generally more reactive towards dienophiles.
  • Di- tri- or tetra-azines are electron deficient when they are substituted with groups or moieties that do not generally hold as electron-donating, or with groups that are electron-withdrawing.
  • R 1 and/or R 2 may denote a substituent selected from the group consisting of H, alkyl, NO2, F, CI, CF3, CN, COOR, CONHR, CONR2, COR, SO2R, SO2OR, SO2NR2, PO3R2, NO, 2-pyridyl, 3- pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl or phenyl, optionally substituted with one or more electron- withdrawing groups such as NO 2 , F, CI, CF 3 , CN, COOR, CONHR, CONR, COR, SO2R, SO2OR, SO2NR2, PO3R2, NO, Ar, wherein R is H or Ci-C 6 alkyl, and Ar stands for an aromatic group, particularly phenyl, pyridyl, or naphthyl.
  • the 1,2,4,5-tetrazines of formula (7) are most preferred as Activator dienes, as these molecules are most reactive in retro Diels-Alder reactions with dienophiles, such as the preferred TCO dienophiles, even when the R 1 and/or R 2 groups are not necessarily electron withdrawing, and even when R 1 and/or R 2 are in fact electron donating.
  • Examples of other electron donating groups are phenyl groups with attached to them one or more of the electron donating groups as mentioned in the list above, especially when substituted in the 2-, 4- and/or 6-position(s) of the phenyl group.
  • 1,2,4,5-tetrazines with two electron withdrawing residues are called electron deficient.
  • 1,2,4,5-tetrazines with two electron donating residues or those with one electron donating residue and one residue that is neither electron withdrawing nor donating, are called electron sufficient.
  • 1,2,4,5-Tetrazines with two residues that are both neither electron withdrawing nor donating, or those that have one electron withdrawing residue and one electron donating residue are neither electron deficient nor electron sufficient.
  • the 1,2,4,5-tetrazines can be asymmetric or symmetric in nature, i.e. the R 1 and R 2 groups in formula (7) may be different groups or may be identical groups, respectively. Symmetric 1,2,4,5-tetrazines are more convenient as these Activators are more easily accessible via synthetic procedures.
  • Electron deficient 1,2,4,5 tetrazines and 1,2,4,5-tetrazines that are neither electron deficient nor electron sufficient are generally more reactive in retro Diels-Alder reactions with dienophiles (such as TCOs), so these two classes of 1,2,4,5-tetrazines are preferred over electron sufficient 1,2,4,5-tetrazines, even though the latter are also capable of inducing Trigger release in Trigger-D D conjugates.
  • Other substitution patterns are also possible, including the use of different substituents, as long as the tetrazine remains symmetric. See below for some examples of these structures.
  • substitutions can be done on the 2- and 3-, 2- and 4-, 2,- and 5-, 2- and 6, 3- and 4-, and the 3- and 5-positions.
  • asymmetric 1,2,4,5-tetrazines are considered, one can choose any combination of given R 1 and R 2 residues that have been highhghted and listed above for the symmetric tetrazines according to formula (7), provided of course that R 1 and R 2 are different.
  • Preferred asymmetric 1,2,4,5-tetrazines are those where at least one of the residues R 1 or R 2 is electron withdrawing in nature. Find below some example structures drawn.
  • Activator Preferred Activators are 1,2-diazines, 1,2,4-triazines and 1,2,4,5-tetrazines, particularly 1,2,4,5-tetrazines, are the preferred diene Activators. In the below, some relevant features of the Activator will be highlighted, where it will also become apparent that there are plentiful options for designing the right Activator formulation for every specific application.
  • the Activator e.g. a 1,2,4,5- tetrazine
  • the Activator has useful and beneficial pharmacological and ph arm aco -kinetic properties, implying that the Activator is non-toxic or at least sufficiently low in toxicity, produces metabolites that are also sufficiently low in toxicity, is sufficiently soluble in physiological solutions, can be applied in aqueous or other formulations that are routinely used in pharmaceutics, and has the right log D value where this value reflects the
  • log D values can be negative (hydrophilic molecules) or positive (hydrophobic molecules), where the lower or the higher the log D values become, the more hydrophilic or the more hydrophobic the molecules are, respectively.
  • the given log D numbers have been calculated from a weighed method, with equal importance of the 'VG' (Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. R.; Robins, R. K., J. Chem. Inf. Comput. Sci., 1989, 29, 163-172), 'KLOP* (according to Klopman, G.; Li, Ju-Yun.; Wang, S.; Dimayuga, M.: J.Chem.Inf.Comput.Sci., 1994, 34, 752) and HYS' (according to the PHYSPROP ⁇ database) methods, based on an aqueous solution in 0.1 M in Na + /K +
  • the Activator according to the invention has an appropriate reactivity towards the Trigger-Construct, and this can be regulated by making the diene, particularly the 1,2,4,5-tetrazines, sufficiently electron deficient. Sufficient reactivity will ensure a fast retro Diels-Alder reaction with the Trigger-Construct as soon as it has been reached by the Activator.
  • the Activator according to the invention has a good bioavailability, implying that it is available inside the (human) body for executing its intended purpose: effectively reaching the Trigger-Construct at the target. Accordingly, the Activator does not stick significantly to blood components or to tissue that is non-targeted.
  • the Activator may be designed to bind to albumin proteins that are present in the blood (so as to increase the blood circulation time, as is known in the art), but it should at the same time be released effectively from the blood stream to be able to reach the Trigger-Construct. Accordingly, blood binding and blood releasing should then be balanced adequately.
  • the blood circulation time of the Activator can also be increased by increasing the molecular weight of the Activator, e.g. by attaching polyethylene glycol (PEG) groups to the Activator ('pegylation').
  • PEG polyethylene glycol
  • the PKPD of the activator may be modulated by conjugating the activator to another moiety such as a polymer, protein, (short) peptide, carbohydrate.
  • the Activator according to the invention may be multimeric, so that multiple diene moieties may be attached to a molecular scaffold, particularly to e.g. multifunctional molecules, carbohydrates, polymers, dendrimers, proteins or peptides, where these scaffolds are preferably water soluble.
  • a molecular scaffold particularly to e.g. multifunctional molecules, carbohydrates, polymers, dendrimers, proteins or peptides, where these scaffolds are preferably water soluble.
  • scaffolds that can be used are (multifunctional) polyethylene glycols, poly (propylene imine) (PPI) dendrimers, PAMAM dendrimers, glycol based dendrimers, heparin derivatives, hyaluronic acid derivatives or serum albumine proteins such as HSA.
  • Trigger-Construct e.g.
  • the Activator is designed to be able to effectively reach this Trigger-Construct. Therefore, the Activator can for example be tailored by varying its log D value, its reactivity or its charge.
  • the Activator may even be engineered with a targeting agent (e.g. a protein, a peptide and/or a sugar moiety), so that the target can be reached actively instead of passively.
  • a targeting agent e.g. a protein, a peptide and/or a sugar moiety
  • it is preferred that it is a simple moiety i.e. a short peptide or a simple sugar.
  • a mixture of different Activators can be applied. This may be relevant for regulation of the release profile of the drug.
  • the Activator that according to the invention will cause and regulate drug release at the target may additionally be modified with moieties giving extra function(s) to the Activator, either for in-vitro and/or for in-vivo studies or applications.
  • the Activator may be modified with dye moieties or fluorescent moieties (see e.g. S.
  • the Activator will not only initiate drug release, but can also be localized inside the (human) body, and can thus be used to localize the Trigger- Construct inside the (human) body. Consequently, the position and amount of drug release can be monitored.
  • the Activator can be modified with DOTA (or DTP A) ligands, where these ligands are ideally suited for complexation with m In 3+ -ions for nuclear imaging.
  • the Activator may be linked to 123 I or 18 F moieties, that are well established for use in SPECT or PET imaging, respectively.
  • beta-emitting isotopes such as Lu-177, or Y-90
  • liposome activation can be combined with localized radiotherapy in a pretargeted format.
  • Preferred activators for use with Triggers based on the cascade mechanism are:
  • Preferred activators for use with Triggers based on the strain release mechanism are
  • the Activator can have a hnk to a Masking Moiety M M such as a peptide, protein, carbohydrate, PEG, or polymer.
  • M M such as a peptide, protein, carbohydrate, PEG, or polymer.
  • these Activators for use with Triggers based on the cascade mechanism satisfy one of the following formulae:
  • R (link to) peptide, protein, carbohydrate, PEG, polymer
  • these Activators for use with Triggers based on the strain release mechanism satisfy one of the following formulae:
  • R (link to) peptide, protein
  • Synthesis routes to the above activators are readily available to the skilled person, based on standard knowledge in the art. References to tetrazine synthesis routes include Lions et al, J. Org. Chem., 1965, 30, 318-319; Horwitz et al, J. Am. Chem. Soc, 1958, 80, 3155-3159; Hapiot et al, New. J. Chem., 2004, 28, 387-392, Kaim et al, Z. Naturforsch., 1995, 50b, 123-127.
  • a masked liposome comprises a conjugate of the species D D and the Trigger T R and comprises a liposome formulation that is capable of release of entrapped drugs after release of D D from the Trigger.
  • Such a masked liposome may optionally have specificity for disease targets.
  • YM and D D are Construct C c and Masking Moiety M M , such that when D D is C c , ⁇ is M M , and such that when D D is M M , ⁇ is C c ; S p is spacer; T R is Trigger, and L D is linker.
  • M M can further comprise T T , optionally via S p .
  • D D can optionally be attached to the TCO derivative through a linker L D or a self-immolative linker L D , or a combination thereof, and which may consist of multiple (self-immolative, or non immolative) units.
  • formula la and lb describe the Trigger and described how the Trigger is attached to D D , C c , L D , YM, S p , M M , but that species D D , C c , L D , Y M , S p , M M are not part of the Trigger and should be viewed as seperate entities, as can be seen in e.g. Scheme 1 and formula 9.
  • the species D D and the Trigger T R - the TCO derivative- can be directly linked to each other. They can also be bound to each other via a linker or a self-immolative linker L D .
  • the D D is linked to the TCO in such a way that the D D is eventually capable of being released after formation of the retro Diels-Alder adduct.
  • the D D and the optional linker is linked via a hetero-atom, preferably via O, N, NH, or S.
  • the cleavable bond is preferably selected from the group consisting of carbamate, thiocarbamate, carbonate, ether, ester, amine, amide, thioether, thioester, sulfoxide, and sulfonamide bonds.
  • lipid derivatives that are comprised in the liposome construct of the present invention are depicted in Scheme 2.
  • Lipids are made up of Lipid chains (or chain), and a Head.
  • “Lipid chains” refers to the hydrophobic moiety of a lipid
  • “Head” refers to the lipid head group to which the lipid chain or chains are attached.
  • the Head group is hydrophilic or polar. If the Head group is further modified with a polar of hydrophilic group, the Head group itself may be hydrophilic or hydrophobic.
  • the invention includes, in one aspect, a liposome composition for interaction with a target membrane of a cell, or the like.
  • the composition includes liposomes designed for interaction with or binding to the target membrane.
  • Each liposome contains a therapeutic agent entrapped in the liposomes, an outer liposome surface having a coating of chemically releasable hydrophilic polymer chains (NP ⁇ ) (Scheme 2, C), and optionally Targeting agents T T on the outer distal end of the hydrophilic polymer chain or on the liposome outer surface (Scheme 2, F).
  • the liposome outer surface contains Targeting agents T A , optionally further modified with a T R -M M (Scheme 2, D and E).
  • the liposome will also comprise one or more of derivatives A or B.
  • the release of the hydrophilic polymer coating of derivative C and/or D facilitates liposome interaction and capture by cells.
  • the T A moieties are initially shielded by the hydrophilic polymer coating, then exposed for interaction with the target membrane when the hydrophilic polymer coating is chemically released.
  • a targeting agent T A or T T as present on the liposome outer surface generally is of a different type than a targeting agent T T present at the end of PEG chain.
  • a targeting agent T T present at the end of PEG chain generally is of a different type than a targeting agent T T present at the end of PEG chain.
  • T A is an hydrophobic polymer it is comprised in derivative D
  • T A is an hydrophilic moiety such as a cell penetrating peptide, it is comprised in derivative E.
  • the hydrophilic polymer M M coating is preferably composed of polymer chains of polyethyleneglycol, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
  • polyhydroxypropyloxazoline polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide,
  • the hydrophilic polymer is polyethyleneglycol (PEG).
  • each liposome contains an outer liposome surface having a coating of chemically releasable hydrophilic polymer chains M M , and hydrophobic polymers T A on the liposome outer surface.
  • the polymers T A are initially shielded by the hydrophilic polymer coating M M , then exposed for fusion with the target membrane when the hydrophilic polymer coating is chemically released.
  • the hydrophilic polymer and hydrophobic polymer preferably form a diblock copolymer in which the two polymer components are joined by the Trigger (Scheme 2, D).
  • the hydrophobic polymer is preferably a chain of polypropylene oxide, polyethylene, polypropylene, polycarbonate, polystyrene, polysulfone, polyphenylene oxide or polytetramethylene ether.
  • the polymer chains have a preferred molecular weight of between of between 100-5,000 daltons, more preferably between 500-3,000 daltons.
  • the hydrophobic polymer is polypropylene oxide (PPO) having a molecular weight between 500-3,000 daltons.
  • composition may further include a shielded T A and/or T T attached to the liposome (Scheme 2, E and F), effective to interact with the cell surface, eg by binding to target cell surface receptor molecules, only after chemical release of the hydrophilic polymer coating.
  • a shielded T A and/or T T attached to the liposome (Scheme 2, E and F), effective to interact with the cell surface, eg by binding to target cell surface receptor molecules, only after chemical release of the hydrophilic polymer coating.
  • each liposome contains an outer liposome surface having a coating of chemically releasable hydrophilic polymer chains M M (Scheme 2, C), and T A and/or T T on the liposome outer surface (Scheme 2, E and F).
  • the T A and/or T T are initially shielded by the hydrophilic polymer coating M M , then exposed for interaction with the target membrane when the hydrophilic polymer coating is chemically released.
  • the liposomes contain a shielded cationic lipid (Scheme 2, A and E) effective to impart a positive liposome-surface charge, to enhance binding of liposomes to target cells only after chemical release of the hydrophilic polymer coating of C.
  • Scheme 2, A and E shielded cationic lipid
  • the composition may further include an unshielded Targeting agent T T attached to the outer end of the hydrophilic polymer coating (Scheme 2, B - D), effective for ligand-specific binding to a receptor molecule on a target cell surface prior to chemical release of the hydrophilic polymer coating.
  • the unshielded ligand may be (i) folate, where the composition is intended for treating tumor cells having cell-surface folate receptors, (ii) pyridoxyl, where the composition is intended for treating virus-infected CD4+ lymphocytes, or (iii) sialyl-Lewis x , where the composition is intended for treating a region of inflammation.
  • this aspect includes a method of delivering a compound to target cells in a subject, by parenterally administering the above liposome composition to a subject, then contacting the liposomes at the target cells with an Activator to release the hydrophilic polymer chains forming the surface coating, to expose T A (e.g. hydrophobic polymers) and/or T T (e.g. receptor binding peptide) on the liposome outer surface for interaction with outer cell membranes of the target cells and thereby promote fusion or interaction of the liposomes with the target cells.
  • T A e.g. hydrophobic polymers
  • T T e.g. receptor binding peptide
  • the hydrophilic polymer chains are releasably attached to the liposome via the Trigger, and the contacting step includes administering an Activator to the subject.
  • the present invention includes a liposome composition for fusion or interaction with a target membrane.
  • Target membrane refers to a lipid bilayer membrane, for example, a bilayer membrane of a biological cell.
  • the liposome composition of the invention is for use in delivery of a liposome-entrapped compound to the cytoplasmic compartment of a target biological cell.
  • the liposome is composed of vesicle-forming lipids, such as lipids A, which each include hydrophilic head groups, and typically two diacyl hydrophobic lipid chains.
  • lipids A which each include hydrophilic head groups, and typically two diacyl hydrophobic lipid chains.
  • Preferred diacyl-chain lipids for use in the present invention include diacyl glycerol, phosphatidyl ethanolamine (PE), diacylaminopropanediols, such as
  • lipids are preferred for use as the vesicle-forming lipid A, the major liposome component, and for use in the polymer-lipid diblock conjugates (D) and lipids with directly linked hydrophilic polymer chains (B), which together are preferably included in the liposome outer layer at a mole ratio between about 1-20 mole percent.
  • D polymer-lipid diblock conjugates
  • B lipids with directly linked hydrophilic polymer chains
  • the liposome has an outer surface coating of hydrophilic polymer chains M M , which are preferably densely packed to form a brushlike coating effective to shield liposome surface components, as described below.
  • the hydrophilic polymer chains are connected to the liposome lipids (Scheme 2, C; and mPEG-TCO-DSPE shown directly below), or to hydrophobic chains connected to liposome lipids (Scheme 2, D; and the three PPO derivatives shown below), by the Trigger that can be released by the Activator, as described further below.
  • hydrophilic polymer chain M M forms the distal end of a diblock copolymer lipid conjugate having a vesicle- forming lipid moiety and a diblock copolymer moiety.
  • the diblock copolymer moiety in turn, consists of a hydrophobic chain T A , which is covalently bound at its proximal end to the polar head group of lipid moiety.
  • Hydrophobic chain T A is bound at its distal end to hydrophilic polymer chain M M through Trigger T R .
  • hydrophilic chain M M is directly linked to the polar head group of a vesicle-forming lipid through a chemically releasably bond T R .
  • hydrophilic polymer chains can be are included in liposomes as part of the diblock polymer moiety of vesicle-forming lipids on the outer surface of the liposomes (Scheme 2, D). It will be appreciated that the hydrophilic polymer segment in a diblock conjugate functions to enhance the water solubility of the associated hydrophobic chain, to prevent destabilization of the liposome membrane by partitioning of the hydrophobic chains into the hposome bilayer region. As will be discussed below, such destabilization is advantageous in promoting liposome/cell membrane fusion, but is undesirable prior to the fusion event, i.e., during liposome storage, administration and biodistribution to a target site. The types and molecular weights of the hydrophilic and hydrophobic segments suitable for achieving these effects are discussed below.
  • the hydrophilic chains In addition to their role in “solubilizing" the hydrophobic chains, and shielding them from interactions with other bilayer membranes, the hydrophilic chains also preferably have a surface density sufficient to create a molecular barrier effective to substantially prevent interaction of serum proteins with the liposome surface. As such, the hydrophilic chain coating is effective to extend the circulation time of liposomes in the blood- stream for periods up to several hours to several days.
  • the hydrophilic chains are preferably present in the outer lipid layer of the liposomes in an amount corresponding to between about 1-20 mole percent of the liposome surface lipids, with lower molecular weight polymers, e.g., 500 daltons, being present at a higher density, e.g., 20 mole percent, and higher molecular weight polymer chains, e.g., 10,000 dalton chains, being present at a lower density, e.g., 1-5 mole percent.
  • the percent of hydrophobic chains, i.e., the percentage of diblock lipid conjugates in the liposomes typically ranges between about 5-100% of the total surface lipids containing conjugated hydrophilic polymers.
  • the hydrophobic polymer would constitute 50% times 5%, or 2.5 mole percent, of the surface lipids.
  • the liposome may further include unshielded Targeting Agents T T , for targeting the liposomes to a specific target membrane-for example to a specific tissue region or cell type bearing appropriate surface receptor molecules.
  • T T is carried at the distal end of a hydrophilic polymer chain,. Means for conjugating T T to the distal end of a hydrophilic polymer chain are well known. The placement of the T T at or near the distal ends of the polymer chains, i.e., unshielded by the hydrophilic polymer coating, allows the ligand to interact with a target cell containing a T T -specific surface receptor, prior to removal of the
  • hydrophilic chains from the liposomes hydrophilic chains from the liposomes.
  • An example of such a liposome- bound T T is folic acid, as shown directly below.
  • the liposomes may further or alternatively include one or more liposome-surface components which are shielded from interaction with target cells until after the removal of the hydrophilic polymers.
  • the shielded component is a Targeting Agent T T , coupled to the polar head group of a vesicle-forming lipid.
  • T T Targeting Agent
  • the purpose of the ligand is to bind specifically with a cell receptor after removal of the hydrophilic polymer coating, to force the liposome into proximity with the cell membrane, to enhance the interaction of hydrophobic polymer chains (T A ) on the liposomes with the target-cell lipid bilayer.
  • the shielded surface component may include vesicle-forming lipids with positively charged polar groups, and are comprised in the general structure of derivative A in Scheme 2.
  • lipids include those typically referred to as cationic lipids, which have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge.
  • the head group of the lipid carries the positive charge.
  • a lipid head group is modified with a cationic moiety, such as a cell-penetrating moiety (T A ), as shown in the structure directly below.
  • the positive surface charge on the surface of the liposomes is shielded by the hydrophilic coating, during liposome biodistribution to the target site. After removal of the hydrophilic coating, electrostatic interaction between the positive liposome surface charge and the negatively charged target cell acts to draw the liposome into more intimate contact with the cell to promote fusion, optionally mediated by hydrophobic polymer chains.
  • the following formula depicts an example of a cationic cell penentrating peptide conjugated to a lipid. As such it is a combination between lipid-targeting agent conjugated and cationic lipid:
  • the liposome is prepared to contain one or more therapeutic or diagnostics agents which are to be delivered to the target cell site.
  • therapeutic or diagnostic agent compound and drug are used interchangeably.
  • the agent may be entrapped in the inner aqueous compartment of the liposome or in the lipid bilayer, depending on the nature of the agent. Exemplary therapeutic agents are described below.
  • the liposomes of the invention may include an unshielded (surface-exposed) ligand effective to bind to specific cell surface receptors on the target cell membrane.
  • the ligand molecules are carried on hydrophilic polymer chains which are anchored to the liposome by covalent attachment to a diacyl hpid.
  • the hydrophilic polymer chains may be covalently attached to a liposome-bound lipid through a conventional bond, e.g. irreversibly attached, or through a chemically releasable bond, such as those described above.
  • the figure below shows the mechanism of cleavage of the mPEG (M M ) moiety from the lipid, unmasking a T A on another lipid. In this example, upon cleavage, the amine-containing lipid is regenerated in its natural, unmodified form.
  • the figure below shows the mechanism of cleavage of the mPEG (M M ) moiety from a lipid-T A conjugate, unmasking a hydrophobic polymer T A on the same lipid.
  • the fusogenic liposome composition described is useful in delivering diagnostic or biologically active therapeutic agents such as drugs, proteins, genetic material or other agents, or receptor molecules, either into a cell membrane, a receptor liposome or the cytoplasm of a cell in vivo or in vitro.
  • the liposome entrapped agent is delivered directly to the cytosol of the target cell by liposome fusion with the cells, rather than via an endocytotic or phagocytic mechanisms.
  • the liposomes are thus particularly advantageous for delivering therapeutic agents, such as gene constructs, oligonucleotides or oligonucleotide analogs, peptides, proteins, and other biological
  • fusogenic liposomes containing encapsulated drug are administered, e.g., intravenously.
  • the fusogenic liposomes, as described above, may include a specific ligand or T T for targeting to cells in need of the entrapped drug.
  • liposomes carrying an antitumor drug, such as doxorubicin can be targeted to the
  • endothelial cells of tumors by including a VEGF ligand in the hposome, for selective attachment to receptors expressed on the proliferating tumor endothelial cells.
  • the hydrophilic coating on the liposomes protects the liposomes from uptake by the reticuloendothelial system, providing a long blood circulation lifetime for more effective targeting.
  • the T T attached to the distal ends of lipid-anchored hydrophilic polymer chains, are exposed for purposes of receptor binding and targeting.
  • the liposomes When the liposomes have reached a selected target site, e.g., by ligand- specific binding of the liposomes to target cells, or accumulation of liposomes in the vicinity of target cells by biodistribution of the injected liposomes, the liposomes are contacted at the target cells with a chemical agent, the Activator, effective to release said chains forming said surface coating. This release exposes T A on the liposome surface to the target cells, promoting fusion of the liposomes with the target cell surface as described below.
  • removal of the hydrophilic polymer chains exposes the hydrophobic polymer on the liposome surface to the target cell membrane surface.
  • the hydrophobic segment now in an aqueous environment, will seek a more favorable, e.g., hydrophobic, environment, both in the liposome bilayer and in the adjacent target cell membrane.
  • the partitioning of the hydrophobic chains into target cells will act both to increase the proximity of the liposome to the target cell membrane, and to destabilize the target cell bilayer, making it more susceptible to fusion with the liposome bilayer.
  • a number of strategies can be employed to optimize or enhance the efficiency of the fusion event. First, it is desirable to increase the tendency of the exposed hydrophobic chain to partitioning into the target cell bilayer rather than the liposome bilayer. This can be done, in part, by increasing the concentration of high phase transition lipids in the liposomes.
  • liposomes are comprised of a pH-sensitive lipid (A) and of a lipid
  • the pH-sensitive lipid is derivatized with a hydrophilic polymer, where the polymer and the lipid are joined by the Trigger (C).
  • the liposomes optionally also include a Targeting Agent T T effective to target the liposomes to a specific cell. Entrapped in the liposomes is a therapeutic agent for delivery.
  • the pH-sensitive liposomes herein described are stabilized by a releasable polymer coating, thus allowing the liposomes to retain an encapsulated compound even at acidic pHs.
  • the pH sensitivity of the liposomes, and therefore destabilization at a specific pH range, is restored by cleaving all or a portion of the polymer coating, to cause destabilization of the liposomes and concomitant release of the liposomal contents.
  • a pH-sensitive lipid is a lipid that forms bilayer vesicles in the absence of a stabilizing component only at specific pH ranges.
  • lipids are typically amphipathic lipids having hydrophobic and polar head group moieties, and when arranged into a bilayer are oriented such that the hydrophobic moiety is in contact with the interior, hydrophobic region of the bilayer membrane, and the polar head group moiety is oriented toward the exterior, polar surface of the membrane.
  • the pH sensitive amphipathic lipids preferably have two hydrocarbon chains, typically acyl chains between about 8-22 carbon atoms in length, and have varying degrees of unsaturation.
  • a preferred pH sensitive lipid is dioleoylphosphatidyl ethanolamine (DOPE), a phospholipid having diacyl chains.
  • DOPE dioleoylphosphatidyl ethanolamine
  • phospholipid having diacyl chains At physiological pH and ionic strengths, DOPE exists in an inverted hexagonal phase incapable of forming bilayers. Bilayer liposomes of DOPE can be made at pHs above the pK a of approximately 8.5 (Allen T. M. et al., Biochemistry, 23:2976 (1990)).
  • DOPE can be stabilized in the bilayer state at pH range between 5.5 - 7.4 by the inclusion of a small mole percent of an amphipathic lipid having a bulky hydrophilic moiety, e.g., a PEG-lipid derivative, as will be described below.
  • an amphipathic lipid having a bulky hydrophilic moiety e.g., a PEG-lipid derivative
  • mPEG is M M and stands for the hydrophilic polymer methoxy -poly ethylene glycol,. CH30(CH2CH20) n where n is from about 10 to about 2300, which corresponds to molecular weights of about 440 Daltons to about 100,000 Daltons.
  • the molecular weight of the polymer depends to some extent on the hpid.
  • a preferred range of PEG molecular weight is from about 750 to about 10,000 Daltons, more preferably from about 2,000 to about 5,000 Daltons.
  • M M can be selected from a variety of hy drophilic polymers, and exemplary polymers are recited herein. It will also be appreciated that the molecular weight of the polymer may depend on the amount of the derivative C included in the liposome composition, where a larger molecular weight polymer is often selected when the amount of derivative C in the composition is small, thus yielding a small number of liposome- attached polymer chains.
  • the figure below shows the mechanism of cleavage of the mPEG moiety from the lipid.
  • the amine-containing hpid is regenerated in its natural, unmodified form.
  • the liposomes also include a lipid derivatized with a hydrophilic polymer.
  • the polymer derivatized lipids serve to stabilize the pH sensitive lipid to facilitate bilayer, and liposome, formation and to form a coating of polymer chains over the hposome surface to extend the blood circulation hfetime of the liposomes. That is, the hydrophilic polymer coating provides colloidal stability and serves to protect the liposomes from uptake by the
  • the polymer chains are attached to the lipid by a releasable bond for cleavage and release of the polymer chains, in order to restore the pH sensitivity of the liposomes, as will be described.
  • the derivatizable lipid is a non-pH sensitive vesicle- forming amphipathic lipid, which can spontaneously form into a bilayer vesicle in water.
  • Vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains and a head group, either polar or non-polar.
  • phosphatidylethanolamine phosphatidic acid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbon chains are typically between about 14 - 22 carbon atoms in length, and having varying degrees of unsaturation.
  • the above-described lipids and phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods.
  • amphipathic lipids for use in the present invention include diacyl glycerol, phosphatidyl ethanolamine (PE) and phosphatidylglycerol (PG), and phosphatidyl ethanolamine (PE) being the most preferred.
  • PE phosphatidyl ethanolamine
  • PG phosphatidylglycerol
  • PE phosphatidyl ethanolamine
  • DSPE ethanolamine
  • the derivatizable lipid is a pH sensitive lipid, such as DOPE.
  • Hydrophilic polymers suitable for derivatizing the amphipathic and other lipids include polyvinylpyrrolidone, polyvinylmethylether,
  • polyhydroxyethylacrylate hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.
  • the hydrophilic polymer is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500- 10,000 Daltons, more preferably between 2,000 10,000 Daltons, and most preferably between 1,000 - 5,000 Daltons.
  • Lipids suitable for use in the M M -Trigger-lipid conjugate are preferably water-insoluble molecules having at least one acyl chain containing at least about eight carbon atoms, more preferably an acyl chain containing between about 8 - 24 carbon atoms.
  • a preferred lipid is a lipid having an amine-containing polar head group and an acyl chain.
  • Exemplary lipids are phospholipids having a single acyl chain, such as stearoylamine, or two acyl chains.
  • Preferred phospholipids with an amine-containing head group include phosphatidylethanolamine and phosphatidylserine.
  • the lipid tail(s) can have between about 12 to about 24 carbon atoms and can be fully saturated or unsaturated.
  • One preferred lipid is
  • DSPE distearoylphosphatidylethanolamine
  • the invention includes a liposome composition comprised of (i) a pH-sensitive lipid; (ii) between 1 - 20 mole percent of a lipid derivatized with a hydrophilic polymer, the polymer attached to the lipid by a bond effective to release the hydrophilic polymer chains in response to reaction between the Activator and the Trigger; (iii) an optional targeting ligand; and (iv) an entrapped therapeutic agent.
  • the liposome composition of the present invention is composed primarily of vesicle-forming lipids.
  • a vesicle-forming lipid is one which (a) can form spontaneously into bilayer vesicles in water, as exemplified by the phospholipids, or (b) is stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its head group moiety oriented toward the exterior, polar surface of the membrane.
  • the vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar.
  • lipids there are a variety of synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids, including the phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin
  • phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods.
  • Other suitable lipids include glycolipids and sterols such as cholesterol.
  • the vesicle-forming lipid is selected to achieve a specified degree of fluidity or rigidity, to control the stability of the liposome in serum and to control the rate of release of the entrapped agent in the liposome.
  • the rigidity of the liposome, as determined by the vesicle- forming lipid, may also play a role in fusion of the liposome to a target cell, as will be described.
  • Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer are achieved by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., up to 60 °C.
  • a relatively rigid lipid e.g., a lipid having a relatively high phase transition temperature, e.g., up to 60 °C.
  • Rigid, i.e., saturated, lipids contribute to greater membrane rigidity in the lipid bilayer.
  • Other lipid components, such as cholesterol are also known to contribute to membrane rigidity in lipid bilayer structures.
  • lipid fluidity is achieved by incorporation of a
  • relatively fluid lipid typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature.
  • the liposomes are prepared with a relatively rigid lipid to impart rigidity to the lipid bilayer.
  • the lipids forming the liposomes have a phase transition temperature of between about 37-70 °C.
  • the vesicle forming lipid is distearyl phosphatidylcholine (DSPC), which has a phase transition temperature of 62 °C.
  • Exemplary cationic lipids include l,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[l-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N- hydroxyethylammonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]- N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE); N-[l-(2,3- dioleyloxy) propyl] - ⁇ , ⁇ , ⁇ -trimethylammonium chloride (DOTMA); [N- ( ⁇ ', ⁇ '-dimethylaminoethane) carbamoyl] cholesterol (DC-Choi); and dimethyl dioctadecylammonium (DDAB).
  • DOTAP l,2-dioleyloxy-3-(trimethylamino) propane
  • DMRIE N-[l-(
  • the cationic vesicle -forming lipid may also be a neutral lipid, such as dioleoylphosphatidyl ethanolamine (DOPE) or an amphipathic lipid, such as a phospholipid, derivatized with a cationic moiety (T A ), such as polylysine.
  • DOPE dioleoylphosphatidyl ethanolamine
  • T A amphipathic lipid
  • T A cationic moiety
  • the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid.
  • the surface coating on the liposome provided by the hydrophilic polymer chains provides colloidal stability and, at a sufficient polymer surface density, serves to protect the liposomes from uptake by the reticuloendothelial system, providing an extended blood circulation lifetime for the liposomes to reach the target cells.
  • the extent of enhancement of blood circulation time is preferably several -fold over that achieved in the absence of the polymer coating, as described in U.S. Pat. No. 5,013,556.
  • kits and method of the invention are very suitable for use in targeted delivery of drugs.
  • a "target” as used in the present invention relates to a target for a targeting agent for therapy.
  • a target can be any molecule, which is present in an organism, tissue or cell.
  • Targets include cell surface targets, e.g. receptors, glycoproteins, peptides, carbohydrates, monosacharides, polysaccharides; structural proteins, e.g. amyloid plaques; abundant extracellular targets such as stroma, extracellular matrix targets such as growth factors, and proteases; enzymes; and/or foreign bodies, e.g. pathogens such as viruses, bacteria, fungi, yeast or parts thereof.
  • targets include compounds such as proteins of which the presence or expression level is correlated with a certain tissue or cell type or of which the expression level is up regulated or down-regulated in a certain disorder.
  • the target is a protein such as a (internalizing or non- internalizing) receptor.
  • targets include somatostatin receptor, transferrin receptor, monoamine oxidase, muscarinic receptors,
  • leukocytes urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti-)angiogenesis marker, gastrin receptor, GPIIb/IIIa receptor and other thrombus related receptors, fibrin,
  • VEGF/EGF and VEGF/EGF receptors TAG72, CEA, CD 19, CD20,CD22, CD40, CD45, CD74, CD79, CD 105, CD 138, CD 174, CD227, CD326, CD340, MUC1, MUC16, GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1 receptor, CD44v6, G250, HLA DR, ED-B, TMEFF2 , EphB2, EphA2, FAP, Mesothelin, GD2, CAIX, 5T4, matrix metalloproteinase (MMP), VCAM-1, ICAM- 1, PECAM-1, P/E/L-selectin receptor, LDL receptor, P-glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, sigma receptors, interleukin receptors, insulin receptor, liver hepatocytes receptor, herpes simplex virus t
  • the targeting agent T T can comprise compounds including but not limited to antibodies, antibody fragments, e.g. Fab2, Fab, scFV, diabodies, triabodies, VHH, antibody (fragment) fusions (eg bi-specific and trispecific mAb fragments), proteins, peptides, e.g. octreotide and derivatives, VIP, MSH, LHRH, chemotactic peptides, bombesin, elastin, peptide mimetics, carbohydrates, monosacharides, polysaccharides, viruses, whole cells, (e.g.
  • the targeting agent T T is an antibody.
  • the target is a receptor and a targeting agent is employed, which is capable of specific binding to the target.
  • Suitable targeting agents include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g. a receptor binding peptide in the case of receptor binding protein ligands.
  • Other examples of targeting agents of protein nature include interferons, e.g. alpha, beta, and gamma interferon, transferrin, interleukins, and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet-derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin.
  • Alternative examples of targeting agents include DNA, RNA, PNA and LNA.
  • the target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.
  • a tissue or disease such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.
  • a tissue or disease such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis
  • folate intracellular accumulation of folate and its analogs, such as methotrexate. Expression is limited in normal tissues, but receptors are overexpressed in various tumor cell types.
  • Targeting agents T A comprise the agents listed for T T and in addition include Hydrophobic Polymers (defined above) and polycationic moieties, including cell penetrating moieties, such as cell-penetrating peptide sequences that facilitates delivery to the intracellular space, e.g., oligo- lysines, oligo-arginines, HIV- derived TAT peptide, penetratins,
  • Masking moieties M M can be a Hydrophilic Polymer (defined above), polymer, protein, peptide, carbohydrate, organic construct, that shields the bound Construct C c . This shielding can be based on eg steric hindrance. Such masking moiety may also be used to affect the in vivo properties (eg blood clearance; recognition by the immunesystem) of the liposome.
  • the Masking Moiety is a Hydrophilic Polymer. Spacers
  • Spacers S p include but are not limited to polyethylene glycol (PEG) chains varying from 2 to 200, particularly 3 to 113 and preferably 5- 50 repeating units.
  • PEG polyethylene glycol
  • Other examples are biopolymer fragments, such as oligo- or polypeptides or polylactides. Further preferred examples are shown in Example 3.
  • the liposomes include a ligand for targeting the liposomes to a selected cell type or another liposome containing the proper receptor.
  • the ligand or T T is bound to the liposome by covalent attachment to the free distal end of a lipid- anchored hydrophilic polymer chain.
  • the hydrophilic polymer chain is PEG, and several methods for attachment of ligands to the distal ends of PEG chains have been described (see e.g. US5891468 and refs therein). In these methods, the inert terminal methoxy group of mPEG is replaced with a reactive functionality suitable for conjugation reactions, such as an amino or hydrazide group.
  • the end functionalized PEG is attached to a lipid, typically DSPE.
  • the functionalized PEG-DSPE derivatives are employed in liposome formation and the desired ligand is attached to the reactive end of the PEG chain before or after liposome formation.
  • the polymer chains are functionalized to contain reactive groups suitable for coupling with, for example, sulfhydryls, amino groups, and aldehydes or ketones (typically derived from mild oxidation of carbohydrate portions of an antibody) present in the antibody.
  • PEG-terminal reactive groups include maleimide (for reaction with sulfhydryl groups), N- hydroxysuccinimide (NHS) or NHS-carbonate ester (for reaction with primary amines), hydrazide or hydrazine (for reaction with aldehydes or ketones), iodoacetyl (preferentially reactive with sulfhydryl groups) and dithiopyridine (thiol-reactive).
  • Another example is the attachment of folic acid to a DSPE-PEG conjugate as described in US6936272. Folic acid is mixed with amino-PEG-DSPE and reacted in the presence of N-hydroxy-s- norbornene-2,3-dicarboxylic acid imide (HONB) and
  • DCC dicyclohexylcarbodiimide
  • the preparation to form liposomes including a folic acid targeting ligand can also be included in the liposomes by means of a lipid-M M conjugate with no releasabe linkage joining the lipid and the M M .
  • the targeting ligand can also be included in the liposomes by means of a lipid-M M conjugate with no releasabe linkage joining the lipid and the M M .
  • any lipids suitable to form the hydrophillic polymer coating of the liposome discussed above may be used to form the T T -modified polymer-lipid conjugate, and any of the hydrophilic polymers described above are suitable.
  • the T T does not suffer any loss of activity.
  • the T T - polymer-lipid conjugate into preformed liposomes by insertion, where the T T -polymer-lipid conjugate is incubated with the preformed liposomes under conditions suitable to allow the conjugate to become incorporated into the hposome lipid bilayer.
  • the insertion technique has been described in the art, for example in U.S. Pat. No. 6,056,973.
  • the liposomes optionally contain a T T or T A bound to the surface of the lipid by attachment to surface lipid components.
  • Such a ligand is initially shielded by the hydrophilic surface coating from interaction with target cells until after the removal of the hydrophilic polymers.
  • such a ligand is coupled to the polar head group of a vesicle-forming lipid and various methods have been described for attachment of ligands to lipids.
  • the Targeting Agent T T or T A is coupled to the lipid, by a coupling reaction described below, to form alipid conjugate.
  • This conjugate is added to a solution of lipids for formation of liposomes, as will be described.
  • a vesicle-forming lipid activated for covalent attachment of an e.g. T T is incorporated into liposomes. The formed liposomes are exposed to the T T to achieve attachment of T T to the activated lipids.
  • a variety of methods are available for preparing a conjugate composed of a Targeting Agent T T or T A and a vesicle-forming lipid.
  • a Targeting Agent T T or T A a Targeting Agent
  • a vesicle-forming lipid For example, water- soluble, amine-containing moieties can be covalently attached to lipids, such as phosphatidylethanolamine, by reacting the amine-containing moiety with a lipid which has been derivatized to contain an activated ester of N-hydroxy-succinimide.
  • biomolecules and in particular large biomolecules such as proteins, can be coupled to lipids according to reported methods.
  • One method involves Schiff-base formation between an aldehyde group on a lipid, typically a phospholipid, and a primary amino acid on the
  • the aldehyde group is preferably formed by periodate oxidation of the lipid.
  • the coupling reaction after removal of the oxidant, is carried out in the presence of a reducing agent, such as dithiothreitol, as described by Heath, (1981).
  • a reducing agent such as dithiothreitol
  • Typical aldehyde-lipid precursors suitable in the method include lactosylceramide, trihexosylceramine, galacto cerebroside, phosphatidylglycerol, phosphatidylinositol and gangliosides.
  • a second general coupling method is applicable to thiol-containing moieties, and involves formation of a disulfide or thioether bond between a lipid and the Targeting Agent.
  • a lipid amine such as phosphatidyl-ethanolamine
  • a pyridyldithio derivative which can react with an exposed thiol group in the Targeting Agent.
  • Reaction conditions for such a method can be found in Martin (1981).
  • the thioether coupling method, described by Martin (1982) is carried out by forming a sulfhydryl-reactive phospholipid, such as N-(4)P- maleimidophenyl(butyryl)phosphatidylethanolamine, and reacting the lipid with the thiol-containing Targeting Agent.
  • Another method for reacting a Targeting Agent with a lipid involves reacting the Targeting Agent with a lipid which has been derivatized to contain an activated ester of N-hydroxysuccinimide.
  • the reaction is typically carried out in the presence of a mild detergent, such as deoxycholate.
  • this coupling reaction is preferably performed prior to incorporating the lipid into the liposome.
  • Liposomes containing an entrapped agent can be prepared according to well-known methods, such as hydration of a lipid film, reverse-phase evaporation, and solvent infusion.
  • the compound to be delivered is either included in the lipid film, in the case of a lipophilic compound, or is included in the hydration medium, in the case of a water-soluble therapeutic agent.
  • the therapeutic agent may be loaded into preformed vesicles, e.g., by loading an ionizable compound against an ion gradient.
  • the liposomes may be prepared by a variety of techniques, such as those detailed in Szoka, et al., 1980.
  • Multilamellar vesicles can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids of the type detailed above dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium. The lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.
  • the lipid components used in forming the fusogenic liposomes of a particular embodiment of the present invention are preferably present in a molar ratio of about 70-90 percent vesicle-forming lipids, 1-20 percent diblock copolymer lipid conjugate and 0.1-5 percent of a lipid having an attached Targeting Agent T T .
  • the hydrophilic polymer added may consist entirely of diblock copolymer lipid conjugate or a combination of diblock copolymer lipid conjugate and polymer directly linked to a lipid. Ideally, the percentage of diblock lipid conjugate in this mixture is the maximum percentage that is consistent with liposome stability.
  • the amount of diblock copolymer lipid conjugate is between 5- 100% of the total hydrophilic polymer lipid included in the lipid
  • One exemplary formulation includes 80-90 mole percent
  • phosphatidylcholine 1-20 mole percent of polymer-lipid conjugates, and 0.1-5 mole percent T T -PEG-DSPE, with the diblock polymer lipid
  • Another procedure suitable for preparation of the fusogenic liposomes of the present invention involves diffusion of polymer-lipid conjugates into preformed liposomes.
  • liposomes with an entrapped therapeutic agent are prepared from vesicle-forming lipids.
  • the preformed liposomes are added to a solution containing a concentrated dispersion of micelles of polymer-lipid diblock conjugates and optionally, T T -PEG-DSPE, and the mixture is incubated under conditions effective to achieve insertion of the micellar lipids into the preformed liposomes.
  • An advantage of this method is that the hydrophobic polymer moiety in the diblock lipid is confined to the outer lipid layer of the liposomes, and is therefore potentially less destabilizing than when the diblock component is
  • the liposomes may be preformed with the directly linked hydrophilic polymer lipid, and incubated under lipid exchange conditions with the diblock polymer conjugate, to exchange the diblock lipid into the outer liposome layer.
  • the therapeutic or diagnostic agent to be administered to cells, in
  • liposomes may be incorporated into liposomes by standard methods, including (i) passive entrapment of a water-soluble compound by hydrating a lipid film with an aqueous solution of the agent, (ii) passive entrapment of a lipophilic compound by hydrating a lipid film containing the agent, and (iii) loading an ionizable drug against an inside/outside liposome pH gradient (U.S. Pat. No. 5, 192,549; Bolotin et al., J. Liposome Res., 4:455 (1994)).
  • Other methods such as reverse
  • the liposomes of the invention are preferably prepared to have
  • substantially homogeneous sizes in a selected size range typically between about 0.01 to 0.5 microns, more preferably between 0.03-0.40 microns.
  • One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns.
  • the pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane.
  • Homogenization methods are also useful for down-sizing liposomes to sizes of 100 nm or less (Martin, 1990).
  • the liposomes are extruded through a series of polycarbonate filters with pore sizes ranging from 0.2 to 0.08 ⁇ resulting in liposomes having diameters in the approximate range of 120 +/-10 nm.
  • Composition for the pH sensitive liposome :
  • Liposomes of the invention are typically prepared with lipid components present in a molar ratio of about 70-90 percent vesicle-forming lipids, 1-20 percent of a (M M )-T R -lipid conjugate for forming the surface coating of releasable polymer chains, and 0.1-5 percent of an end-functionalized T T - M M -lipid conjugate.
  • the polymer-lipid conjugate with the releasable hnkage can be end-functionalized to couple a T T , or the liposomes can include two different M M -lipid species-one M M -lipid conjugate with a releasable linkage and another M M -lipid conjugate with no releasable linkage but with an attached T T .
  • the liposome is administered first, and it will take a certain time period before the liposome has reached the Target. This time period may differ from one application to the other and may be minutes, days or weeks.
  • the Activator is administered, will find and react with the liposome and will thus activate Drug release at the Target.
  • the reaction between the Trigger and the Activator may occur extracellularly or intracellulary, or both.
  • compositions of the invention can be administered via different routes including subcutaneous, intramuscular, interlesional (to tumors), intertracheal by inhalation, topical, internasal, intraocular, via direct injection into organs and intravenous.
  • Formulations suitable for these different types of administrations are known to the skilled person.
  • Liposomal compositions or Activators according to the invention can be administered together with a pharmaceutically acceptable carrier.
  • a suitable pharmaceutical carrier as used herein relates to a carrier suitable for medical or veterinary purposes, not being toxic or otherwise
  • Such carriers are well known in the art and include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the preferred mode of administration is intravenous injection.
  • the chemical entities administered viz. the liposome and the activator, can be in a modified form that does not alter the chemical functionality of said chemical entity, such as salts, hydrates, or solvates thereof.
  • a Clearing Agent is an agent, compound, or moiety that is administered to a subject for the purpose of binding to, or complexing with, an administered agent (in this case the Liposome) of which excess is to be removed from circulation.
  • the Clearing Agent is capable of being directed to removal from circulation. The latter is generally achieved through hver receptor-based mechanisms, although other ways of secretion from circulation exist, as are known to the skilled person.
  • the Clearing Agent for removing circulating Liposome preferably comprises a diene moiety, e.g. as discussed above, capable of reacting to the TCO moiety of the Liposome.
  • the Trigger and Activator can be selected such to achieve a specific release kinetics, which is a feature that can advantageously utilized to vary and tailor the release rate of an entrapped agent. In this manner one can choose to effect a slow drug release or a burst release.
  • Entrapped in the liposomes is a therapeutic agent or drug for delivery to the target.
  • a variety of therapeutic agents can be entrapped in lipid vesicles, including water-soluble agents that can be stably encapsulated in the aqueous compartment of the vesicles, lipophilic compounds that stably partition in the lipid phase of the vesicles, or agents that can be stably attached, e.g., by electrostatic attachment to the outer vesicle surfaces.
  • Exemplary water-soluble compounds include small, water-soluble organic compounds, peptides, proteins, DNA plasmids, oligonucleotides and gene fragments.
  • the hposome-entrapped compound may also be an imaging agent for tracking progression of a disease.
  • the entrapped agent may also be a reporter molecule, such as an enzyme or a fluorophore, for use in assays.
  • the drug or agent to be delivered may be a polynucleotide capable of expressing a selected protein, whe taken up by a target cell, an
  • oligonucleotide or oligonucleotide analog designed for binding to a specific- sequence nucleic acid in the target cells e.g. siRNA, antisense
  • oligonucleotide any other therapeutic polymer or small-molecule therapeutic or diagnostic agent.
  • Liposomes can contain an entrapped gene (cDNA plasmid) to be delivered to target cells, for gene therapy.
  • cDNA plasmid a variety of genes for treatment of various conditions have been described .
  • arid coding sequences for specific genes of interest can be retrieved from DNA sequence databanks, such as GenBank or EMBL.
  • the selected coding sequences may encode any of a variety of different types of proteins or polypeptides, depending on the particular application.
  • the fusogenie liposome ma be used to introduce sequences encoding enzymes into, e.g., stem cells or lymphocytes of individuals suffering from an enzyme deficiency.
  • sequences encoding ADA may be transfeeted into stem cells or lymphocytes of such individuals.
  • the liposomes may contain genes encoding any of a variety of circulating proteins, such as c i- antitry sin, clotting factors (e.g., Factor VIII, Factor IX) and globins (e.g., ⁇ -glohin,
  • gene coding sequences suitable for use with the present invention include sequences encoding structural proteins; receptors, such as low density lipoprotein receptor (LDL-R) for transfection of hepatocytes to treat LDL-deficient patients, human CD4 and soluble forms thereof, and the like; transmembrane proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) for treatment of cystic fibrosis patients; signalling molecules; cytokines, such as various growth factors (e.g., TGF-alpha, TGF-beta, EGF, FGF, IGF, NGF, PDGF, CGF, CSF, SCF), inteiieuldns, interferons, erythropoietin, and the like, as well as receptors for such cytokines; antibodies including chimeric antibodies; genes useful in targeting malignant tumors (e.g.
  • the liposomes may also encode enzymes to convert a noncytotoxic prodrug into a cytotoxic drug in tumor cells or tumor-adjacent endothelial cells.
  • the liposomes contain a polynucleotide designed to be incorporated into the genome of the target cell or designed for autologous replication within the cell.
  • the compound entrapped in the lipid vesic!es is an
  • oligonucleotide segment designed for sequence-specific binding to cellular RNA or DNA include but are not limited to: antibodies, antibody derivatives, antibody fragments, e.g. Fab2, Fab, scFV, diabodies, triabodies, antibody (fragment) fusions (eg bi-specific and trispecific mAb fragments), proteins, aptamers, oligopeptides, oligosaccharides, as well as peptides, peptoids, steroids, organic drug compounds, toxins (e.g.
  • ricin A diphtheria toxin, cholera toxin
  • hormones viruses, antiproliferative/antitumor/cytotoxic agents, antibiotics, cytokines, anti-inflammatory agents, anti-viral agents, antihypertensive agents, chemosensitizing and radiosensitizing agents.
  • Some embodiments use auristatins, maytansines, cahcheamicin, duocarmycins, maytansinoids DM1 and DM4, auristatin MMAE, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs.
  • cytotoxic agents include antimetabolites, natural products and their analogs, enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA alkylators, radiation sensitizers, DNA intercalators, DNA cleavers, anti-tubulin agents, topoisomerases inhibitors, platinum-based drugs, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, taxanes, lexitropsins, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins,
  • maytansinoids maytansinoids, differentiation inducers, and taxols.
  • Particularly useful members of those classes include, for example, duocarmycin , methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil DNA minor groove binders, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin,
  • exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No. 4,486,414), dolastatin B (U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No. 4,486,444, 5,410,024, 5,504, 191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663, 149,
  • the drug moiety is a mytomycin, vinca alkaloid, taxol, anthracycline, a cahcheamicin, maytansinoid or an auristatin.
  • hydrophilic small molecules that are envisaged to be provided in the liposomes of the present invention include, but are not limited to, peptides and proteins that modulate the immune response such as interleukins; potent inhibitors of protein synthesis in human cells such as Diphteria toxin (fragment); activators of immune system for
  • tumour cells such as muramyl dipeptide
  • drugs for the treatment of lung" fibrosis such as Cis-4- hydroxyproline
  • compounds for cancer treatment such as Cisplatin and. derivatives thereof, cytosine arabinose, carboplatin, methotrexate, 1-SD- arabino-furanyl-cytosine (ara-C),5-fl.uoro-uracil, floxuridine, and gemcitabine
  • antibacterial agents such as phospb.onopeptid.es
  • activator of prodrugs such as Glucuronidase for the activation of e.g. epirubicin- glueuronide
  • small therapeutic proteins and peptides such as insulin, growth factors and chemokines.
  • the drug is selected so as to target and or address a disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.
  • a disease such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.
  • the compound is useful for treatment of a plasma cell disorder, such as multiple myeloma, which is characterized by neoplasms of B -lymphocyte lineage cells.
  • a plasma cell disorder such as multiple myeloma
  • Therapeutic agents preferred, for treatment of multiple myeloma include melphalan, cyclophosphamide, prednisone, chlorambucil, carmustine, dexamethasone, doxorubicin, cisplatin, paelitaxel, vincristine, lomustine, and interferon.
  • intracytoplasmic delivery of plasmids, antisense oligonucleotides, and ribozymes for the treatment of cancer and. viral. infections.
  • the released drag is in fact a prodrug designed to release a further drug.
  • Drugs optionally include a membrane translocation moiety (adamantine, poly-Iysine/argine, TAT) and/or a targeting agent (against eg a tumor eel receptor) optionally linked through a stable or labile linker.
  • the Activator can have a hnk to a Masking Moiety M M such as a peptide, protein, carbohydrate, PEG, or polymer.
  • M M such as a peptide, protein, carbohydrate, PEG, or polymer.
  • Activators for use with Triggers based on the cascade mechanism satisfy one of the following formulae:
  • R (link to) peptide, protein, carbohydrate, PEG, polymer
  • Exemplary lipids to be used in activatable liposomes include:
  • Exemplary liposomal formulations that release a contained drug upon reaction with a tetrazine at a pH within the range of 4.5-8.0:
  • preferred formulations are those with lipid A: 0.5 - 10 mol%
  • DOPE 50-100 mol% Cholesterol (Choi) and/or cholesterolhemisuccinate (CHEMS): total 0-50 mol%
  • compositions include:
  • DOPE/DSPE-TCO-mPEG 90 10 molar ratio
  • DOPE/DSPE-TCO-mPEG 99 1 molar ratio
  • the DSPE-TCO-mPEG used above embodiments is either DSPE-TCO-mPEG I or DSPE-TCO-mPEG II.
  • the DOPE-TCO-mPEG used above is either DOPE-TCO- mPEG I or DOPE-TCO-mPEG II.
  • the mPEG is preferably mPEG-2000 or mPEG- 5000.
  • IR spectra were recorded on a Perkin Elmer 1600 FT-IR (UATR).
  • LC-MS was performed using a Shimadzu LC-10 AD VP series HPLC coupled to a diode array detector (Finnigan Surveyor PDA Plus detector, Thermo Electron Corporation) and an Ion-Trap (LCQ Fleet, Thermo Scientific).
  • Size exclusion (SEC) HPLC was carried out on an Agilent 1200 system equipped with a Gabi radioactive detector. The samples were loaded on a Superdex-200 10/300 GL column (GE Healthcare Life Sciences) and eluted with 10 mM phosphate buffer, pH 7.4, at 0.35-0.5 niL/min. The UV wavelength was preset at 260 and 280 nm. The concentration of antibody solutions was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) from the absorbance at 322 nm and 280 nm, respectively.
  • SEC Size exclusion
  • the invention can be exemplified with the same combinations of TCO and diene as included in applications WO2012156919A1 (e.g. Examples 9 - 14) and WO2012156920A1 (e.g. Examples 8 - 11), except that a Construct as defined hereinbefore is taken in lieu of a drug as disclosed therein.
  • 3-PNP-TCO was synthesized following WO2012156919A1.
  • 3-PNP-TCO (41.9 mg; 1.44* 10 "4 mol) was dissolved in dichloromethane (1.5 mL), and DIPEA (55.7 mg; 4.32* 10 "4 mol) and 1-naphthylmethylamine (27.2 mg; 1.73* 10 " 4 mol) were added.
  • the reaction mixture was stirred at 20°C under and atmosphere of argon and slowly turned yellow. After 20 h the solvent was removed by evaporation in vacuo, and the mixture was redissolved in dichloromethane and washed with subsequently, 1 M aqueous sodium hydroxide (5 times 2.5 mL) and 1 M aqueous citric acid (2 times 1.5 mL).
  • Axial-(E)-cyclooct-2-en-l-ol (152 mg, 1.20 mmol) was dissolved in 10 mL dichloromethane.
  • 4-(N,N-dimethylamino)pyridine (306 mg, 2.50 mmol) was added and the solution was cooled in an ice-bath.
  • a solution of 4-nitrobenzoyl chloride (201 mg, 1.08 mmol) in 5 mL dichloromethane was added in portions over a 5 min period. The solution was stirred for 3 days. The solvent was partially removed by rotary evaporation. The remaining solution (a few mL) was chromatographed on 19 g silica, using dichloromethane as the eluent. The product fractions were rotary evaporated yielding a colourless solid (144 mg, 0.52 mmol, 48%).
  • Equatorial-(E)-cyclooct-2-en-l-ol 154 mg, 1.22 mmol was dissolved in 10 mL dichloromethane.
  • 4-(N,N-dimethylamino)pyridine 300 mg, 2.46 mmol was added and the solution was cooled in an ice-bath.
  • a solution of 4-nitrobenzoyl chloride (268 mg, 1.44 mmol) in 5 mL dichloromethane was added in portions over a 5 min period. The solution was stirred for 4 days. The solvent was removed by rotary evaporation and the residue was chromatographed on 19 g silica, using dichloromethane as the eluent.
  • Cyclooct-2-en-l-ol (5.002 g, 39.64 mmol) was dissolved in 100 niL THF. Phenol (3.927 g, 41.78 mmol ) was added to the solution. Triphenylphosphine (10.514 g, 40.01 mmol) was added and the resulting solution was cooled in an ice-bath. A solution of diethyl azodicarboxylate (6.975 g, 40.01 mmol) in 50 mL THF was added over a 30 min period. The reaction mixture was stirred for 24 h and then rotary evaporated. The residue was stirred with heptane, the mixture was filtered and the filtrate was rotary evaporated.
  • 3-phenoxycyclooctene (5.5 g, 27.23 mmol) was dissolved in heptane - ether (ca.1/2). The solution was irradiated for 7 days while the solution was continuously flushed through a 42 g silver nitrate impregnated silica column (containing ca. 4.2 g silver nitrate). The column was rinsed twice with TBME, then with TBME containing 5% methanol, then with TBME containing 10% MeOH. The product fractions were washed with 100 mL 15% ammonia (the same ammonia being used for each fraction), then dried and rotary evaporated. The column material was stirred with TBME and 15% ammonia, then filtered, and the layers were separated.
  • the organic layer was dried and rotary evaporated.
  • the first two TBME fractions were combined, and all other fractions were separately rotary evaporated, then examined for the presence of the product (none of the fractions contained a pure trans-cyclooctene isomer, however).
  • the product fractions were combined and chromatographed on 102 g silica, using heptane as the eluent.
  • the first fractions yielded the pure minor (believed to be axial) isomer as an oil (144 mg, 0.712 mmol, 2.6%).
  • the next fractions contained a mixture of minor and major isomer. Pure major (believed to be equatorial) isomer was eluted last, yielding a colourless solid (711 mg, 3.52 mmol, 13%).
  • Axial (E)-cyclooct-2-en- l-ol (102 mg, 0.81 mmol) was dissolved in 7.5 mL dichloromethane with 4-(N,N-dimethylamino)pyridine (303 mg, 2.70 mmol).
  • a solution of phenylacetyl chloride (155 mg, 1.00 mmol) in 2.5 mL dichloromethane was added in portions over a 5 min period to the ice-cooled solution. The reaction mixture was stirred for 4 days, then washed with water. The aqueous layer was extracted with 10 mL dichloromethane. The combined organic layers where dried and rotary evaporated, followed by chromatography yielding a colourless powder (22 mg) which was identified as the depicted byproduct.
  • the iodolactone was dissolved in 250 mL toluene, and DBU (65.2 g, 0.428 mol) was added. The mixture was allowed to stand overnight, after which it was heated under reflux for 75 min (NMR indicated full conversion). After cooling the reaction mixture, it was washed with 150 and 100 mL water. The successive aqueous layers were extracted with 250 mL toluene. The organic layers were dried and rotary evaporated and the residue was purified by Kugelrohr distillation to yield 38.86 g of the bicyclic olefin (0.234 mol, 94%, containing a trace of toluene).
  • the silica column was successively flushed with 600 mL TBME, 500 mL TBME / 5% methanol, 500 mL TBME / 10% methanol, and 500 mL TBME / 20% methanol.
  • the first 3 eluates were rotary evaporated.
  • the first eluate contained methyl benzoate and the starting hydroxy ester in a ca. 2/3 ratio.
  • the fourth eluate was washed with 300 mL 10% ammonia solution, then dried and rotary evaporated (axial/equatorial ratio of the trans-cycloctene was ca. 5/4).
  • the residues from the second and third eluate were combined, dissolved in TBME and washed with the ammonia layer of above.
  • the combined aqueous layers were treated with 30 mL TBME, and then with 4.5 g citric acid. The layers were separated and the aqueous layer was extracted with 30 mL TBME. The organic layers were dried and rotary evaporated at 55°C to afford 0.34 g (1.85 mmol, 75%) of the pure axial isomer of the trans-cyclooctene acid.
  • DSPE l,2-Distearoyl-sn-glycero-3-phosphoethanolamine
  • axial TCO-2 5.0 mg; 11.84* 10 ⁇ 6 mol
  • DBU l,8-Diazabicyclo[5.4.0]undec-7-ene
  • Axial TCO-2 (5.0 mg; 11.84* 10 "6 mol) was added, and the homogeneous solution was stirred under an atmosphere of argon at -15°C for 30 min.
  • DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • the tetrazines featured in Figure 1 were tested with respect to their ability to release doxorubicin from TCO-2-doxorubicin. It shall be understood that the tetrazine- induced release in this experiment can be considered representative of the cleavage of lipid-TCO-PEG constructs.
  • PBS/MeCN (1 mL, 3/1) preheated at 37°C and TCO-2-doxorubicin (10 of a 2.5 mM solution in DMSO, 1 eq.) were added to a preheated injection vial.
  • Tetrazine (10 ⁇ L ⁇ of a 25 mM solution in DMSO, 10 eq.) was added and the vial was vortexed. After incubation for 1 hour at 37°C, the vial was placed in LC-MS autosampler at 10°C. LC- MS analysis was performed using a 5% to 100% H 2 0/MeCN gradient over 11 minutes with a C18 reverse-phase column at 35°C. A control sample containing only TCO-2-doxorubicin (1 eq), as well as a sample containing only doxorubicin (1 eq.), was analyzed under the same conditions.
  • TCO-l-doxorubucin (6.25 xlO "8 mol) was dissolved in DMSO (0.050 mL), and PBS (0.475 mL) was added slowly in aliquots of 0.010 mL, followed by mouse serum (0.475 mL). A portion of this mixture (0.200 mL) was equilibrated at 37 °C, and a solution of tetrazine (1.25 xlO " mol) in DMSO (0.005 mL) was added, and the solution was thoroughly mixed and incubated at 37 °C in the dark for 4 h.
  • the TCO stock solution (10 iL 25 mM; 2.5* 10 " ' mol) was added to a solution of the specific condition (100 ⁇ ). The mixture was stirred at the specific condition for a certain amount of time, and then the fate of the TCO compound was monitored by HPLC-MS/PDA analysis and/or GC-MS analysis, and an estimation of its stability was made.
  • the TCO stock solution (10 25 niM in acetonitrile; 2.5* 10 " mol) was added to a solution of the specific condition (100 A solution of 3,6-dimethyl-l,2,4,5- tetrazine (8, 20 uL 25 niM in acetonitrile; 5.0* 10 " mol) was added, and the mixture was stirred at the specific condition for a certain amount of time.
  • the reaction was monitored by HPLC-MS/PDA analysis and/or GC-MS analysis, and the percentage of deprotection was estimated.
  • Activatable liposomes comprising DSPE-TCO-2-PEG2000
  • Lipomes containing a quenched dye in the interior and DSPE-TCO-2-PEG2000 in the membrane were prepared. Subsequenly, the tetrazine-induced cleavage of DSPE- TCO-2-PEG2000 and the resulting release of the liposomal contents was
  • Multilamellar liposomes were prepared by hydrating the lyophilized lipid mixtures with 2.5 ml of 30 mM trisodium 8-hydroxypyrene-l,3,6-trisulfonic acid (HPTS) / 30 mM p-xylene-bis-pyridinium bromide (DPX) (20 mM 4-(2-hydroxyethyl)-l- piperazine-ethanesulfonic acid (HEPES), pH 9.0, adjusted to 290 mOsmol with NaCl] via five freeze-thaw vortex cycles.
  • HPTS trisodium 8-hydroxypyrene-l,3,6-trisulfonic acid
  • DPX p-xylene-bis-pyridinium bromide
  • HEPES 4-(2-hydroxyethyl)-l- piperazine-ethanesulfonic acid

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Abstract

La présente invention concerne un liposome réactif comprenant une bicouche lipidique, la bicouche comprenant une liaison à un groupe alcénylène cyclique non aromatique octogonal, de préférence un groupe cyclo-octénique et mieux encore un groupe trans-cyclo-octénique. Les liposomes sont utilisés dans un kit comprenant le liposome lié, directement ou indirectement, à un déclencheur et un activateur pour le déclencheur, le déclencheur comportant un groupe alcénylène cyclique non aromatique octogonal et l'activateur comprend un diène.
PCT/NL2013/050846 2012-11-22 2013-11-22 Liposome pouvant être actif WO2014081299A1 (fr)

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