WO2014081299A1

WO2014081299A1 - Activatable liposomes

Info

Publication number: WO2014081299A1
Application number: PCT/NL2013/050846
Authority: WO
Inventors: Marc Stefan Robillard; Sander Martinus Johannes VAN DUIJNHOVEN; Maarten Jozef Pouderoijen; Ronny Mathieu Versteegen
Original assignee: Tagworks Pharmaceuticals BV
Current assignee: Tagworks Pharmaceuticals BV
Priority date: 2012-11-22
Filing date: 2013-11-22
Publication date: 2014-05-30
Anticipated expiration: 2015-05-22

Abstract

Disclosed are 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 liposomes are use in a kit comprising the 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, and the Activator comprises a diene.

Description

Title: ACTIVATABLE LIPOSOMES

Field of the invention

The invention pertains to reactive (activatable) liposomes, and particularly to liposomal compositions designed for improved target delivery of an entrapped agent.

Background of the invention

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). There are two liposomal targeting strategies: passive and specific. Passive targeting refers to the preferential accumulation of liposomes in tumours and at sites of infection and inflammation. Small sterically stabilized liposomes extravasate through leaky blood vessels that are formed through tumour angiogenesis or damaged by infection and inflammation. The liposomes accumulate in tumour interstices and at sites of infection and inflammation, where they gradually release their encapsulated drugs. This sequesters potentially toxic agents from susceptible non-target sites such as the brain, liver and heart. 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. In principle, 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. Hence, 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.

There are two caveats, however: 1) At the target site, the drugs must be released from the liposomes efficiently enough to have a clinically significant effect, and 2) The target cells must be accessible.

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.

Alternatively, these Constructs stay in the extracellular domain and are slowly degraded, depending on the specific environment. When liposomes are taken up by cells through endocytosis, the liposome needs to be degraded and the drug has to be able to escape the endosomal or lysosomal compartment. In this respect it should be noted that endothelial cells do not possess a machinery to degrade liposomes and as a result these

Constructs are expulsed back to the extracellular environment. In addition, when liposomes are entrapped in macrophages, one needs a burst release of the drug to achieve an effective peak concentration, which allows diffusion from the macrophage.

Currently the targeting and localization of liposomes can be controlled by passive or active methods, but there is only an indirect, unpredictable and non-universal control over the subsequent release from these liposomes. The release pathway, the extent of release, the release profile (slow or bolus), and the timing, to a large extent depend on the specifics and peculiarities of the target physiology and biology. To address this issue, liposomal delivery systems capable of release of their content under the influence of e.g. pH, thiols, and light have been developed.

For example, liposomes that destabilize under mildly acidic conditions, so-called 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.,

84:7851 (1987), Liu et al., Biochim. Biophys. Acta, 981:254 (1989)).

These liposomes are primarily composed of a lipid, such as

dioleoylphosphatidylethanolamine (DOPE), that forms a lipid bilayer in a defined pH range. Outside this pH range, the lipid bilayer destabilizes. After such liposomes enter cells via endocytosis, the acidic pH inside the endosomes causes the pH-sensitive liposomes to destabilize and release the entrapped agent.

Because pH-sensitive liposomes, like "conventional", "non-pH- sensitive liposomes", have short circulation lifetimes, addition of PEG- derivatized lipids to extend the blood circulation time has been proposed (Slepushkin et al.). However, addition of PEG-derivatized lipids

attenuates the pH-sensitivity of the liposomes, resulting in a loss of the desired rapid destabilization of the liposome bilayer and accompanying rapid release of the entrapped agent into the cell. One approach to providing pH-sensitive liposomes having a long blood circulation lifetime and retaining the ability of the liposome to rapidly destabilize is the use PEG-derivatized lipids where the PEG is attached to the lipid by a thiol- cleavable linkage for release at the target site, restoring the pH sensitivity and effecting drug release (Kirpotin et al., FEBS Letters, 388: 115 (1996); and US7108863).

Also described in the art are liposomes capable of fusion with a target cell (U.S. Pat. No. 5,891,468). 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.

An alternative liposome release technology is based on a well- studied bacterial channel protein "Mechanosensitive channel of large conductance", MscL, from E. coli. In its native form 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). In nature, 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

weakening of the hydrophobic van der Waals forces responsible for the close packing of the inner membrane helices in the closed state of the channel. The effect is reinforced if charged or bulky groups are introduced because of electrostatic repulsion and steric factors, respectively. This is reflected in the energetics of the gating transitions and leads to the opening of the channel even in the absence of tension. On the basis of this principle, the 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

nitrophenol moiety, which is removed upon illumination. This affords a free amino moiety which, depending on the pH, can get protonated and trigger the opening of the channel. The ability to control the release of liposome content with reversible channel opening and closing was demonstrated under the influence of UV and visible light, respectively (Kocer et al. (2005) Science 309, 755-758) and in response to a decrease in pH using channels modified to respond directly to pH as well as channels engineered (using masked reagents) to respond to pH only after

illumination (Kocer et al. (2006) Angew. Chem. Int. Ed. 45, 3126-3130). Rationally designed chemical modulators convert a bacterial channel protein into a pH-sensory valve. This methodology is also disclosed in PCT patent applications WO2005051902, WO03084508 and WO03000233. The activateable liposomal drug delivery systems discussed above allow an increased level of control over drug release from liposomes using light-, pH-, or thiol-mediated release. These mechanisms provide an additional selectivity for a specific local environment (e.g. low pH in certain tumors, high intracellular thiol concentration) or localized illumination. However, the drawback of this additional selectivity is that these tools cannot be universally applied. Due to the low penetration depth of light, the technology of controlled drug delivery is hmited to disorders situated at or near the body surface or in combination with a catheter-light pipe. With respect to the pH-activateable liposomes, many potential targets do not have an extracellular pH that is significantly different from surrounding non-target tissue. Intracellular pH-mediated activation is possible, but does requires efficient cellular uptake, which may not be feasible as it depends on several variables, composition and targets. Likewise, thiol- based activation requires efficient cellular uptake of the liposome.

However, this requires that ideally most target cells can be reached by the administered liposome. Following intracellular release, the drug needs to be able to escape from the endosome or lysosome intact and, in the case of heterogeneous targeted tissue, it is advantageous if the drug can escape from the cell to target neighbouring cells that have not bound liposomes. Extracellular drug distribution may be of further importance in tumors that are difficult to penetrate due e.g. to elevated interstitial pressure, which impedes convectional flow. This is especially a problem for large Constructs like liposomes.

In addition, the approach to use Constructs that should be cleavable by endogenous elements in the target while stable to endogenous elements en route to the target may have limited applicability.

In order to avoid the drawbacks of current liposome activation, it has been proposed in WO2009144659 to make use of an abiotic, bio- orthogonal chemical reaction, viz. the Staudinger reaction, to provoke activation of a masked liposome. Briefly, in the introduced concept, 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 species that reacts with the Trigger moiety in the masked liposome, to induce release of the Trigger from the protein, leading to opening up of the channel protein and release of the liposome- entrapped drugs. In more detail, a cysteine in the channel protein was modified with moiety comprising an amine, which was masked by an azide Trigger. Upon Staudinger reaction with a phosphine Activator, the Trigger is removed, unveiling the amine moiety, which due to its pKa is

protonated, leading to drug release from the liposome. The presented

Staudinger approach for this concept, however, 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.

It is desirable to be able to activate 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. The use of a biocompatible chemical reaction that does not rely on endogenous activation mechanisms (eg pH, thiols) for selective liposome activation would represent a powerful new tool in cancer therapy. 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.

Summary of the invention

In order to better address one or more of the foregoing desires, the invention presents, in one aspect, 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. In another aspect, 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.

In a further aspect, the invention is a liposomal composition, comprising:

(a) a Construct of a liposome comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a lipid linkage and, optionally, a Targeting Agent attached to the lipid linkage

(b) a Masking Moiety for the liposome;

(c) an eight-membered non-aromatic cyclic alkenylene group,

preferably a cyclooctene group, and more preferably a trans- cyclooctene group, linked to both the Construct and the Masking Moiety.

In a still further aspect, 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.

Detailed description of the invention

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. As will be clear from the explanation that follows, the present use of the rDA reaction is irrespective of the precise chemical components further present. Thus, e.g., liposomes are known, as is their use as a carrier for therapeutic agents. Also 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

activated. Or, alternatively, results in eliminating the deactivation of the liposome.

It should be noted that where, in this disclosure, it is spoken of "a cycloalkenylene group", "a cyclooctene group", or "a trans -cyclooctene group" these expressions refer to such a group by itself, or linked to a further chemical structure. In the art of chemistry, this is also indicated with the term "moiety". E.g., a cyclooctene group as present on the liposomes of the invention, can itself be attached to a further structure (e.g. polyethylene glycol), and can also be referred to as a "cyclooctene moiety."

Retro Diels-Alder reaction

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

Reactive Group), 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).

The exceptionally fast reaction of e.g. electron- deficient

(substituted) tetrazines with a TCO moiety results in a ligation

intermediate that rearranges to a dihydropyridazine retro Diels-Alder adduct by eliminating N2 as the sole by-product in a [4+2] Retro Diels- Alder cycloaddition. In aqueous environment, 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. Thus, the compounds and the method of the invention can be used in a living organism. Moreover, 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; Wallfahrer, U; Sauer, J,

Tetrahedron Letters, 1990, 31 (47), 6851-6854; Wijnen, JW; Zavarise, S; Engberts, JBFN, Journal Of Organic Chemistry, 1996, 61, 2001-2005; Blackman, ML; Royzen, M; Fox, JM, Journal Of The American Chemical Society, 2008, 130 (41), 13518-19), R. Rossin, P. Renart Verkerk, Sandra M. van den Bosch, R. C. M. Vulders, 1. Verel, J. Lub, M. S. Robillard, Angew Chem Int Ed 2010, 49, 3375, N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R. Weissleder, Angew Chem Int Ed 2009, 48, 7013, and Devaraj et al., Angew.Chem.Int.Ed., 2009, 48, 1-5.

It will be understood that, in a broad sense, according to the invention 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. I.e. 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.

Trigger

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.

Optionally, 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. E.g., the at least two exocyclic bonds can be the two bonds of a double bond to an oxygen (i.e. C=O). 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. Examples of the latter include strained rings such as cyclopropyl and cyclobutyl. Without wishing to be bound by theory, the inventors believe that the presence of at least two exocyclic bonds in the same plane will result in an at least partial flattening of the TCO ring, which can lead to higher reactivity in the retro-Diels-Alder reaction.

In this invention, the TCO satisfies the following formula

(la):

(la) wherein A and P each independently are CR¾ or CR^aX^D provided that at least one, and preferably not more than one, is CR^aX^D. X^D is (O-C(O))p-(LD)_n-(DD), S-C(O)-(LD)_n-(DD), O-C(S)-(LD)_n-(E>D), S-C(S)-(LD)_n- (D^D), O-S(O)-(L^D)_n-(D^D), wherein p = 0 or 1. Preferably, X^D is (O-C(O))_p- (L^D)_n-(D^D), where p = 0 or 1, preferably 1, and n = 0 or 1.

In an interesting embodiment, Y,Z,X,Q each independently are selected from the group consisting of CR¾,

C=NR^b, S, SO, SO₂, O, NR^b, and SiR¾ with at most three of Y, Z, X, and Q being selected from the group consisting of C=CR¾, C=O, C=S, and C=NR^b, wherein two R moieties together may form a ring, and with the proviso that no adjacent pairs of atoms are present selected from the group consisting of O-O, O-NR^b, S-NR^b, O-S, O-S(O), O-S(O)₂, and S-S, and such that Si is only adjacent to CR¾ or O.

In another interesting embodiment, one of the bonds PQ, QX, XZ, ZY, YA is part of a fused ring or consists of CR^a=CR^a, such that two exocyclic bonds are fixed in the same plane, and provided that PQ and YA are not part of an aromatic 5-or 6-membered ring, of a conjugated 7- membered ring, or of CR^a=CR^a; when not part of a fused ring P and A are independently CR¾ or CR^aX^D provided that at least one, and preferably not more than one, is CR^aX^D ; when part of a fused ring P and A are independently CR^a or CX^D provided that at least one, and preferably not more than one, is CX^D; the remaining groups (Y,Z,X,Q) being

independently from each other CR¾, C=CR¾, C=O, C=S, C=NR^b, S, SO, SO₂, O, NR^b, SiR¾ such that at most 1 group is C=CR¾, C=O, C=S, C=NR^b, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-NR^b, S-NR^b, O-S, O-S(O), O-S(O)₂, and S-S, and such that Si, if present, is adjacent to CR^a ₂ or O, and the CR^a ₂=CR^a ₂ bond, if present, is adjacent to CR^a ₂ or C=CR^a ₂ groups;

In some embodiments 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:

Therein 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, and D is CR¾,C=0, C=S, C=NR^b,

NR^b, O, S; or E-G is CR^a-N or CX^D-N, and D is CR¾, C=0, C=S, C=NR^b, NR^bO, or S.

Fused 4-membered rings are:

\

E^D

I I

G-M

s

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 CR^a, CX^D or N, and D,M independently from each other are CR¾, C=0, C=S, C= NR^b, C=CR¾, S, SO, SO₂, O, NR^b but no adjacent O-O or S-S groups; or

E-D is C=CR^a and G is N, CR^a, CX^D and M is CR¾, S, SO, SO₂, O, NR^b; or E-D is C=N and G is N, CR^a, CX^D and M is CR¾, S, SO, SO₂, O; or

D-M is CR^a=CR^a and E, G each independently are CR^a, CX^D or N; or D-M is CR^a=N and E is CR^a, CX^D, N, and G is CR^a or CX^D; or

E is C, G is CR^a, CX^D or N, and D, M are CR¾, S, SO, SO₂, O, NR^b, or at most one of C=O, C=S, C= NR^b, C=CR^a ₂, but no adjacent O-O or S-S groups; or E and G are C, and D and M independently from each other are CR¾, S, SO, SO₂, O, NR^b but no adjacent O-O, or S-S groups. 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 or N; K, L are CR^a; D,M form a CR^a=CR^a or CR^a=N, or D,M independently from each other are CR¾, C=0, C=S, C= NR^b, C=CR¾, S, SO, SO₂, O, NR^b but no adjacent O-O, S-S, N-S groups; J is CR¾, C=O, C=S, C= NR^b, C=CR¾, S, SO, SO₂, O, NR^b ; at most 2 N groups; or

E,G are C, CR^a, CX^D; K is N and L is CR^a; D,M form a

CR^a=CR^a bond or D,M independently from each other are CR¾, C=O, C=S, C= NR^b, C=CR¾, NR^b but no adjacent O-O, S-S, N-S groups; J is CR¾, C=O, C=S, C= NR^b, C=CR¾, S, SO, SO₂, O, NR^b ; at most 2 N groups; or E,G are C, CR^a, CX^D; K and L are N; D,M, J independently from each other are CR¾, C=O, C=S, C= NR^b, C=CR¾ groups;

Fused aromatic 5-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 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 is C; L, K, D , M are independently from each other CR^a or N.

Fused planar conjugated 7-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 CR^a; J is S, O, CR¾, NR^b.

(L^D)_n is an optional linker, with n = 0 or 1, preferably linked to T^R via S, N, NH, or O, wherein these atoms are part of the linker, which may consist of multiple units arranged linearly and/or branched. 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, F each independently denotes H, or a substituent selected from the group consisting of alkyl, F, CI, Br, or I.

Without wishing to be bound by theory, 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).

In several alternative embodiments, with reference to formula (la), said release or elimination is believed to be mediated by a strain release mechanism.

Therein, in Embodiment 1, one of the bonds PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY consists of -CR^aX^D-CR^aY^D-, the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR¾, S, O, SiR^c2, such that P and A are CR^a2, 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 CR¾ 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),

S-C(S)-(L^D)_n-(D^D), NR^d-C(O)-(L^D)_n-(D^D), NR^d-C(S)-(L^D)_n-(D^D), and then is NHR^d, OH, SH; or X^D is C(O)-(L^D)_n-(D^D), C(S)-(L^D)_n-(D^D); and then Y° is CR^d ₂NHR^d, CR^d ₂OH, CR^d ₂SH, NH-NH₂, O-NH₂, NH-OH.

Preferably X^D is NR^d-C(O)-(LD)_N-(D^D), and Y^D is NHR^d.

In this Embodiment 1, 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^aX^D-CR^aY^D-, then X^D and Y^D are preferably positioned trans relative to each other; if ZX or XZ is -CR^aX^D-CR^aY^D -, then X^D and Y^D are preferably positioned cis relative to each other.

In Embodiment 2, A is CR^aX^D and Z is CR^aY^D, or Z is CR^aX^D and A is CR^aY^D, or P is CR^aX^D and X is CR^aY^D, or X is CR^aX^D and P is CR^aY^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 CR¾, S, O, SiR^c2, such that P and A are CR^a2, 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 CR¾ or O; X^D is O-C(O)-(L^D)_n-(D^D), S-C(O)-(L^D)_n-(D^D), O-C(S)-(L^D)_n-(D^D), S-C(S)-(L^D)_n-(D^D), NR^d-C(O)-(L^D)_n-(D^D), NR^d-C(S)-(L^D)_n-(D^D), and then ^ is NHR^d, OH, SH, CR^d ₂NHR^d, CR^d ₂OH, CR^d ₂SH, NH-NH₂, O-NH₂, NH- OH; or X^D is CR^d ₂-O-C(O)-(LD)_N-(D^D), CR^d ₂-S-C(O)-(LD)_N-(D^D), CR^d ₂-O-C(S)- (L^D)_n-(D^D), CR^d ₂-S-C(S)-(LD)_N-(D^D), CR^d ₂-NR^d-C(O)-(LD)_N-(D^D), CR^d2-NR^d- C(S)-(L^D)_n-(D^D); and then Y° is NHR^d, QH, SH; or X^D is C(O)-(L^D)_n-(D^D), C(S)-(L^D)_n-(D^D); and then Y° is CR^d ₂NHR^d, CR^d ₂OH, CR^d ₂SH, NH-NH₂, O- NH₂, NH-OH. Preferably X^D is NR^d-C(O)-(L^D)_n-(D^D), and Y^D is NHR^d.

In Embodiment 3, A is CR^aY° and one of P, Q, X, Z is CR^aX^D, or P is CR^aY^D and one of A, Y, Z, X is CR^aX^D, or Y is CR^aY^D and X or P is CR^aX^D, or Q is CR^aY^D and Z or A is CR^aX^D, or either Z or X is CR^aY^D and A or P is CR^aX^D, such that X^D and Y¹³ 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 CR¾, 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 CR¾ or O.

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); Y^D is CR^d ₂NHR^d, CR¾OH, CR¾SH, NH-NH₂, O- NH₂, NH-OH; p = 0 or 1.

Preferably X^D is (O-C(O))_p-(L^D)_n-(D^D), with p=l, and Y^D is CRd₂NHRd.

In Embodiment 4, P is CR^aY^D and Y is CR^aX^D, or A is CR^aY^D and Q is CR^aX^D, or Q is CR^aY^D and A is CR^aX^D, or Y is CR^aY^D and P is CR^aX^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.

X^D is (O-C(O))p-(LD)_n-(DD), S-C(O)-(LD)_n-(DD), O-C(S)-(LD)_n- (D^D), S-C(S)-(L^D)_n-(D^D); Y^D is NHR^d, QH, SH; p = 0 or 1.

Preferably X^D is (O-C(O))_p-(L^D)_n-(D^D), with p=l, and Y^D is

NHRd.

In Embodiment 5, Y is Y^D and P is CR^aX^D, or Q is Y¹⁵ and A is CR^aX^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-

(D^D), S-C(S)-(L^D)n-(E>^D), NR^d-C(0)-(L^D)_n-(DD), NR^d-C(S)-(L^D)_n-(DD), C(0)- (L^D)_n-(D^D), C(S)-(L^D)_n-(DD); Y^D is NH; p = 0 or 1.

Preferably X^D is NR^d-C(0)-(L^D)_n-(DD) or (0-C(0))_p-(L^D)_n-(DD), with p = 0 or 1.

In Embodiment 6, Y is Y^D and P or Q is X^D, or Q is Y⁰ and A or Y is X^D; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR¾, S, O, SiR¾ such that P and A are CR^a2, 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 CR¾ or O.

X^D is N-C(O)-(L^D)_n-(DD), N-C(S)-(L^D)_n-(DD); Y^ is NH;

Preferably X^D is N-C(O)-(L^D)_n-(D^D)

Also herein, (L^D)_n is an optional linker, with n = 0 or 1, preferably linked to T^Rvia S, N, NH, or O, wherein these atoms are part of the linker, which may consist of multiple units arranged linearly and/or branched. 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.

It is preferred that when D^D is bound to T^R or L^D via NH, this NH is a primary amine (-NH2) residue from D^D, and when D^D is bound via N, this N is a secondary amine (-NH-) residue from D^D. Similarly, it is preferred that when D^D is bound via O or S, said O or S are, respectively, a hydroxy! (-OH) residue or a sulfhydryl (-SH) residue from D^D.

It is further preferred that said S, N, NH, or O moieties comprised in D^D are bound to an aliphatic or aromatic carbon of D^D.

It is preferred that when L^D is bound to T^R via NH, this NH is a primary amine (-NH2) residue from L^D, and when L^D is bound via N, this N is a secondary amine (-NH-) residue from L^D. Similarly, it is preferred that when L^D is bound via O or S, said O or S are, respectively, a hydroxyl (-OH) residue or a sulfhydryl (-SH) residue from L^D.

It is further preferred that said S, N, NH, or O moieties comprised in L^D are bound to an aliphatic or aromatic carbon of L^D.

Where reference is made in the invention to a 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.

By way of further clarification, if p=0 and n=0, the species D^D directly constitutes the leaving group of the elimination reaction, and if p=0 and n=l, the self-immolative linker constitutes the leaving group of the elimination. 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).

Nevertheless, typical but non-limiting examples of self-immolative linkers L^D are benzyl-derivatives, such as those drawn below. On the right, 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.

By substituting the benzyl groups of aforementioned self-immolative linkers L^D , preferably on the 2- and/or 6-position, it may be possible to tune the rate of release of the species D^D, caused by either steric and/or electronic effects on the intramolecular elimination reaction. Synthetic procedures to prepare such substituted benzyl-derivatives are known to the skilled person (see for example Green wald et al, J. Med. Chem., 1999, 42, 3657-3667 and Thornthwaite et al, Polym. Chem., 2011, 2, 773-790). Some examples of substituted benzyl-derivatives with different release

In a preferred embodiment, the TCO of formula (la) is an all-carbon ring. In another preferred embodiment, the TCO of formula (la) is a

heterocyclic carbon ring, having of one to two oxygen atoms in the ring, and preferably a single oxygen atom. Each R^a as above-indicated can independently be H, alkyl, aryl, OR', SR', S(=O)R"', S(=O)₂R'", S(=O)₂NR'R", Si-R'", Si-O-R'", OC(=O)R"', SC(=O)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, SO₂H, SO₃H, SO₄H, PO₃H, PO₄H, NO, NO₂, CN, OCN, SCN, NCO, NCS, CF₃, CF₂-R', NR'R", C(=O)R', C(=S)R', C(=O)O-R', C(=S)O-R', C(=O)S-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'C(=O)-R"', NR'C(=S)-R"', NR'C(=O)O-R"', NR'C(=S)O-R"', NR'C(=O)S-R"', NR'C(=S)S-R"', OC(=O)NR'-R"', SC(=O)NR'-R"',

OC(=S)NR'-R"', SC(=S)NR'-R"', NR'C(=O)NR"-R", NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R'" independently being aryl or alkyl;

Each R^b as above indicated is independently selected from the group consisting of H, alkyl, aryl, O-aryl, O-alkyl, OH, C(=O)NR'R" with R' and R" each independently being H, aryl or alkyl, R'CO-alkyl with R' being H, alkyl, and aryl;

Each R^c as above indicated is independently selected from the group consisting of H, alkyl, aryl, O-alkyl, O-aryl, OH;

Each R^d as above indicated is independently selected from H, Ci-6 alkyl and Ci-6 aryl;

wherein two or more R^a >^b >^c >^d moieties together may form a ring;

Preferably, each R^a is selected independently from the group consisting of H, alkyl, O-alkyl, O-aryl, OH, C(=O)O-R', C(=O)NR'R", NR'C(=0)-R"', with R' and R" each independently being H, aryl or alkyl, and with R'" independently being alkyl or aryl.

In all of the above embodiments, 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. The synthesis of 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.

In a preferred embodiment, the ircms-cyclooctene moiety satisfies formula (lb):

(lb)

wherein, in addition to the optional presence of at most two exocyclic bonds fixed in the same plane, each R^a independently denotes H, or, in at most four instances, a substituent selected from the group consisting of alkyl, aryl, OR', SR', S(=O)R"', S(=O)₂R'", S(=O)₂NR'R", Si-R'", Si-O-R'", OC(=O)R"', SC(=O)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, SO₂H, SO₃H, SO₄H, PO3H, PO4H, NO, NO₂, CN, OCN, SCN, NCO, NCS, CF₃, CF₂-R', NR'R", C(=O)R', C(=S)R', C(=O)O-R', C(=S)O-R', C(=O)S-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'C(=O)-R"', NR'C(=S)-R"', NR'C(=O)O-R"', NR'C(=S)O-R"', NR'C(=O)S-R"', NR'C(=S)S-R"', OC(=O)NR'-R"', SC(=0)NR'-R"', OC(=S)NR'-R"', SC(=S)NR'-R"', NR'C(=0)NR"-R",

NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R'" independently being aryl or alkyl;

Each R^e as above indicated is independently selected from the group consisting of H, alkyl, aryl, OR', SR', S(=0)R"', S(=O)₂R'", Si-R'", Si-O-R'", OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, SO₂H, SO₃H, PO₃H, NO, NO2, CN, CF₃, CF2-R', C(=O)R', C(=S)R', C(=O)O-R', C(=S)0-R', C(=0)S-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'C(=0)-R"', NR'C(=S)- R'", NR'C(=0)0-R"', NR'C(=S)0-R"', NR'C(=0)S-R"', NR'C(=S)S-R"', NR'C(=O)NR"-R", NR'C(=S)NR"-R", CR'NR", with each R and each R" independently being H, aryl or alkyl and R'" independently being aryl or alkyl;

wherein two R^{a e} moieties together may form a ring; wherein one R^{a e} or the self-immolative linker L^D, is bound, optionally via a spacer or spacers S^p, to the species Y^M, and wherein T and F each independently denote H, or a substituent selected from the group consisting of alkyl, F, CI, Br, and I, and X^D is (0-C(0))_p-(LD)_n-(DD), S-C(O)- (L^D)_n-(D^D), O-C(S)-(LD)_n-(DD), S-C(S)-(LD)_n-(D^D), O-S(O)-(LD)n-(E>D), wherein p = 0 or 1. Preferably, X^D is (O-C(O))_p-(L^D)_n-(D^D), where p = 0 or 1, preferably 1, and n = 0 or 1.

Preferably, each R^a and each R^e is selected independently from the group consisting of H, alkyl, O-alkyl, O-aryl, OH, C(=O)O-R', C(=O)NR'R", NR'C(=O)-R", with R and R" each independently being H, aryl or alkyl, and with R'" independently being alkyl or aryl.

In the foregoing dienophiles, it is preferred that 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. In this respect, reference is made to 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. In these isomers the OH substituent is either in the equatorial or axial position.

In a preferred embodiment, with reference to formula (la), for structures where the substituents of A and/or P, such as X^D and Y^D, can be either in the axial or the equatorial position, the substituent is in the 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:

= rest of attached L^D-D^D, comprising Y^M or S^p-Y^M

In a further preferred embodiment, the dienophile is a compound selected

= rest of attached L^D-D^D, wherein L^D comprises Y or S^P-Y In alternative embodiments, the dienophile is a compound selected from

^•™^w = rest of attached L^D-D^D, wherein L^D comprises Y^M or S^P-Y^M

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.

Below a reaction scheme is given for a [4+2] Diels-Alder reaction between the (3,6)-di-(2-pyridyl)-s-tetrazine diene and a trans- cyclooctene dienophile, followed by a retro Diels Alder reaction in which the product and dinitrogen is formed. The reaction product may

tautomerize, and this is also shown in the scheme. Because the trans- cyclooctene derivative does not contain electron withdrawing groups as in the classical Diels Alder reaction, this type of Diels Alder reaction is distinguished from the classical one, and frequently referred to as an "inverse electron demand Diels Alder reaction". In the following text the sequence of both reaction steps, i.e. the initial Diels-Alder cyclo-addition (typically an inverse electron demand Diels Alder cyclo-addition) and the subsequent retro Diels Alder reaction will be referred to in shorthand as "retro Diels Alder reaction" or "retro-DA". It will sometimes be abbreviated as "rDA" reaction. The product of the reaction is then the retro Diels-Alder adduct, or the rDA adduct.

In a general sense, 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. The dienophiles of formula (la) have the advantage that they react (and effectuate D^D release) with substantially any diene.

Without wishing to be bound by theory, 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. Particularly, with reference to the aforementioned cascade-mediated mechanism, 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.

Alternatively, with reference to the aforementioned strain release mechanism, 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. Particularly, 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.

The general concept of using the retro-Diels Alder reaction in liposome activation is illustrated in Scheme 1.

Scheme 1: general scheme of activation of a masked liposome according to this invention.

Tri er

Trigger-Construct conjugate

Activation, retro Diels-Alder reaction (-N2)

retro Diels-Alder adduct

D^D release

Herein the "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°.

In this scheme "TCO" stands for ircms-cyclooctene. The term trans -cyclooctene is used here as possibly including one or more hetero- atoms, and particularly refers to a structure satisfying formula (la). In a broad sense, 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.

It will be understood that in Scheme 1 in the retro Diels- Alder adduct as well as in the end product, the indicated TCO group and the indicated diene group are the residues of, respectively, the TCO and diene groups after these groups have been converted in the retro Diels- Alder reaction.

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. Ideally, 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, however, 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. 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.

Reflecting the suitability of the rDA reaction, 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. In connection herewith, 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. pyridazine) aromatic group, (c) the nature of the rDA adduct that may bear a dihydro pyridazine group that is weakly basic and that may therefore catalyze elimination reactions. Alternatively, 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. A nucleophilic site in the dienophile prior to rDA reaction is locked within the specific conformation of the dienophile and is therefore not properly positioned to react intramolecularly and to thereby release D^D. In contrast, and due to the changed nature of the eight membered ring, this

nucleophilic site is properly positioned within the rDA adduct and will react intramolecularly, thereby releasing D^D. According to the above, but without being limited by theory, we believe that D^D release is mediated by strain-release of the TCO-dienophile after and due to the rDA reaction with the diene Activator.

In a broad sense, 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.

It is to be emphasized that the invention is thus of a scope well beyond specific chemical structures. In a broad sense, 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.

Reflecting this, 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 fact that the reaction is bio-orthogonal, and that many structural options exist for the reaction pairs, will be clear to the skilled person. E.g., 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. Hereinafter this eight-membered ring moiety will be defined as a ircms-cyclooctene moiety, for the sake of legibility, or abbreviated as "TCO" moiety. It will be understood that the essence resides in the possibility of the eight-membered ring to act as a dienophile and to be released from its conjugated D^D upon reaction. The skilled person is familiar with the fact that the dienophile activity is not necessarily dependent on the presence of all carbon atoms in the ring, since also heterocyclic monoalkenylene eight-membered rings are known to possess dienophile activity.

Thus, in general, 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. I.e., 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.

Other than is the case with e.g. medicinally active substances, where the in vivo action is often changed with minor structural changes, the present invention first and foremost requires the right chemical reactivity combined with an appropriate design of the D^D- conjugate. Thus, the possible structures extend to those of which the skilled person is familiar with that these are reactive as dienophiles.

It should be noted that, depending on the choice of

nomenclature, the TCO dienophile may also be denoted E -cyclooctene. With reference to the conventional nomenclature, it will be understood that, as a result of substitution on the cyclooctene ring, depending on the location and molecular weight of the substituent, the same cyclooctene isomer may formally become denoted as a Z-isomer. In the present invention, 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. The terms "trans -cyclooctene" (TCO) as well as E-cyclooctene are used interchangeably and are maintained for all dienophiles according to the present invention, also in the event that substituents would formally require the opposite nomenclature. I.e., the invention relates to cyclooctene in which carbon atoms 1 and 6 as numbered below are in the E (entgegen) or trans position.

Formula (1)

The present invention will further be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-hmiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

It is furthermore to be noticed that the term "comprising", used in the description and in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

In several chemical formulae below reference is made to

"alkyl" and "aryl." In this respect "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, and "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" groups also include "alkylaryl" or "arylalkyl" groups (simple example: benzyl groups). The number of carbon atoms that an "alkyl", "aryl", "alkylaryl" and "arylalkyl" contains can be indicated by a designation preceding such terms (i.e. 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. In several formulae, groups or substituents are indicated with reference to letters such as "A", "B", "X", "Υ', 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.

In all embodiments of the invention as described herein, alkyl is preferably lower alkyl (C 1.4 alkyl), and each aryl preferably is phenyl.

The TCO is preferably an all-carbon TCO.

Activator-induced release

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^Dor several D^Ds, 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.

Without wishing to be bound by theory, the inventors believe that 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). This

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^Ds. 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.

Without wishing to be bound by theory, 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. In one scenario, 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. Then, 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 skilled person will understand that 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. Thus, the formation of a species more stable than the retro Diels- Alder adduct is the driving force for the (cascade) elimination reaction. In any case, and in whatever way the process is viewed, the D^D (here the amine D^D-NH2) 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. Without wishing to be bound by theory, 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. Here, 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.

As discussed above, in this invention, 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) reacts with an electrophilic site on the same TCO (particularly from the X^D group in this Trigger, vide supra) after this TCO reacts with the Activator to form an rDA adduct. 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

conformational freedom compared to the eight membered ring in the TCO prior to the rDA reaction, allowing the nucleophilic reaction to occur, thereby releasing the D^D as a leaving group. The intramolecular

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,

conformation ally restrained TCO ring. 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. In a preferred embodiment the TCO ring is in the crown conformation. The example below illustrates the release mechanism pertaining to this invention.

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. As 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. It may also be possible that the 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. In this case, 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

reformation of the nucleophilic site.

Without wishing to be bound by theory, the above example illustrates how the conformational restriction and the resulting

unfavorable positioning of the nucleophilic and electrophilic site in the TCO trigger is relieved following rDA adduct formation, leading to an elimination/cyclization reaction and release.

With respect to the nucleophilic site on the TCO, one has to consider that the site must be able to act as a nucleophile under conditions that may exist inside the (human) body, so for example at physiological conditions where the pH = ca. 7.4, or for example at conditions that prevail in malignant tissue where pH-values may be lower than 7.4. Preferred nucleophiles are amine, thiol or alcohol groups, as these are generally most nucleophilic in nature and therefore most effective.

The combination of and reaction between the TCP-Trigger and the

Activator

It should be noted that in cases of release of 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.

Release of D^D with other functionalities may also be possible (e.g. thiol functinalized 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.

Hereunder, some nonlimiting model combinations of 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, whether or not via a linker, is preferably attached to a carbon atom that is adjacent to the double bond in the TCO ring.

L^D = self-immolatoive linker comprising Υ'

The above example of urethane (or carbamate) substituted TCOs gives release of an amine functional D^D from the adduct. The tetrazine Activator is symmetric and electron deficient.

L^D = self-immolatoive linker comprising Y^M R_i = H, and R₂ = Bn-NH₂, or R-, = Bn-NH₂ and R₂ = H

The above examples of 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

The above example of urethane (or carbamate) TCOs gives release of an amine functional D^D from the adduct. The tetrazine Activator is symmetric and electron sufficient.

The following schemes depict non-limiting examples illustrative for the various strain release mechanisms that can be made to apply on the basis of the choice for the rDA reaction for activating a Trigger- D^D conjugate.

embodiment 2

alternative:

embodiment 3 tetrazine

embodiment 4

tetrazine

embodiment 5

embodiment 6

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).

Generally, the Activator is a molecule comprising a

heterocyclic moiety comprising at least 2 conjugated double bonds.

Preferred dienes are given below, with reference to formulae

(2)-(4).

In formula (2) R¹ is selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', OR', SR', C(=O)R', C(=S)R', C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R", C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", NR'C(=O)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl; A and B each independently are selected from the group consisting of alkyl-substituted carbon, aryl substituted carbon, nitrogen, N⁺O-, N⁺R with R being alkyl, with the proviso that A and B are not both carbon; X is selected from the group consisting of O, N-alkyl, and C=O, and Y is CR with R being selected from the group consisting of H, alkyl, aryl, C(=O)OR', C(=O)SR', C(=S)OR', C(=S)SR', C(=O)NR'R" with R' and R" each independently being H, aryl or alkyl.

(3)

A diene particularly suitable as a reaction partner for cyclooctene is given in formula (3), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R', NO2, OR', SR', C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=O)₂R'", S(=O)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=O)NR"R",

NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl; A is selected from the group consisting of N-alkyl, N-aryl, C=O, and CN-alkyl; B is O or S; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(=O)R', CC(=S)R', CS(=0)R', CS(=0)₂R"', CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R', CC(=O)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and

(4)

Another diene particularly suitable as a reaction partner for cyclooctene is diene (4), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R, NO, NO₂, OR, SR, CN, C(=O)R, C(=S)R, OC(=O)R", SC(=O)R", OC(=S)R", SC(=S)R", S(=O)R, S(=O)₂R", S(=O)₂OR, PO3RR", S(=O)₂NRR", C(=O)O-R', C(=0)S- R, C(=S)O-R, C(=S)S-R, C(=O)NRR", C(=S)NRR", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R",

NR'C(=O)NR"R", NR'C(=S)NR"R" with each R and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl; A is selected from the group consisting of N, C-alkyl, C-aryl, and N⁺0-; B is N; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(=0)R', CC(=S)R', CS(=0)R', CS(=0)₂R"', CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R', CC(=0)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and

(5) (6) (7)

According to the invention, 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.

The 1,2-diazine is given in (5), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R, NO2, OR, SR, C(=O)R, C(=S)R, OC(=O)R", SC(=O)R", OC(=S)R", SC(=S)R", S(=O)R, S(=O)₂R", S(=O)₂NRR", C(=O)O-R, C(=O)S-R,

C(=S)O-R, C(=S)S-R, C(=O)NRR", C(=S)NRR", NRR", NRC(=O)R", NRC(=S)R", NRC(=O)OR", NRC(=S)OR", NRC(=O)SR", NRC(=S)SR", OC(=O)NRR", SC(=O)NRR", OC(=S)NRR", SC(=S)NRR",

NRC(=O)NR"R", NRC(=S)NR"R" with each R and each R" independently being H, aryl or alkyl, and R" independently being aryl or alkyl; X and Y each independently are selected from the group consisting of O, N-alkyl, N-aryl, C=O, CN-alkyl, CH, C-alkyl, C-aryl, CC(=O)R, CC(=S)R, CS(=O)R, CS(=O)₂R", CC(=O)O-R, CC(=O)S-R', CC(=S)O-R, CC(=S)S-R,

CC(=O)NRR", CC(=S)NRR", with R and R" each independently being H, aryl or alkyl and R" independently being aryl or alkyl, where X-Y may be a single or a double bond, and where X and Y may be connected in a second ring structure apart from the 6-membered diazine. Preferably, X-Y represents an ester group (X = O and Y = C=0; X-Y is a single bond) or X- Y represents a cycloalkane group (X = CR' and Y = CR"; X-Y is a single bond; R' and R" are connected), preferably a cyclopropane ring, so that R' and R" are connected to each other at the first carbon atom outside the 1,2-diazine ring.

The 1,2,4-triazine is given in (6), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=0)₂R"', S(=O)₂NR'R", C(=O)O-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R",

NR'C(=O)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl; X is selected from the group consisting of CH, C-alkyl, C-aryl, CC(=O)R',

CC(=S)R', CS(=0)R', CS(=0)₂R"', CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R', CC(=O)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R" independently being aryl or alkyl.

The 1,2,4,5-tetrazine is given in (7), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R, NO, NO2, OR, SR, CN, C(=O)R, C(=S)R, OC(=O)R", SC(=O)R", OC(=S)R", SC(=S)R", S(=O)R, S(=O)₂R", S(=O)₂OR, PO₃R^*R",

S(=O)₂NRR", C(=O)O-R, C(=O)S-R, C(=S)O-R, C(=S)S-R', C(=O)NRR", C(=S)NRR", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR",

NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=O)NR"R", NR'C(=S)NR"R" with each R and each R" independently being H, aryl or alkyl, and R" independently being aryl or alkyl.

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. For example, R¹ and/or R² 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 NO₂, F, CI, CF₃, CN, COOR, CONHR, CONR, COR, SO2R, SO2OR, SO2NR2, PO3R2, NO, Ar, wherein R is H or Ci-C₆ 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 R¹ and/or R² groups are not necessarily electron withdrawing, and even when R¹ and/or R² are in fact electron donating. Electron donating groups are for example OH, OR', SH, SR', NH₂, NHR', NR'R", NHC(=O)R", NR'C(=O)R", NHC(=S)R", NR'C(=S)R", NHSO2R", NR'SO₂R" with R' and R" each independently being alkyl or aryl groups. 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.

According to the invention, 1,2,4,5-tetrazines with two electron withdrawing residues, or those with one electron withdrawing residue and one residue that is neither electron withdrawing nor donating, are called electron deficient. In a similar way, 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 R¹ and R² 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.

We have tested several 1,2,4,5-tetrazines with respect to their ability as Activator to release a model D^D (e.g. benzyl amine, phenol) from a Trigger- D^D conjugate, and we have found that tetrazines that are electron deficient, electron sufficient or neither electron deficient nor electron sufficient are capable to induce the D^D release. Furthermore, both symmetric as well as asymmetric tetrazines were effective.

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.

In the following paragraphs specific examples of 1,2,4,5- tetrazine Activators according to the second embodiment of this invention will be highlighted by defining the R¹ and R² residues in formula (7).

Symmetric electron deficient 1,2,4,5-tetrazines with electron withdrawing residues are for example those with R¹ = R² = H, 2-pyridyl, 3- pyridyl, 4-pyridyl, 2,4-pyrimidyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,3,4-triazyl or 2,3,5-triazyl. Other examples are those with R¹ = R² = phenyl with COOH or COOMe carboxylate, or with CN nitrile, or with CONH₂,

CONHCH3 or CON(CH₃)₂ amide, or with SO₃H or SO₃Na sulfonate, or with SO2NH2, SO2NHCH3 or SO₂N(CH₃)2 sulfonamide, or with PO3H2 or PO3Na2 phosphonate substituents in the 2-, 3- or 4- position of the phenyl group, or in the 3- and 5-positions, or in the 2- and 4-positions, or in the 2,- and 6-positions of the phenyl group. 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.

Symmetric electron sufficient 1,2,4,5-tetrazines with electron donating residues are for example those with R¹ = R² = OH, OR', SH, SR', NH₂, NHR', NR'2, NH-CO-R', NH-SO-R', NH-SO2-R', 2-pyrryl, 3-pyrryl, 2- thiophene, 3-thiophene, where R' represents a methyl, ethyl, phenyl or tolyl group. Other examples are those with R¹ = R² = phenyl with OH, OR', SH, SR', NH₂, NHR', NR'₂, NH-CO-R', NR"-CO-R', NH-SO-R' or NH-SO2-R' substituent(s), where R' represents a methyl, ethyl, phenyl or tolyl group, where R" represents a methyl or ethyl group, and where the substitution is done on the 2- or 3- or 4- or 2- and 3- or 2- and 4- or 2- and 5- or 2- and 6- or 3- and 4- or 3- and 5- or 3-, 4- and 5-position(s). See below for some examples of these structures.

Symmetric 1,2,4,5-tetrazines with neither electron withdrawing nor electron donating residues are for example those with R = R² = phenyl, methyl, ethyl, (iso)propyl, 2,4-imidazyl, 2,5-imidazyl, 2,3- pyrazyl or 3,4-pyrazyl. Other examples are those where R¹ = R² = a hetero(aromatic) cycle such as a oxazole, isoxazole, thiazole or oxazoline cycle. Other examples are those where R¹ = R² = a phenyl with one electron withdrawing substituent selected from COOH, COOMe, CN, CONH₂, CONHCH3, CON(CH₃)₂, SO3H, SO₃Na, SO2NH2, SO2NHCH3, SO2N(CH3)2, PO3H2 or PO3Na2 and one electron donating subsituent selected from OH, OR', SH, SR', NH₂, NHR', NR'₂, NH-CO-R', NR"-CO-R', NH-SO-R' or NH-SO2-R' substituent(s), where R' represents a methyl, ethyl, phenyl or tolyl group and where R" represents a methyl or ethyl group. 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. Yet other examples are those where R¹ = R² = a pyridyl or pyrimidyl moiety with one electron donating subsituent selected from OH, OR', SH, SR', NH₂, NHR', NR'₂, NH-CO-R', NR"-CO-R', NH-SO-R' or NH-SO2-R' substituents, where R' represents a methyl, ethyl, phenyl or tolyl group and where R" represents a methyl or ethyl group. See below for some examples.

In case asymmetric 1,2,4,5-tetrazines are considered, one can choose any combination of given R¹ and R² residues that have been highhghted and listed above for the symmetric tetrazines according to formula (7), provided of course that R¹ and R² are different. Preferred asymmetric 1,2,4,5-tetrazines are those where at least one of the residues R¹ or R² is electron withdrawing in nature. Find below some example structures drawn.

X = CH₃ X = CH₃ X = CH₃ X = CH₃ X = CH₃

Y = C, or Y = C, or

Y = N Y = N

Further considerations regarding the 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.

According to the invention, the Activator, e.g. a 1,2,4,5- tetrazine, 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

hydrophilic/hydrophobic balance of the Activator molecule at physiological pH. As is known in the art, 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. Log D values can be predicted fairly adequately for most molecules, and log D values of Activators can be tuned by adding or removing polar or apolar groups in their designs. Find below some Activator designs with their corresponding calculated log D values (at pH = 7.4). Note that addition of methyl, cycloalkylene, pyridine, amine, alcohol or sulfonate groups or deletion of phenyl groups modifies the log D value, and that a very broad range of log D values is accessible.

3.02 1 .33 0.58 ^■ 2.22 0.69 ^■ 2.85 1 .1 ?

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'). Alternatively, 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. Examples of 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.

Depending on the position of the Trigger-Construct (e.g.

inside the cell or outside the cell; specific organ that is targeted) 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. In case a targeting agent is applied, it is preferred that it is a simple moiety (i.e. a short peptide or a simple sugar). According to the invention, 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. For example, the Activator may be modified with dye moieties or fluorescent moieties (see e.g. S. Hilderbrand et al., Bioconjugate Chem., 2008, 19, 2297-2299 for 3-(4-benzylamino)- 1,2,4,5-tetrazine that is amidated with the near -infrared (NIR) fluorophore VT680), or they may be functionalized with imaging probes, where these probes may be useful in imaging modalities, such as the nuclear imaging techniques PET or SPECT. In this way, 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. For example, the Activator can be modified with DOTA (or DTP A) ligands, where these ligands are ideally suited for complexation with ^mIn³⁺-ions for nuclear imaging. In other examples, the Activator may be linked to ¹²³I or ¹⁸F moieties, that are well established for use in SPECT or PET imaging, respectively. Furthermore, when used in combination with e.g. 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:

The 1,2,4,5-tetrazine given in Formula (8a) and (8b), wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', NO₂, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R"', OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)₂R'", S(=O)₂NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR",

NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=O)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

Other preferred activators for use with Triggers based

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. Preferably, 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

Preferably, these Activators for use with Triggers based on the strain release mechanism, satisfy one of the following formulae:

R = (link to) peptide, protein,

carbohydrate, PEG, polymer

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.

Masked liposomes 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.

The general formula of the masked liposome is shown below in Formula (9a) and (9b).

(Y^M)_k— (S^p)t— (T^R)_m— (L°)_n— (D°)_r or (Y^M)k— (S^p),— (L°)n— (D°)_r

(T^R)n

(9a) (9b)

YM and D^D are Construct C^c and Masking Moiety M^M, such that when D^D is C^c, ΥΜ is M^M, and such that when D^D is M^M, ΥΜ is C^c; S^p is spacer; T^R is Trigger, and L^D is linker.

Formula (9a): r, m, k = 1; n, t = 0 or 1.

Formula (9b): r, m, n, k = 1; t = 0 or 1.

Although it has been omitted for the sake of clarity in the above formula, M^M can further comprise T^T, optionally via S^p.

It will be understood that 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.

It will be understood that 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. In the Trigger-Construct conjugate, 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. It will be understood that the invention encompasses any conceivable manner in which the dienophile Trigger is attached to the D^D. The same holds for the attachment of species Y^M to the Trigger. Methods of affecting conjugation to these species are known to the skilled person.

It will be understood that 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. Generally, this means that the bond between the D^D and the TCO, or in the event of a linker, the bond between the TCO and the linker L^D, or in the event of a self-immolative linker L^D, the bond between the linker and the TCO and between the D^D and the linker, should be cleavable. Predominantly, 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.

Exemplary 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, and "Head" refers to the lipid head group to which the lipid chain or chains are attached. Preferably, 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.

Scheme 2:

Lipid chains

Head T^T

Aspect 1 - unmasking of cell interacting moieties

With reference to Scheme 2, 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). In addition the liposome outer surface contains Targeting agents T^A, optionally further modified with a T^R-M^M (Scheme 2, D and E). In addition 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.

For completeness' sake, it is noted that 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. E.g., in the former case, one could use non-specific cell penetrating moieties, whilst in the latter case, one should use specific targeting agents. Both concepts are well-known to the skilled person and, by itself, do not affect the

functioning of the invention (which is directed to the rDA reaction using the TCO). It is preferred that when T^A is an hydrophobic polymer it is comprised in derivative D, and when T^A is an hydrophilic moiety such as a cell penetrating peptide, it is comprised in derivative E.

Where the liposomes are designed to have an extended blood circulation time, the hydrophilic polymer M^M coating is preferably composed of polymer chains of polyethyleneglycol, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,

polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide,

polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,

hydroxymethylcellulose, hydroxyethylcellulose, or polyaspartamide. The polymer chains have a preferred molecular weight of between about 500- 10,000 daltons, more preferably between 2,000 10,000 Daltons, and most preferably between 1,000 - 5,000 Daltons. In a preferred embodiment, the hydrophilic polymer is polyethyleneglycol (PEG).

In one embodiment, 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. In one preferred embodiment, the hydrophobic polymer is polypropylene oxide (PPO) having a molecular weight between 500-3,000 daltons. In addition, the 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.

In an alternative embodiment, 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.

In another embodiment, 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.

In all above described embodiments 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. As examples, 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.

In summary, 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.

In one general embodiment, the hydrophilic polymer chains are releasably attached to the liposome via the Trigger, and the contacting step includes administering an Activator to the subject.

Detailed description of Aspect 1

The present invention includes a liposome composition for fusion or interaction with a target membrane. Target membrane, as used herein, refers to a lipid bilayer membrane, for example, a bilayer membrane of a biological cell. In a preferred embodiment, 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.

With reference to Scheme 2, 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. Preferred diacyl-chain lipids for use in the present invention include diacyl glycerol, phosphatidyl ethanolamine (PE), diacylaminopropanediols, such as

disteroylaminopropanediol (DS), and phosphatidylglycerol (PG). These 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. Further exemplary hposome-forming lipids are given below.

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.

According to an important feature of the invention, 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.

mPEG-TCO-DSPE

In one embodiment (Scheme 2, D), 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.

mPEG-TCO-PPO-distearyl

PPO = polypropyleneoxide mPEG-TCO-PPO-DSPE By contrast, in derivative C (Scheme 2) hydrophilic chain M^M is directly linked to the polar head group of a vesicle-forming lipid through a chemically releasably bond T^R.

As indicated above, 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.

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.

In the latter embodiment, 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. Thus, for example, in a liposome formulation containing 5 mole percent hydrophilic polymer liposome-surface lipids, and 50% diblock lipid conjugates, 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. As seen best in Scheme 2 (B-D) , 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. An example of such a liposome- bound T^T is folic acid, as shown directly below.

Folic acid-PEG-DSPE In addition to the liposome components just described, 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. In one general embodiment, and with reference to Scheme 2, the shielded component is a Targeting Agent T^T, coupled to the polar head group of a vesicle-forming lipid. 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.

Alternatively, or in addition, 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. Such 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. Preferably, the head group of the lipid carries the positive charge. Alternatively, as comprised in derivative E in Scheme 2, 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:

poly-(D)-Arginine-DSPE; n= 8

Finally, the liposome is prepared to contain one or more therapeutic or diagnostics agents which are to be delivered to the target cell site. As used herein, 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.

As noted above, 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 procedure

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.

In accordance with one embodiment of the invention, 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

macromolecules, that do not readily penetrate a cell membrane by passive or active transport. In this general application, 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. For example, liposomes carrying an antitumor drug, such as doxorubicin, can be targeted to the vascular

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. At the same time, the T^T, attached to the distal ends of lipid-anchored hydrophilic polymer chains, are exposed for purposes of receptor binding and targeting. 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.

In one embodiment, removal of the hydrophilic polymer chains, in whole or in part, 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.

Second, it is desirable to bring the liposomes into close proximity with the target membrane. This may be done, as discussed above, by providing a shielded ligand or positively charged lipid component capable of

interacting with the target membrane, after release of the hydrophobic polymers, thus forcing the two bilayers closer together.

Aspect 2 -pH sensitive liposomes

In another aspect of the invention, and with reference to Scheme 2, liposomes are comprised of a pH-sensitive lipid (A) and of a lipid

derivatized with a hydrophilic polymer M^M, where the polymer M^M and the lipid are joined by the Trigger (Scheme 2, C). In addition or

alternatively, 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. These 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. 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.

Detailed description of Aspect 2

mPEG-TCO-DSPE

The structure directly above is an exemplary compound in accord with the invention, where 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. In embodiments where the lipid is an amine-containing lipid for use in a liposome 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. It will be appreciated that 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. In this example, upon cleavage, 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

mononuclear phagocyte system, providing a longer blood circulation lifetime for the liposomes to distribute in the organism. 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.

In one embodiment, 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. There are a variety of synthetic and naturally - occurring vesicle-forming amphipathic lipids, including and not limited to 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 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. Preferred diacyl-chain

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. In one preferred embodiment of the invention, distearolyl phosphatidyl

ethanolamine (DSPE) is used.

In an alternative embodiment, the derivatizable lipid is a pH sensitive lipid, such as DOPE.

Hydrophilic polymers suitable for derivatizing the amphipathic and other lipids include polyvinylpyrrolidone, polyvinylmethylether,

polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropyhnethacrylamide, polymethacrylamide,

polydimethylacrylamide, polyhydroxypropylmethacrylate,

polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.

In a preferred embodiment, 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

distearoylphosphatidylethanolamine (DSPE), however those of skill in the art will appreciate the wide variety of lipids that fall within this description. It will also be appreciated that the lipid can naturally include an amine group or can be derivatized to include an amine group. Other lipid moieties that do not have an acyl tail, such as cholesterolamine, are also suitable.

Accordingly, in one aspect 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.

Vesicle-Forming Lipid Component

The liposome composition of the present invention is composed primarily of vesicle-forming lipids. Such 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. 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. The above-described lipids and

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.

Additionally, 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. 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.

On the other hand, 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.

In one embodiment of the invention, the liposomes are prepared with a relatively rigid lipid to impart rigidity to the lipid bilayer. In this embodiment, the lipids forming the liposomes have a phase transition temperature of between about 37-70 °C. In a preferred embodiment, the vesicle forming lipid is distearyl phosphatidylcholine (DSPC), which has a phase transition temperature of 62 °C.

Cationic lipids

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] -Ν,Ν,Ν-trimethylammonium chloride (DOTMA); [N- (Ν',Ν'-dimethylaminoethane) carbamoyl] cholesterol (DC-Choi); and dimethyl dioctadecylammonium (DDAB). 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. For example, the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid.

Hydrophilic Polymers

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. Targeting agents T^T

The 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. For example, 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. Examples of 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. According to a particular embodiment of the present invention, the target is a protein such as a (internalizing or non- internalizing) receptor.

Examples of targets include somatostatin receptor, transferrin receptor, monoamine oxidase, muscarinic receptors,

myocardial sympatic nerve system, leukotriene receptors, e.g. on

leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti-)angiogenesis marker, gastrin receptor, GPIIb/IIIa receptor and other thrombus related receptors, fibrin,

calcitonin receptor, tuftsin receptor, integrin receptor, fibronectin,

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 tyrosine kinase, human tyrosine kinase.

In order to allow specific targeting of the above-listed targets, 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. bone marrow stem cells), drugs, polymers, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, vitamins. Examples of organic compounds envisaged within the context of the present invention are, or are derived from, estrogens, e.g. estradiol, androgens, progestins, corticosteroids, methotrexate, folic acid, and cholesterol. In a preferred embodiment, the targeting agent T^T is an antibody. According to a particular embodiment of the present invention, 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.

According to a further particular embodiment of the invention, 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. This can be achieved by selecting targets with tissue-, cell- or disease- specific expression. For example, membrane folic acid receptors mediate

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

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,

transportans, or hCT derived cell-penetrating peptides, see, e.g., Caron et al„ (2001) Mol Ther. 3(3):310-8; Langel, Cell-Penetrating Peptides:

Processes and Applications (CRC Press, Boca Raton Fla. 2002); El- Andaloussi et aL, (2005) Curr Pharm Des. 11(28):3597-611; and Deshayes et al., (2005) Cell Mol Life Sci. 62(16): 1839-49.

Masking Moieties

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.

Preferably 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. Other examples are biopolymer fragments, such as oligo- or polypeptides or polylactides. Further preferred examples are shown in Example 3.

Liposome Preparation

A. Preparation of Releasable Polymer Coating: see for example US6936272. B. Attachment of a lipid to a Hydrophilic Polymer

As described above, in one embodiment of the invention, 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.

In one embodiment of the invention, 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. For example, where the targeting moiety is an antibody or an antibody fragment, 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. Examples of such 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

dicyclohexylcarbodiimide (DCC) to form a folic acid-PEG-DSPE conjugate. This conjugate is included in the lipid mixture during liposome

preparation to form liposomes including a folic acid targeting ligand. It will be appreciated that 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. It will also be appreciated that 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. Preferably, in attaching a Targeting Agent T^T to a PEG-functionalized lipid, the T^T does not suffer any loss of activity. It is also possible to incorporate 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. As described above, 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. Generally, 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. In one preferred method, 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. In another method, 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. 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.

As another example, 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

Targeting Agent. 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). 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. In the disulfide reaction, a lipid amine, such as phosphatidyl-ethanolamine, is modified to contain 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. Like the reactions described above, this coupling reaction is preferably performed prior to incorporating the lipid into the liposome.

The above-described coupling techniques are exemplary and it will be appreciated that other suitable methods are known in the art and have been described, for example in U.S. Pat. Nos. 4,605,630, 4,731,324, 4,429,008, 4,622,294 and 4,483,929.

C. Liposome Preparation

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. Alternatively, 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 (MLVs) 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 . As noted above, 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. Thus, to optimize the formulation for a particular diblock lipid composition, one would select various ratios of the two types of hydrophilic polymer lipids, and use the highest ratio that gave good hposome stability. Preferably, the amount of diblock copolymer lipid conjugate is between 5- 100% of the total hydrophilic polymer lipid included in the lipid

preparation.

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

conjugate making up 20-100 percent of the total hydrophilic polymer lipid conjugates. Cholesterol may be included in the formulation at between about 1-50 mole percent. Another procedure suitable for preparation of the fusogenic liposomes of the present invention involves diffusion of polymer-lipid conjugates into preformed liposomes. In this method, 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

incorporated into all of the lipid layers forming the liposomes.

Alternatively, 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

accordance with the invention, 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

evaporation phase liposome preparation, are also available.

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). In a preferred embodiment of the present invention, the liposomes are extruded through a series of polycarbonate filters with pore sizes ranging from 0.2 to 0.08 μιη 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. It will be appreciated that 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.

Administration

In the context of the invention, 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. After the time period of choice has elapsed, 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.

The 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

unacceptable. 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.

It will be understood that 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.

After administration of the liposome, and before the administration of the Activator, it is possible to remove excess Liposome by means of a Clearing Agent in cases when Liposome activation in circulation is undesired and when natural Liposome clearance is insufficient. 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. In the invention, 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.

Drugs

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), or 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. 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. For example, 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. For instance, in the case of individuals with adenosine deaminase (ADA) deficiency, sequences encoding ADA may be transfeeted into stem cells or lymphocytes of such individuals.

In related applications, 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., β-glohin,

hemoglobin), for the treatment of hemophilia, sickle-ceil anemia and other blood-related diseases. Other examples of 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., malignant melanoma by transformation of, e.g., tumor-infiltrating lymphocytes, TIL), tumor suppressor genes such as p53 or KB genes that regulate apoptosis such as Be .1.-2 gene for thymidine kinase followed by ganciclovir gene for cytosine deaminase followed by 5-fluorocytosine gene for over expression of MDR-l. gene product to protect normal cells from cytotoxic chemotherapy, with genes deleterious to tumors, such as tumor necrosis factor, leukemia inhibitory factor, or various other toxic genes; hormones, such as insulin and growth hormone; transcriptional and.

translational regulatory elements; and the like. The liposomes may also encode enzymes to convert a noncytotoxic prodrug into a cytotoxic drug in tumor cells or tumor-adjacent endothelial cells.

In one embodiment of the invention, the liposomes contain a polynucleotide designed to be incorporated into the genome of the target cell or designed for autologous replication within the cell. In another embodiment, the compound entrapped in the lipid vesic!es is an

oligonucleotide segment designed for sequence-specific binding to cellular RNA or DNA. Other drugs relevant to this invention 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.

Exemplary classes of 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, 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,

calicheamicin, esperamicin, ene-diynes, and their analogues. 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,

5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat No. 4,986,988), dolastatin 14 (U.S. Pat No. 5, 138,036), dolastatin 15 (U.S. Pat No. 4,879,278), dolastatin 16 (U.S. Pat No. 6,239, 104), dolastatin 17 (U.S. Pat No. . 6,239,104), and dolastatin 18 (U.S. Pat No. . 6,239, 104), each patent incorporated herein by reference in their entirety.

In exemplary embodiments of the invention, the drug moiety is a mytomycin, vinca alkaloid, taxol, anthracycline, a cahcheamicin, maytansinoid or an auristatin.

Examples of 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

macrophage-medi ated destruction of 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.

According to a further particular embodiment of the invention, 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.

In one embodiment, 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. Therapeutic agents preferred, for treatment of multiple myeloma include melphalan, cyclophosphamide, prednisone, chlorambucil, carmustine, dexamethasone, doxorubicin, cisplatin, paelitaxel, vincristine, lomustine, and interferon. Also contemplated is intracytoplasmic delivery of plasmids, antisense oligonucleotides, and ribozymes for the treatment of cancer and. viral. infections.

In some embodiments 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. Additional Embodiment 1

With reference to formula (la) and (lb) for Triggers that function via cascade-mediated release or elimination (i.e. cascade mechanism), when p = 1 and n = 1 it is preferred that L^D is linked to T^R via N or NH or an aliphatic or aromatic carbon, wherein these atoms are part of the linker; and when p = 1 and n = 0 it is preferred that D^D is linked to T^R via N or NH or an aliphatic or aromatic carbon, wherein these atoms are part of D^D. It is further preferred that said N and NH moieties comprised in L^D or D^D are bound to an aliphatic or aromatic carbon of L^D or D^D.

With reference to formula (la) and (lb) for Triggers that function via cascade-mediated release or elimination (i.e. cascade mechanism), when p = 0 and n = 1 it is preferred that L^D is linked to T^R via S or O, wherein these atoms are part of the linker; and when p = 0 and n = 0 it is preferred that D^D is linked to T^R via S or O, wherein these atoms are part of D^D. It is further preferred that said S and O moieties comprised in L^D or D^D are bound to an aliphatic or aromatic carbon or carbonyl or thiocarbonyl of L^D or D^D.

With reference to formula (la) and (lb) for Triggers that function via cascade-mediated release or elimination (i.e. cascade mechanism), in particular embodiments when is S-C(0)-(LD)_n-(E)D), 0-C(S)-(LD)_n-(E)D), S-C(S)-(L^D)_n-(D^D) and n = 1 it is preferred that L^D is linked to T^R via N or NH or an aliphatic or aromatic carbon, wherein these atoms are part of the linker; and when n = 0 it is preferred that D^D is linked to T^R via N or NH or an aliphatic or aromatic carbon, wherein these atoms are part of D^D. It is further preferred that said N and NH moieties comprised in L^D or D^D are bound to an aliphatic or aromatic carbon of L^D or D^D. Additional Embodiment 2

Further preferred activators for use with Triggers based cascade mechanism are:

The 1,2,4,5-tetrazine given in Formula (8c-g), wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', NO₂, OR', SR', C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=0)₂R"', S(=O)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR",

NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=O)NR"R", NR'C(=S)NR"R" with each R' and each R' independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

Other preferred activators for use with Triggers based on the cascade mechanism are:

Other 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. Preferably, 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

Additional embodiment 3:

Some embodiments satisfy the one of the following formulas:

= rest of attached Y^M or S^P-Y^M

= rest of attached D^D or L^D-D^D

•~>~ = rest of attached L^D-D^D, comprising Y^M or S^P-Y^M

Additional embodiment 4:

Exemplary lipids to be used in activatable liposomes include:

DAG-TCO-mPEG I DAG-TCO-mPEG II

Exemplary liposomal formulations that release a contained drug upon reaction with a tetrazine at a pH within the range of 4.5-8.0:

With reference to Scheme 2, 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%

With reference to Scheme 2 other exemplary formulations are:

DSPC/Chol/lipid E or F/DOPE-TCO-mPEG (55:39.5:5:0.5)

DSPC/Chol/lipid E or F/DOPE-TCO-mPEG (54.5:39.5:5: 1)

DSPC/Chol/lipid E or F/DOPE-TCO-mPEG (52.5:39.5:5:3)

Particular preferred formulations include:

DOPE/CHEMS/DSPE-TCO-mPEG 60:40:3 molar ratio

DOPE/CHEMS/ DSPE-TCO-mPEG 70:30:3 molar ratio

DOPE/CHEMS/ DSPE-TCO-mPEG 80:20:3 molar ratio

DOPE/CHEMS/ DSPE-TCO-mPEG 90: 10:3 molar ratio

DOPE/CHEMS/ DSPE-TCO-mPEG 60:40: 1 molar ratio

DOPE/CHEMS/ DSPE-TCO-mPEG 70:30: 1 molar ratio

DOPE/CHEMS/ DSPE-TCO-mPEG 80:20: 1 molar ratio

DOPE/CHEMS/ DSPE-TCO-mPEG 90: 10: 1 molar ratio

DOPE/DSPE-TCO-mPEG 90: 10 molar ratio

DOPE/DSPE-TCO-mPEG 95:5 molar ratio

DOPE/DSPE-TCO-mPEG 97:3 molar ratio

DOPE/DSPE-TCO-mPEG 98:2 molar ratio

DOPE/DSPE-TCO-mPEG 99: 1 molar ratio

Phosphatidylcholine/CHEMS/Chol (5:4: 1 molar ratio) containing 10-40 mol% DOPE-

TCO-mPEG

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.

For suitable liposomal formulations reference is made to, amongst others: Ishida et al. BBA 2001;1515:144-158; Zhang et al. Pharm. Res.2004;49:185-198; Shin et al. J

Cont Release 2003; 91 : 187-200; Shin et al. Mol. Pharm. 2012;9:3266-3276; Kim et al. Bioconjug Chem. 2012;23:2071-2077; Guo X, Szoka FC. Bioconjugate

Chem. 2001;12:291-300.; Guo X, MacKav JA, Szoka FC. Biophvs. J. 2003:84:1784- 1795; Wan et al., Biomaterials 2013 Apr ;34( 12): 3020-30; and Terada et al. J Cont Release 2006;111:333-342.

EXAMPLES

The following examples demonstrate the invention or aspects of the invention, and do not serve to define or limit the scope of the invention or its claims.

Methods. 1 H-NMR and 13 C-NMR spectra were recorded on a Varian Mercury (400 MHz for 1H-NMR and 100 MHz for ¹³C-NMR) spectrometer at 298 K. Chemical shifts are reported in ppm downfield from TMS at room temperature.

Abbreviations used for splitting patterns are s = singlet, t = triplet, q = quartet, m = multiplet and br = broad. 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). Analyses were performed using a Alltech Alltima HP C₁₈ 3μ column using an injection volume of 1-4 μί, a flow rate of 0.2 mL min ¹ and typically a gradient (5% to 100% in 10 min, held at 100% for a further 3 min) of CH₃CN in H₂0 (both containing 0.1% formic acid) at 25 °C. Preparative RP-HPLC (CH₃CN / H₂0 with 0.1% formic acid) was performed using a Shimadzu SCL-IOA VP coupled to two Shimadzu LC-8A pumps and a Shimadzu SPD-10AV VP UV-vis detector on a Phenomenex Gemini 5μ Ci8 110A column. 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.

Materials. All reagents, chemicals, materials and solvents were obtained from commercial sources, and were used as received: Biosolve, Merck and Cambridge Isotope Laboratories for (deuterated) solvents; and Aldrich, Acros, ABCR, Merck and Fluka for chemicals, materials and reagents. All solvents were of AR quality.

General examples

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.

Example 1

Synthesis of tetrazine Activators

For previously synthesized tetrazines see WO2012156919A1 and WO2012156920A1 Bis-pyridyl-tetrazine-NHS derivative was described in J. Nucl. Med. 2013, 54,1989- 1995.

3 6-dibenzyl-l,2,4, 5 -tetrazine (4)

Hydrazine hydrate (2.43 mL, 50.0 mmol) was added to a solution of benzyl cyanide (1.16 mL, 10.0 mmol) and Znl₂ (160 mg, 0.5 mmol) in DMF (20 mL) and the solution was stirred overnight at 60°C under argon. NaN0₂ (3.45 g, 50.0 mmol in 10 mL H₂0) was added dropwise to the suspension at room temperature. 1M HC1 (ca. 80 mL) was added until gas formation stopped and pH=2. The mixture was extracted with CH₂C1₂ (3x80 mL) and the combined organic fractions were dried with Na₂S0₄ and concentrated. 4 was obtained after silica gel column chromatography (EtOAc/heptanes, 1/20) as purple oil. Yield: 0.64 g (2.44 mmol, 45%). 1H NMR (400 MHz, CDC1₃): δ 7.38-7.26 (m, 8H), 3.75 (s, 4H) ppm. ¹³C NMR (400 MHz, CDC1₃): δ 129.9, 129.1, 128.0, 127.9, 23.6 ppm. No MS data available due to poor ionization.

Synthesis of 3,6-diisopropyl-l,2,4,5-tetrazine (5)

Hydrazine hydrate (13.2 mL, 312 mmol) was added to isobutyronitrile (3.59 mL, 40.0 mmol) and Znl₂ (0.64 g, 2.0 mmol) and the mixture was stirred overnight at 60°C under argon. NaN0₂ (13.45 g, 200 mmol in 200 mL H₂0) was added dropwise to the light colored suspension at room temperature over a cold-water bath. 1M HCl (ca. 400 mL) was added to the pink solution until gas formation stopped and pH=2. The mixture was extracted with CH₂C1₂ (4x100 mL) and the combined organic fractions were dried with Na₂S0₄ and concentrated. 5 was obtained after silica gel column chromatography (EtOAc/hexanes, 1/9) as volatile purple oil. Yield: 3.6 g (21.4 mmol, quantitative yield). R_f: 0.25 (EtOAc/hexanes, 1/9). 1H NMR (400 MHz, CDC1₃): δ 3.63 (sep, 7=7.2 Hz, 2H), 1.52 (d, 7=7.0 Hz, 12H) ppm. ¹³C NMR (400 MHz, CDCI3): δ 173.8, 34.2, 21.3 ppm. ESI-MS [M+H⁺]: calc: 167.13 Da, found: 167.08 Da.

3,6-dimethyl-l,2,4,5-tetrazine (8)

Acetamidine hydrochloride (3.97 mg; 42.0 mmol) was dissolved in water (20 mL), and hydrazine hydrate (4.0 mL; 84.0 mmol) was added. The mixture was stirred at 20°C under an atmosphere of argon for 5 h. Water (20 mL) was added, followed by sodium nitrite (14.4 g; 210 mmol). The reaction mixture was cooled on an icebath and acidified to pH=3 by careful addition of acetic acid (15.0 g; 250 mmol). The dark pink, aqueous solution was extracted with dichloromethane (2 times 50 mL), and the combined organic layers were washed with 1 M hydrochloric acid (50 mL), dried over magnesium sulfate, and the solvent was removed by evaporation. The product was obtained as dark red crystals (750 mg; 33%). 1H-NMR (CDCI3): δ = 3.04 (s, 6H) ppm. ¹³C-NMR (CDCI₃): δ = 167.2, 21.0 ppm. GC-MS: m/z = +110 M⁺ (calcd 110.06 for C₄H₆N₄).

3-meth l-6-(pyridin-3-yl)-l,2,4,5-tetrazine (10)

Hydrazine hydrate (2.68 niL, 55.2 mmol) was added to 3-cyanopyridine (500 mg, 4.8 mmol), acetamidine hydrochloride (2.00 g, 21.2 mmol) and sulfur (78 mg, 2.4 mmol) and the mixture was stirred overnight under argon at room temperature. The reaction mixture was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (2.76 g, 40.0 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (80 mL) and CHC1₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x100 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 10 contaminated with a small amount of the bis-pyridyl side product. Recrystallization from EtOAc yielded 10 as long needles (70 mg, 0.40 mmol, 8%). Concentration of the EtOAc filtrate yielded another crop (170 mg) of almost pure 10. 1H NMR (400 MHz, CDCI3): δ 9.80 (dd, =0.8 Hz, J₂=1.9 Hz), 8.88-8.84 (m, 2H), 7.55 (m, 1H), 3.14 (s, 3H) ppm. ¹³C NMR (400 MHz, CDCI₃): δ 168.0, 163.1, 153.2, 149.3, 135.1, 127.9, 123.9, 21.3 ppm. ESI-MS [M+H⁺] calc: 174.08 Da, found: 174.08 Da.

3-meth l-6-(pyridin-4-yl)-l,2,4,5-tetrazine (11)

Hydrazine hydrate (2.68 mL, 55.2 mmol) was added to 4-cyanopyridine (500 mg, 4.8 mmol), acetamidine hydrochloride (2.00 g, 21.2 mmol) and sulfur (78 mg, 2.4 mmol) and the mixture was stirred overnight under argon at room temperature. The reaction mixture was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (2.76 g, 40.0 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (80 mL) and CHCI₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x100 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 11 with a ca. 20% contamination of a thiadiazole compound. The crude material (220 mg) was recrystallized from diisopropylether to yield 11 as pink crystals. Yield: 135 mg (0.78 mmol, 16%). R_f: 0.07 (acetone/hexanes, 1/4). 1H NMR (400 MHz, CDC1₃): δ 8.91 (dd, J₁₌ 1.5 Hz, J₂= 4.7 Hz, 2H), 8.44 (dd, =1.8 Hz, ₂=4.5 Hz, 2H), 3.17 (s, 3H) ppm. ¹³C NMR (400 MHz, CDCI₃): δ 168.5, 163.0, 151.1, 139.2, 121.2, 21.4 ppm. ESI-MS [M+H⁺] : calc: 174.08 Da, found: 174.08 Da. -methyl-6-(3-methylpyridin-2-yl)-l,2,4,5-tetrazine (12)

Hydrazine hydrate (2.76 mL, 56.2 mmol) was added to 3-methylpicolinonitrile (0.57 g, 4.8 mmol), acetamidine hydrochloride (2.00, 21.2 mmol) and sulfur (155 mg, 4.8 mmol) and the mixture was stirred under argon at room temperature for 40 hours. EtOH (10 mL) was added and the mixture was filtered. The filtrate was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (2.76 g, 40.0 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (80 mL) and CHCI₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x100 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 12 as purple liquid. Yield: 110 mg (0.59 mmol, 12%). 1H NMR (400 MHz, CDCI₃): δ 8.73 (dd, =0.8 Hz, ₂=4.6 Hz, 1H), 7.76 (ddd, =0.8 Hz, ₂=l -5, 3= 7.8 Hz, 1H), 7.43 (dd, =4.7 Hz, ₂=7.8 Hz, 1H), 3.17 (s, 3H), 2.60 (s, 3H) ppm. ¹³C NMR (400 MHz, CDCI3): δ 167.3, 166.0, 149.8, 148.0, 139.7, 134.5, 125.2, 21.4, 19.8 ppm. ESI-MS [M+H⁺] calc: 188.09 Da, found: 188.08 Da.

3-meth l-6-phenyl-l,2,4,5-tetrazine (14)

Hydrazine hydrate (3.24 mL, 66.7 mmol) was added to benzonitrile (600 mL, 5.8 mmol), acetamidine hydrochloride (2.41 g, 25.5 mmol) and sulfur (94 mg, 2.9 mmol) and the mixture was stirred overnight under argon at room temperature. The reaction mixture was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (3.33 g, 28.3 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (50 mL) and CHCI₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x70 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 14 with some contamination (ca. 75 mg). The crude product could not be purified by recrystallization from numerous solvents. 1H NMR (400 MHz, CDC1₃): δ 8.59 (dd, =1.6 Hz, /₂=8.2 Hz, 2H), 7.68- 7.60 (m, 2H), 7.50-7.46 (m, 1H), 3.10 (s, 3H) ppm. ¹³C NMR (400 MHz, CDC1₃): δ 167.3, 164.2, 132.6, 131.8, 129.3, 127.9, 21.2 ppm. MALDI-TOF-MS: [M+H⁺]: calc: 173.08 Da, found 173.30 Da.

Example 2

TCO synthesis

The following TCO constructs have been prepared according to WO2012156920A1

3-PNP-TCO was synthesized following WO2012156919A1.

Axial-TCO-1 -Doxorubicin

The synthesis of Axial- TCO-1 -Doxorubicin is described in WO2012156919A1.

Synthesis of (E)-cyclooct-2-enyl naphthalen-1 -ylmethylcarbamate

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). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. The product was further purified by preparative RP-HPLC, and isolated by lyophilization, to yield a white powder (32.0 mg; 72%). 1H-NMR (CDC1₃): δ = 8.04 (d, 1H), 7.89 (d, 1H), 7.81 (d, 1H), 7.54 (m, 2H), 7.45 (m, 2H), 5.79 (m, 1H), 5.56 (d, 1H), 5.40 (m, 1H), 5.03 (br. s, 1H), 4.85 (m, 2H), 2.44 (m, 1H), 2.2 - 1.6 (br. m, 6H), 1.43 (m, 1H), 1.02 (m, 1H), 0.79 (m, 1H) ppm. ¹³C-NMR (CDCI3): δ = 131.7, 131.4, 128.8, 128.5, 126.5, 126.4, 125.9, 125.4, 123.5, 74.2, 43.2, 40.7, 35.9, 29.1, 29.0, 24.1 ppm. FT-IR (ATR): v = 3322, 2927, 2857, 1692, 1533, 1258, 1070, 1025, 987 cm^"1. LC-MS: m/z = +310.25 [M+H]⁺ (calcd 309.17 for C₂oH₂₃N02). Axial-(E)-cyclooct-2-en-l- l 4-nitrobenzoate

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%).

1H-NMR (CDC1₃): δ 8.4-8.2 (m, 4H), 5.9 (m, 1H), 5.6 (m, 2H), 2.2 (dd, 1H), 2.5 (m, 1H), 2.15-1.7 (m, 6H), 1.55 (m, 1H), 1.2 (dt, 1H), 0.9 (dt, 1H).

Equatorial-(E)-cyclooct-2-en-l-yl 4-nitrobenzoate

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. The product fractions were rotary evaporated yielding a colourless solid. 1H-NMR (CDCI₃): δ 8.4-8.1 (m, 4H), 5.9 (m, 1H), 5.7 (m, 1H), 5.4 (m, 1H), 2.5 (m, 1H), 2.3 (m, 1H), 2.1-1.8 (m, 3H), 2.8-2.4 (m, 4H), 1.8-1.4 (m, 4H), 1.0-0.8 (m, 1H).

(E)-3-phenoxycyclooct-l -ene

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. The residue was chromatographed on 50 g silica, using heptane as eluent. Product fractions were rotary evaporated and the residue was stirred with methanol until homogeneous, then filtered, and rotary evaporated. The residue was purified by Kugelrohr distillation to yield the product as an oil (3.5 g, 17.33 mmol, 44%).

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%).

(Z)-3-phenoxycyclooct-l-ene: 1H-NMR (CDC1₃): δ 7.25 (m, 2H), 6.9 (m, 3H), 5.7 (m, 1H), 5.5 (m, 1H), 5.1 (m, 1H), 2.5-2.0 (m, 3H), 1.3-1.9 (m, 7H). (E)-3-phenoxycyclooct-l-ene (axial isomer): 1H-NMR (CDC1₃): δ 7.25 (m, 2H), 6.9 (m, 3H), 5.9 (m, 1H), 5.6 (m, 1H), 4.9 (s, 1H), 2.4 (m, 1H), 2.2 (m, 1H), 2.0-0.8 (m, 8H)

(E)-3-phenoxycyclooct-l-ene (equatorial isomer): 1H-NMR (CDCI₃): δ 7.25 (m, 2H), 6.9 (m, 3H), 5.9 (m, 1H), 5.55 (m, 1H), 4.8 (m, 1H), 2.45-2.25 (m, 2H), 2.05- 1.4 (m, 6H), 1.0-0.8 (m, 2H) -cyclooct-2-en-l-yl 2-phenylacetate

by product

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.

Axial- (E)-3-( benzyloxy )cyclooct- 1-ene

Axial (E)-cyclooct-2-en- l-ol (131 mg, 1.04 mmol) was dissolved in 5 mL THF.

Sodium hydride (60 % dispersion in oil, 80 mg, 2 mmol) was added. The mixture was stirred for 5 min, then heated at 55 °C for 1 h, and then stirred at rt for 4 h. Benzyl bromide (210 μί, 300 mg, 1.9 mmol ) was added in 5 small portions. The reaction mixture was stirred for 4 days, after which 10 mL water was added carefully. The mixture was extracted with 2 x 10 mL dichloromethane and the successive organic layers were washed with 10 mL water, dried and rotary evaporated. The residue was heated at ca. 40°C under high vacuum in order to remove most of the benzyl bromide The residue was purified by chromatography on 20 g silica using heptane as eluent, followed by elution with toluene. The latter solvent eluted the product. The product fractions were rotary evaporated, leaving a colourless oil, which contained traces of dibenzyl ether (69 mg, 0.32 mmol, 31%).

1H-NMR (CDC1₃): δ 7.4-7.2 (m, 5H), 6.0 (m, 1H), 5.45 (d, 1H), 4.7-4.4 (dd, 2H), 4.2 (s, 1H), 2.5 (m, 1H), 2.2-1.8 (m, 4H), 1.7-1.5 (m, 3H), 1.3-1.1 (m, 1H), 0.8 (m, 1H)

Axial-(E)-2,5-dioxopyrrolidin-l-yl 5-((((2,5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)- l-methylcyclooct-3-enecarboxylate TCO-2

( -5-bromocyclooct-l-ene

1,5-cyclooctadiene (225 mL, 1.83 mol) was added to ice-cooled 310 mL 33% hydrogen bromide in acetic acid over a 30 min period at ca. 10°C. The mixture was stirred for 2 days, then 300 mL water was added, and the mixture was extracted with 2 x 300 mL pentane containing some TBME. The successive organic layers were washed with 75 mL water and with 75 mL sodium bicarbonate solution. Drying and rotary evaporation left 325 g residue which was used as such in the next step. (Z)-cyclooct-4-enecarbonitrile

A mixture of 700 mL DMSO and sodium cyanide (117.3 g, 2.39 mol) was heated to 90°C. The bromide obtained above was added over a 4 h period at 90 - 96°C. The mixture was subsequently heated at 98°C for 16 h, then it was cooled and 200 mL water was added during this cooling process. The mixture was extracted with 3 x 300 mL pentane containing some TBME. Washing with 50 mL water, drying and rotary evaporation resulted in 170 g residue which was used as such in the next step. See J. Org. Chem. 1988, 53, 1082 for a similar procedure.

(Z)-cyclooct-4-enecarboxylic acid

The product obtained above was treated with 100 mL ethanol, 160 mL 35% hydrogen peroxide, and 400 mL 30% sodium hydroxide solution, via the method described by D. Hartley in J. Chem. Soc. 1962, 4722. After acidification, further workup and

Kugelrohr distillation, the distillate (94.4 g) appeared to be mainly the starting nitrile. This distillate, combined with ca. 25 g of the solid residue from the Kugelrohr distillation, was stirred with 400 mL ethanol. Potassium hydroxide (155 g, 2.35 mol) was added, and the mixture was cooled with cold water (reaction mixture attained 40°C). When the temperature had dropped to 25°C, 35 mL water was added, followed by the portion-wise addition of 140 mL 35% hydrogen peroxide (foaming,

temperature around 30°C). After the addition was complete and the temperature had dropped, the cooling-bath was removed and replaced by a heating mantle. The mixture was warmed up slowly, resulting in an exothermal reaction and foaming. Hereby the temperature gradually reached 63°C (some cooling was necessary). When the temperature had decreased to 55°C, 100 mL 30% sodium hydroxide solution was added. The mixture was then heated for 4 h, while distilling off ca. 350 mL of solvent. Another 30 mL 30% sodium hydroxide solution was added and the mixture was heated under reflux for 10 h. The reaction mixture was cooled to rt, 400 mL heptane was added and the layers were separated. The organic layer was washed with a small amount of water. The combined aqueous layers were acidified with cone.

hydrochloric acid and extracted with 3 x 250 mL TBME. Drying, rotary evaporation and Kugelrohr distillation gave 109.77 g of the desired acid (0.713 mol, 39% yield based on 1,5-cyclooctadiene). (Z)-l -methylcyclooct-4-enecarboxylic acid

A mixture of diisopropylamine (90.2 g, 0.893 mol) and 300 mL THF was cooled below -20°C. n-Butyllithium in hexanes (2.5 N, 360 mL, 0.900 mol) was added in a slow stream, keeping the temperature below -20°C. The solution was stirred for 15 min, then cooled to -50°C. (Z)-cyclooct-5-enecarboxylic acid (54.0g, 0.351 mol), dissolved in 150 mL THF, was added over a 20 min period at temperatures between - 50 and -25°C. The mixture was stirred for an additional 40 minutes, allowing the temperature to rise to -5°C. The mixture was subsequently heated for 3 h at 50°C, then cooled to -50°C. lodomethane (195.5 g, 1.377 mol) was added over a 20 min period at temperatures between -50 and -30°C. The mixture was stirred overnight, heated for 1 h at 40°C, then rotary evaporated in order to remove most of the solvents. Toluene (250 mL) was added to the residue, followed by 500 mL dilute hydrochloric acid. The layers were separated and the organic layer was washed with 100 mL 2 N

hydrochloric acid. The successive aqueous layers were extracted with 2 x 250 mL toluene. The organic layers were dried and rotary evaporated. The residue was purified by Kugelrohr distillation to yield 59.37 g of the methylated acid (0.353 mol,

100%), which was sufficiently pure to be used as such in the next step.

1H-NMR (CDC1₃): δ 5.75-5.60 (m, 1H), 5.55-5.40 (m, 1H), 2.4-1.5 (m, 10H), 1.27 (s,

3H).

¹³C-NMR (CDCI₃): δ 185.5 (C=0), 131.9 (=CH), 126.5 (=CH), 46.2, 35.3, 32.3, 27.1 (CH₃), 26.1, 24.8, 24.7. re lR,5R)-5-methyl-9-oxabicyclo[3.3.2]dec-7-en-10-one

To a mixture of the methylated acid (42.0 g, 0.25 mol), 300 mL dichloromethane, and 300 mL water sodium bicarbonate was added (68.9 g, 0.82 mol). The mixture was stirred for 10 min, then it was cooled in ice. A mixture of potassium iodide (125.2 g, 0.754 mol) and iodine (129 g, 0.508 mol) was added over a 1 h period in 6 equal portions. The reaction mixture was stirred for 3½ h. Sodium bisulfite was added slowly, until the dark colour had disappeared. The layers were separated and the cloudy aqueous layer was extracted with 2 x 250 mL dichloromethane. Drying and rotary evaporation gave the desired iodolactone.

1H-NMR (CDC1₃, product signals): δ 5.65-5.5 (m, 2H), 4.8 (dt, 1H), 3.95 (dt, 1H), 2.6-1.95 (m, 8H).

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).

1H-NMR (CDCI₃): δ 5.95-5.85 (m, 1H), 5.45-5.35 (dm, 1H), 5.05 (bs, 1H), 2.5-2.3 (m, 1H), 2.2-2.0 (m, 2H), 1.95-1.6 (m, 5H), 1.27 (s, 3H).

¹³C-NMR (CDCI₃): δ 177.2 (C=0), 129.1 (=CH), 127.9 (=CH), 79.2 (CH), 45.2, 43.0, 31.9, 29.5 (CH₃), 26.6, 24.0.

(Z)-methyl 5-hydroxy-l -methylcyclooct-3-enecarboxylate

The bicyclic olefin obtained above (38.86 g, 0.234 mol), plus another batch of 1.5 g bicyclic olefin, was stirred for 64 h at 25 - 30°C with 250 mL methanol and potassium bicarbonate (100.0 g, 1.0 mol). Another 50.0 g potassium bicarbonate (0.5 mol) was added because NMR indicated the presence of ca. 35% starting olefin. The mixture was stirred for an additional 64 h, but the amount of starting material remained unchanged. Filtration, washing with methanol and rotary evaporation of the filtrate gave a residue, which was chromatographed on 200 g silica using dichloromethane as the eluent. The starting olefin eluted first, then a mixture of starting olefin and product eluted. Further elution with dichloromethane / methanol gave 6.69 g of product, contaminated with ca. 15% of starting olefin, and then 17.53 g of pure product (total 0.117 mmol, 48%).

1H-NMR (CDC1₃): δ 5.6-5.5 (m, 1H), 5.35-5.25 (m, 1H), 5.0-4.85 (m, 1H), 3.63 (s, 3H), 2.90 (d, 1H, OH), 2.35-1.90 (m, 5H), 1.75-1.45 (m, 3H), 1.20 (s, 3H). ¹³C-NMR (CDCI₃): δ 178.8 (C=0), 132.7 (=CH), 129.0 (=CH), 68.0 (CH), 52.0 (CH₃), 46.1, 35.9, 33.7, 30.4, 26.8, 24.7 (CH₃).

(E)-methyl 5-hydroxy-l-methylcyclooct-3-enecarboxylate

The two portions of hydroxy ester obtained above, plus 2.29 g of hydroxy ester from another experiment (total amount 26.51 g, 133.8 mmol) were mixed with 25.0 g methyl benzoate and heptane / ether (ca. 4/1). The solution was irradiated, the irradiated solution being continuously flushed through a silver nitrate impregnated silica column (213.6 g, containing ca. 126 mmol silver nitrate). During the irradiation process some solvent was lost due to evaporation; this solvent was replaced by ether. The irradiation and flushing were stopped when the irradiated solution contained hardly any starting material. 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. Drying and rotary evaporation gave a residue which consisted of the axial /equatorial isomers of the trans-cycloctene in a ratio of ca. 5/4. The residual column material was stirred with TBME, 100 mL water and the ammonia layer of above. Filtration, layer separation, drying and rotary evaporation gave a residue. The process was repeated twice to give a residue which consisted of the axial/equatorial isomers of the trans cycloctenes in a ratio of ca. 1/7. All fractions of the trans cyclooctenes were combined to give a total yield of 19.1 g (96.5 mmol, 72%).

Note: The axial/equatorial assignment is based on the the stereochemistry of the hydroxy group, in similar fashion as for trans-cycloocten-2-ol. In both isomers the hydroxy and methylester substituents are positionsed cis relative to each other. In the axial isomer, these czs-positioned subsituents are both in the axial position.

1H-NMR (CDCI3) (mixture of isomers): axial isomer: δ 5.8 (m, 1H), 5.35 (m, 1H), 4.2 (m, 1H), 3.72 (s, 3H), 2.7 (m, 1H), 2.3-1.7 (m, 6H), 1.5 (m, 1H), 1.3 (m, 1H), 1.19 (s, 3H). ¹³C-NMR (CDCI₃): δ 177.6 (C=0), 136.1 (=CH), 132.3 (=CH), 74.8 (CH), 51.5 (CH₃), 47.5, 46.0, 39.9, 38.9, 34.8 (CH₃), 31.0.

1H-NMR (CDC1₃) (mixture of isomers): equatorial isomer: δ 6.05 (m, 1H), 5.6 (dd, 1H), 4.45 (bs, 1H), 3.62 (s, 3H), 2.35-1.7 (m, 8H), 1.5 (m, 1H), 1.08 (s, 3H). ¹³C- NMR (CDC1₃): δ 180.7 (C=0), 135.2 (=CH), 130.3 (=CH), 69.6 (CH), 52.1 (CH₃), 44.9, 44.7, 38.3, 30.9, 29.8, 18.3 (CH₃).

Axial-(E)-5-hydroxy-l-methylcyclooct-3-enecarboxylic acid

A solution of 1.60 g potassium hydroxide in 5 mL water was added over a 5 min period to a water-cooled solution of the trans-cyclooctene ester isomer mixture (0.49 g, 2.47 mmol, ratio of the axial/equatorial isomer ca. 2½/l) in 11 mL methanol. The solution was stirred for 18 h at 28°C. 15 mL water was added and the mixture was extracted with 2 x 30 mL TBME. The combined organic layers were washed with 10 mL water, then dried and rotary evaporated to give the non-hydrolyzed equatorial ester. 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.

1H-NMR (CDC1₃): δ 6.15-5.95 (m, 1H), 5.6 (d, 1H), 4.45 (bs, 1H), 2.4-1.7 (m, 7H), 1.6 (dd, 1H), 1.18 (s, 3H).

¹³C-NMR (CDC1₃): δ 185.4 (C=0), 134.8 (=CH), 130.7 (=CH), 69.8 (CH), 44.8, 38.2, 31.0, 29.8 (CH₂), 18.1 (CH₃).

Note: The hydrolysis of the axial/equatorial ester appears to be extremely selective. Whereas the axial isomer hydrolyzes surprisingly easily at rt, the major isomer remains unaffected, thus enabling an straightforward separation between both isomers (the equatorial isomer hydrolyzes upon overnight heating at ca. 60°C). In both isomers the hydroxy and carboxylic substituents are positionsed cis relative to each other. In the axial isomer, these czs-positioned subsituents are both in the axial position.

Axial-(E)-2,5-dioxopyrrolidin-l-yl 5-((((2,5-dioxopyrrolidin-l- yl)oxy )carbonyl )oxy )-l -methylcyclooct-3-enecarboxylate TCO-2

To a solution of Axial-(E)-5-hydroxy-l-methylcyclooct-3-enecarboxylic acid obtained above (375 mg, 2.04 mmol) in 10.1 g acetonitrile there was added N,N- diisopropylethylamine (1.95 g, 15.07 mmol), followed by N,N'-disuccinimidyl carbonate (2.25 g, 8.79 mmol). The mixture was stirred for 3 days at rt, and subsequently rotary evaporated at 55 °C. The residue was chromatographed on 20 g silica, elution being done with dichloromethane, followed by elution with dichloromethane containing some TBME. The latter solvent mixture eluted the product. The product fractions were combined and rotary evaporated. The resulting residue was stirred with TBME until a homogeneous suspension was obtained. Filtration and washing gave 400 mg of product.

1H-NMR (CDC1₃): δ 6.15-6.0 (m, 1H), 5.6 (dd, 1H), 5.25 (bs, 1H), 2.8 (2s, 8H), 2.5- 1.85 (m, 8H), 1.25 (s, 3H).

Axial-TCO-2-Doxorubicin

Doxorubicin hydrochloride (133 mg; 2.30* 10^"4 mol) and TCO-2 (97.0 mg; 2.30* 10^"4 mol) were dissolved in DMF (5 niL), and DIPEA (148 mg; 1.15* 10 ³ mol) was added. The solution was stirred under an atmosphere of argon at 20°C for 18 h. Acetonitrile (6.5 mL), formic acid (0.2 mL), and water (6.5 mL), were added and the suspension was filtered. The filtrate was purified by preparative RP-HPLC (50 v% acetonitrile in water, containing 0.1 v% formic acid). The product was isolated by lyophilization, dissolved in chloroform (3 mL), and precipitated in diethyl ether (20 mL), to yield 134 mg of an orange powder (68%). 1H-NMR (CDC1₃): δ = 13.97 (s, 1H), 13.22 (s, 1H), 8.03 (d, =7.9 Hz, 1H), 7.78 (t, =8.0 Hz, 1H), 7.38 (d, =8.6 Hz, 1H), 5.85 (m, 1H), 5.59 (m, 1H), 5.51 (s, 1H), 5.29 (s, 1H). 5.16 (d, =8.4 Hz, 1H), 5.12 (s, 1H), 4.75 (d, =4.8 Hz, 2 H), 4.52 (d, =5.8 Hz, 1H), 4.15 (q, =6.5 Hz, 1H), 4.08 (d, =3.6 Hz, 3H), 3.87 (m, 1H), 3.69 (m, 1H), 3.26 (d, =18.8 Hz, 1H), 3.00 (m, 2H), 2.81 (s, 4H), 2.4 - 1.7 (br. m, 13H), 1.62 (s, 2H), 1.30 (d, =6.5 Hz, 3H), 1.23 (s, 3H) ppm. ¹³C-NMR (CDCI3): δ = 213.89, 187.07, 186.68, 174.30, 169.27, 161.03, 156.15, 155.64, 154.66, 135.73, 135.49, 133.58, 131.70, 131.10, 120.88, 119.83, 118.43, 111.58, 111.40, 100.73, 72.09, 69.65, 67.28, 65.54, 56.67, 46.87, 44.38, 35.75, 34.00, 30.49, 30.39, 30.20, 25.61, 17.92, 16.84 ppm. LC-MS: m/z = +873.42 [M+Na]⁺ , - 849.58 [Μ-ΗΓ (calcd 850.28 for C^H^NiOn).

DSPE- TCO-2-mPEG2000

Procedure A )

l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) (7.4 mg; 9.88* 10^"6 mol) and axial TCO-2 (5.0 mg; 11.84* 10^~6 mol) were added to chloroform (0.25 mL), and the suspension was cooled to -15°C. l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (7.51 mg; 49.3* 10^"6 mol) was added and the homogeneous solution was stirred under an atmosphere of argon at -15°C for 15 min. Subsequently, poly(ethylene glycol) methyl ether amine (mPEG amine M_n=2000 g/mol) (19.73 mg; 9.86* 10^"6 mol) was added, and the reaction mixture was allowed to warm to room temperature and stirred overnight. Then, it was diluted with chloroform (2 mL), washed with aqueous citric acid (1.5 mL 0.5 M), and the product was purified by size exclusion chromatography using Biobeads SX-1 as the stationary phase and chloroform as the eluent. The product was isolated by evaporation of the chloroform. Yield: 22.3 mg of a white powder (76%). 1H-NMR (CDC1₃):□ = 7.99 (br. s, 1H), 5.94 (m, 1H), 5.59 (m, 1H), 5.18 (m, 2H), 4.40 (m, 1H), 4.18 (m, 2H), 4.00 (m, 4H), 4.0 - 3.4 (br. m, 200 H), 3.39 (s, 3H), 3.15 (m, 2H), 2.75 - 2.35 (br. m), 2.28 (q, 4H), 2.2 - 1.3 (br. m), 1.25 (m, 60H), 1.10 (s, 3H), 0.88 (t, 6H) ppm. ESI-MS: m z = - 2998.4 +/- 44.0 Da. Procedure B ) :

Poly(ethylene glycol) methyl ether amine (mPEG amine M_n=2000 g/mol) (19.73 mg; 9.86* 10^"6 mol) and l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (7.51 mg; 49.3* 10^"6 mol) were dissolved in chloroform (0.25 mL), and the suspension was cooled to - 15°C. 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. Subsequently, l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) (7.4 mg; 9.88* 10^"6 mol) was added and the reaction mixture was stirred under an atmosphere of argon at -15°C for 60 min, and then allowed to warm to room temperature and stirred overnight. Then, it was diluted with chloroform (2 mL), washed with aqueous citric acid (1.5 mL 0.5 M), and the product was purified by size exclusion chromatography using Biobeads SX-1 as the stationary phase and chloroform as the eluent. The product was isolated by evaporation of the chloroform. Yield: 21.7 mg of a white powder (74%). 1H-NMR (CDC1₃):□ = 5.94 (m, 1H), 5.59 (m, 1H), 5.20 (m, 2H), 4.39 (m, 1H), 4.18 (m, 2H), 3.98 (m, 4H), 4.0 - 3.4 (br. m, 200 H), 3.39 (s, 3H), 3.12 (m, 2H), 2.78 - 2.35 (br. m), 2.28 (q, 4H), 2.2 - 1.3 (br. m), 1.25 (m, 60H), 1.10 (s, 3H), 0.88 (t, 6H) ppm. ESI-MS: m/z = - 2997.91 +/- 44.0 Da. MALDI-TOF MS: m/z = - 2997.05 +/- 44.0 Da.

Example 3

Tetrazine induced release of doxorubicin from TCO-l-doxorubicin

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. The relative release yield for each tetrazine is given in Figure 1 (+++ = highest). 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 μL· 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% H₂0/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. All tetrazine containing samples were measured twice and the doxorubicin control sample was run after every three other samples during an overnight program. The peak area of released dox was divided by the peak area of TCO-2-doxorubicin or doxorubicin reference signals and multiplied by 100 to calculate the percentage of release. The calculated percentage of release was corrected when it was observed that the TCO-2-doxorubicin was not fully converted to inv-DA adduct(s). This was done by quantification of remaining dox-TCO, but full conversion was almost always observed. Peak areas (used for doxorubin quantification) were determined at λ^470-500 nm where characteristic doxorubicin absorption takes place and peak integration was done by hand.

Example 4

In a similar fashion as Example 3, the release of doxorubicin from TCO-1- doxorubucin as induced by tetrazines 1,8,9 in PBS/ACN and in serum was measured. From Table 1 it is clear that tetrazine 8 affords the highest release and that the release yields are retained when testing in serum.

Serum experiments were conducted as followes:

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.

Subsequently, cold MeCN (0.200 mL) was added, followed by centrifugation at 13400 rpm for 5 min. The supernatant was used for further analysis by

HPLCMS/PDA analysis to determine the release of doxorubicin. Table 1. Doxorubicin release (%) from Axial-TCO-l-Doxorubicin following addition of 10 equiv. tetrazine 1,9,8 in 25% MeCN in PBS or 50% serum at 37 °C; measured with LCMS at 4 h (n=3).

Probe PBS/MeCN (3/1) Serum

Ϊ 7+3 Ϊ2+Ϊ

9 55+4 46+3

8 79+3 75+4

-^[a] 0 0

[a] no release of doxorubicin from Axial- TCO-l-Doxorubicin at 37 °C in PBS (72 h) and serum (24 h).

Example 5

Versatility of the TCO linker

To demonstrate the versatility of the TCO linker, the stability of a range of TCO derivatives as model compounds was tested under various conditions. In addition, the tetrazine-induced TCO activation was studied under the same conditions. The results in Tables 2-4 support the versatility of the TCO linker and at the same time demonstrate that in addition to aromatic and aliphatic carbamates also carbonates and aromatic and aliphatic esters and ethers are effectively cleaved from the TCO upon tetrazine reaction. In addition to amines, also hydroxy and carboxylic acids form stable conjugates with TCO and can subsequently be cleaved in a range of conditions.

Typical example for testing the stability of a TCO compound

The TCO stock solution (10 iL 25 mM; 2.5* 10^"' mol) was added to a solution of the specific condition (100 μί). 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. Typical example for testing the feasibility of the deprotection

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.

Conditions:

A) in acetonitrile with 5 equivalents of pyridine per TCO at 20°C

B) in acetonitrile with 5 equivalents of DIPEA per TCO at 20°C

C) in acetonitrile with 5 equivalents of piperidine per TCO at 20°C

D) in acetonitrile with 5 equivalents of n-butylamine per TCO at 20°C

E) in acetonitrile with 5 equivalents of 2-mercaptoethanol per TCO at 20°C

F) in tetrahydrofuran with 5 equivalents of triphenylphosphine per TCO at 20°C

G) in acetonitrile with 5 equivalents of DCC per TCO at 20°C

H) in acetonitrile with 5 equivalents of PyBOP per TCO at 20°C

I) in acetonitrile with 1 v% of formic acid at 20°C

J) in chloroform at 20°C

K) in chloroform with 1 v% of formic acid at 20°C

L) in chloroform with 1 v% of trifluoroacetic acid at 20°C

M) in chloroform with 10 v% of trifluoroacetic acid at 20°C

N) in chloroform with 33 v% of trifluoroacetic acid at 20°C

Z) in 25% acetonitrile in water at 20°C

Table 2

somer

Table 3

Table 4

): study is performed at 20°C, unless stated otherwise

Example 6

Reaction of DSPE-TCO-2-PEG2000 with 3,6-dimethyl-l,2,4,5-tetrazine (8)

dissolved in THF (0.5 mL) and water (0.5 mL). Subsequently, a solution of 3,6- dimethyl-l,2,4,5-tetrazine in water (30* 10^"6 L 25 mM; 7.5* 10^"7 mol) was added and the mixture was stirred at 20°C for 1 h. Analysis of the reaction mixture by ESI-MS revealed the cleavage of mPEG from the inv-DA adduct, to yield the DSPE-derived 3,6-dimethyl-pyridazine product: ESI-MS: m/z = 978.67 Da (M+H⁺ )(calculated: 978.72 Da).

Comparable results were obtained when using DSPE-TCO-2-PEG2000 as prepared via procedure B.

Example 7

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

demonstrated by the de-quenching of the released dye.

Liposome preparation

Stock solutions of DOPE lipid, DSPE-TCO-2-PEG2000 (procedure B) and DSPE- PEG2000 in benzene-MeOH (95:5) were both prepared at 20 mg/ml. The lipid stock solutions were combined at respectively 99: 1 and 97:3 molar ratio (25 μιηοΐ total lipid). The solutions were then frozen in liquid N₂ for 5 min and lyophilized overnight. 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. These multilamellar liposomes were then extruded 2x through 200 nm, 2x through 100 nm, and 6x through 50 nm polycarbonate membrane filters at 37 °C to produce large unilamellar liposomes. Free HPTS / DPX dye was removed from the liposome suspension by passing it through two Sephadex G-25 columns equilibrated with 20 mM HEPES, 140 mM NaCl, pH 7.4.

HPTS release assay

An aliquot of the liposome stock solution (-40 μΐ) was added to 950 μΐ 20 mM HEPES, 140 mM NaCl, pH 5.5 or 20 mM HEPES, 140 mM NaCl, pH 7.4 (final -67 mM lipid). Tetrazine 8 (10 ul; 200 mM stock in MQ) or 10 uL MQ were added to the liposomes and the solutions were maintained at 37 °C for the duration of the experiment. At each time point, 300 μΐ aliquots were removed and diluted with 1.7 ml HEPES, 140 mM NaCl, pH 7.4. The fluorescence

nm) was then measured. 5 uL 10% Triton X-100 was added to the last aliquot and the solution was gently shaken to ensure complete liposome disruption (100% release). The fluorescence was then measured and the % HPTS release was calculated for all samples.

Results

The results are depicted in Figure 2. In summary, tetrazine 8 afforded effective and selective release of HPTS from DSPE-TCO-2-PEG2000 containing liposomes at pH 5.5 and pH 7.4. No release was observed for DSPE-PEG control liposomes without DSPE-TCO-2-PEG2000.

Claims

Claims:

1. 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 ircms-cyclooctene group.

2. A liposome according to claim 1, wherein eight-membered non-aromatic cyclic alkenylene group satisfies the following formula (la):

(la) wherein A and P each independently are CR¾ or CR^aX^D provided that at least one, and preferably not more than one, is CR^aX^D. X^D is (O-C(O))_p-

(L^D)_n-(D^D), S-C(0)-(LD)_n-(DD), O-C(S)-(LD)_n-(DD), S-C(S)-(LD)_n-(DD), O-S(O)- (L^D)n-(D^D), wherein p = 0 or 1; wherein (L^D)_n is a linker, which linker is optionally self-immolative, with n = 0 or 1, preferably linked to T^R via S, N, NH, or O, wherein these atoms are part of the linker, which may consist of multiple units arranged linearly and/or branched; D^D is either a masking moiety M^M or a Construct C^c and 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; and wherein T and F each independently denote H, or a substituent selected from the group

consisting of alkyl, F, CI, Br, and I; Y,Z,X,Q each independently are selected from the group consisting of CR¾, C=CR¾, C=0, C=S, C=NR^b, S, SO, SO2, O, NR^b, and SiR^c2, with at most three of Y, Z, X, and Q being selected from the group consisting of C=CR¾, C=O, C=S, and C=NR^b, wherein two R moieties together may form a ring, and with the proviso that no adjacent pairs of atoms are present selected from the group consisting of O-O, O-NR^b, S-NR^b, O-S, O-S(O), 0-S(0)₂, and S-S, and such that Si is only adjacent to CR¾ or O.

3. A liposome according to claim 1, wherein eight-membered non-aromatic cyclic alkenylene group satisfies the following formula (la):

(L^D)_n-(D^D), S-C(0)-(LD)n-(E>D), 0-C(S)-(LD)_n-(IP), S-C(S)-(LD)_n-(DD), O-S(O)- (L^D)n-(D^D), wherein p = 0 or 1; wherein (L^D)_n is a linker, which linker is optionally self-immolative, with n = 0 or 1, preferably linked to T^R via S, N, NH, or O, wherein these atoms are part of the linker, which may consist of multiple units arranged linearly and/or branched; D^D is either a masking moiety M^M or a Construct C^c and 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; and wherein T and F each independently denote H, or a substituent selected from the group

consisting of alkyl, F, CI, Br, and I; wherein one of the bonds PQ, QX, XZ, ZY, YA is part of a fused ring or consists of CR^a=CR^a, such that two exocyclic bonds are fixed in the same plane, and provided that PQ and YA are not part of an aromatic 5-or 6-membered ring, of a conjugated 7- membered ring, or of CR^a=CR^a; when not part of a fused ring P and A are independently CR¾ or CR^aX^D provided that at least one, and preferably not more than one, is CR^aX^D ; when part of a fused ring P and A are independently CR^a or CX^D provided that at least one, and preferably not more than one, is CX^D; the remaining groups (Y,Z,X,Q) being

independently from each other CR¾, C=CR¾, C=O, C=S, C=NR^b, S, SO, SO₂, O, NR^b, SiR¾, such that at most 1 group is C=CR¾, C=O, C=S, C=NR^b, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-NR^b, S-NR^b, O-S, O-S(O), O-S(O)₂, and S-S, and such that Si, if present, is adjacent to CR¾ or O, and the

bond, if present, is adjacent to CR¾ or C=CR¾ groups.

4. A liposome according to any one of the preceding claims, wherein the eight-membered non-aromatic cyclic alkenylene group is a ircms-cyclooctene moiety that satisfies formula (lb):

wherein, in addition to the optional presence of at most two exocyclic bonds fixed in the same plane, each R^a independently denotes H, or, in at most four instances, a substituent selected from the group consisting of alkyl, aryl, OR', SR', S(=0)R"', S(=0)₂R'", S(=O)₂NR'R", Si-R'", Si-O-R'", OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, SO₂H, SO₃H, SO₄H, PO3H, PO4H, NO, NO₂, CN, OCN, SCN, NCO, NCS, CF₃, CF₂-R', NR'R", C(=O)R', C(=S)R', C(=O)O-R', C(=S)0-R', C(=0)S-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'C(=0)-R"', NR'C(=S)-R"', NR'C(=0)0-R"', NR'C(=S)0-R"', NR'C(=0)S-R"', NR'C(=S)S-R"', OC(=0)NR'-R"',

SC(=0)NR'-R"', OC(=S)NR'-R"', SC(=S)NR'-R"', NR'C(=O)NR"-R",

NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being

H, aryl or alkyl and R" independently being aryl or alkyl;

wherein each R^e as above indicated is independently selected from the group consisting of H, alkyl, aryl, OR, SR, S(=O)R", S(=O)₂R", Si-R", Si- O-R", OC(=O)R", SC(=O)R", OC(=S)R", SC(=S)R"', F, CI, Br, I, N₃, SO₂H, SO3H, PO3H, NO, NO₂, CN, CF₃, CF₂-R, C(=O)R, C(=S)R, C(=O)O-R, C(=S)O-R, C(=O)S-R', C(=S)S-R, C(=O)NRR", C(=S)NRR", NRC(=O)-R", NRC(=S)-R", NRC(=O)O-R", NRC(=S)O-R", NRC(=O)S-R", NRC(=S)S- R", NRC(=O)NR"-R", NRC(=S)NR"-R", CR'NR", with each R and each R" independently being H, aryl or alkyl and R" independently being aryl or alkyl; wherein two R^{a e} moieties together may form a ring; wherein one R^{a e} or a self-immolative linker L^D, is bound, optionally via a spacer or spacers S^p, to the species Y^M, and wherein T and F each independently denote H, or a substituent selected from the group consisting of alkyl, F, CI, Br, and I, and X^ is (0-C(0))p-(LD)n-(E>D), S-C(0)-(LD)_n-(D^D), 0-C(S)-(LD)_n-(D^D), S- C(S)-(L^D)_n-(D^D), 0-S(0)-(L^D)_n-(D^D), wherein p = 0 or 1.

5. A liposome according to any one of the preceding claims, wherein the ircms-cyclooctene moiety satisfies any one of the following

= rest of attached Y^M or S^P-Y^M

= rest of attached D^D, L^D-D^D, wherein L^D may optionally comprise Y^M or S^P-Y^M

6. A liposome according to any one of the claims 1-4, wherein the trans -cyclooctene moiety satisfies any one of the following formulae:

= rest of attached L -D , com rising Y^M or S^P-Y^M

·^ΛΛ™ = rest of attached L^D-D^D, wherein L^D comprises Y^M or S^P-Y

7. A liposome according to any one of the claims 1-4, wherein the trans -cyclooctene moiety satisfies any one of the following formulae:

rest of attached Y or S^P-Y^M

rest of attached D^D or L^D-D^D

8. A liposome according to any one of the claims 1-4, wherein the trans -cyclooctene moiety comprises the structure:

9. A liposome according to any one of the claims 1-4, wherein the trans -cyclooctene moiety comprises either of the following structures:

10. A liposome according to any one of the preceding claims, wherein the hposome formulation is selected from the group consisting of:

DOPE/CHEMS/DSPE-TCO-I-mPEG2000 60:40:3 molar ratio

DOPE/CHEMS/DSPE-TCO-II-mPEG2000 60:40:3 molar ratio

DOPE/CHEMS/DSPE-TCO-I-mPEG5000 90: 10:3 molar ratio

DOPE/CHEMS/DSPE-TCO-II-mPEG5000 90: 10:3 molar ratio

DOPE/CHEMS/DSPE-TCO-I-mPEG5000 60:40: 1 molar ratio

DOPE/CHEMS/DSPE-TCO-II-mPEG5000 60:40: 1 molar ratio

DOPE/CHEMS/DSPE-TCO-I-mPEG5000 90: 10: 1 molar ratio

DOPE/CHEMS/DSPE-TCO-II-mPEG5000 90: 10: 1 molar ratio

DOPE/DSPE-TCO-I-mPEG5000 97:3 molar ratio

DOPE/DSPE-TCO-II-mPEG5000 97:3 molar ratio

DOPE/DSPE-TCO-I-mPEG5000 99: 1 molar ratio

DOPE/DSPE-TCO-II-mPEG5000 99: 1 molar ratio

DOPE/DSPE-TCO-I-mPEG2000 97:3 molar ratio

DOPE/DSPE-TCO-II-mPEG2000 97:3 molar ratio

DOPE/DSPE-TCO-I-mPEG2000 99: 1 molar ratio

DOPE/DSPE-TCO-II-mPEG2000 99: 1 molar ratio Phosphatidylcholine/CHEMS/Chol (5:4: 1 molar ratio) containing 10- 40 mol% DOPE-TCO-I-mPEG2000; and

Phosphatidylcholine/CHEMS/Chol (5:4: 1 molar ratio) containing 10- 40 mol% DOPE-TCO-II-mPEG2000,

wherein the abbreviations have the following meanings:

DOPE: l,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine

CHEMS: Cholesterol hemisuccinate

DSPE: l,2-Distearoyl-sn-glycero-3-phosphoethanolamine

PEG: Polyethyleneglycol

and wherein TCO is a an eight-membered non-aromatic cyclic alkenylene group, preferably a ircms-cyclooctene moiety, as defined in any one of the preceding claims.

11. 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 defined in any one of the preceding claims, and the Activator comprises a diene.

12. A kit according to claim 11, wherein the diene satisfies any one of the following formulae (2) to (4):

wherein R¹ is selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', OR', SR', C(=O)R', C(=S)R', C(=O)O-R', C(=0)S-R', C(=S)0-R', C(=S)S-R", C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR",

NR'C(=O)NR"R", and NR'C(=S)NR"R" with each R' and each R"

independently being H, aryl or alkyl; A and B each independently are selected from the group consisting of alkyl-substituted carbon, aryl substituted carbon, nitrogen, N⁺O-, N⁺R with R being alkyl, with the proviso that A and B are not both carbon; X is selected from the group consisting of O, N-alkyl, and C=0, and Y is CR with R being selected from the group consisting of H, alkyl, aryl, C(=0)OR', C(=0)SR', C(=S)OR', C(=S)SR', C(=0)NR'R" with R' and R" each independently being H, aryl or alkyl;

(3)

wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R, N0₂, OR, SR, C(=O)R, C(=S)R, OC(=O)R", SC(=O)R", OC(=S)R", SC(=S)R", S(=O)R, S(=O)₂R",

S(=O)₂NRR", C(=O)O-R, C(=O)S-R, C(=S)O-R, C(=S)S-R', C(=O)NRR", C(=S)NRR", NRR", NRC(=O)R", NRC(=S)R", NRC(=O)OR",

NRC(=S)OR", NRC(=O)SR", NRC(=S)SR", OC(=O)NRR", SC(=O)NRR", OC(=S)NRR", SC(=S)NRR", NRC(=O)NR"R", and NRC(=S)NR"R" with each R and each R" independently being H, aryl or alkyl, and R" independently being aryl or alkyl; A is selected from the group consisting of N-alkyl, N-aryl, C=O, and CN-alkyl; B is O or S; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(=O)R, CC(=S)R, CS(=O)R, CS(=O)₂R", CC(=O)O-R, CC(=O)S-R', CC(=S)O-R, CC(=S)S-R,

CC(=O)NRR", and CC(=S)NRR", R and R" each independently being H, aryl or alkyl and R" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and

(4) wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', NO, NO₂, OR', SR', CN, C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R"', OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)₂R'", S(=O)₂OR', PO₃R*R", S(=O)₂NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=O)NR"R", and

NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl; A is selected from the group consisting of N, C-alkyl, C-aryl, and N⁺O-; B is N; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(=O)R', CC(=S)R', CS(=O)R', CS(=O)₂R'", CC(=O)O-R', CC(=O)S-R', CC(=S)O-R', CC(=S)S-R', CC(=O)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R'" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and

13. A kit according to claim 12, wherein the diene satisfies formula (7) as defined in the description.

14. A kit according to claim 12, wherein the diene satisfies formula (8 a) or (8b):

wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', NO₂, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R"', OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)₂R"', S(=O)₂NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", and NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

15. A kit according to claim 12, wherein the diene is satisfies a formula selected from the group consisting of (8c), (8d), (8e), (8f), and (8g):

wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', N0₂, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R"', OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)₂R'", S(=O)₂NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR",

NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=O)NR"R", and NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

16. A kit according to claim 12, wherein the diene satisfies any one of the formulae:

17. A kit according to claim 12, wherein the diene satisfies any one of the formulae:

18. A kit according to claim 12, wherein the diene satisfies any one of the formulae:

19. A kit according to claim 11, wherein the diene satisfies the formul

20. A kit according to claim 11, wherein the diene satisfies the formul

21. A liposomal composition, comprising:

(a) a Construct of a liposome comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a lipid linkage and, optionally, a Targeting Agent T^T attached to the lipid linkage

(b) a Masking Moiety for the liposome; (c) an eight-membered non-aromatic cyclic alkenylene group, preferably a cyclooctene group, and more preferably a trans- cyclooctene group, linked to both the Construct and the Masking Moiety.

wherein the liposome is as defined in any one of the claims 1 to 10.

22. The use of an eight-membered non-aromatic cyclic alkenylene dienophile group, preferably a cyclooctene group, and more preferably a trans -cyclooctene group, as a Trigger on an activatable liposome, wherein the reaction of the dienophile with a diene results in activation of the liposome, wherein the liposome is as defined in any one of the claims 1 to 10 and the diene is as defined in any one of the claims 11 to 20.