WO2017046602A1 - Tétrazine comme déclencheur afin de libérer une charge enfermée dans une cage - Google Patents

Tétrazine comme déclencheur afin de libérer une charge enfermée dans une cage Download PDF

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Publication number
WO2017046602A1
WO2017046602A1 PCT/GB2016/052895 GB2016052895W WO2017046602A1 WO 2017046602 A1 WO2017046602 A1 WO 2017046602A1 GB 2016052895 W GB2016052895 W GB 2016052895W WO 2017046602 A1 WO2017046602 A1 WO 2017046602A1
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active agent
masked
moiety
tetrazine
monovinyl ether
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PCT/GB2016/052895
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English (en)
Inventor
Mark Bradley
Sarthak Jain
Jin GENG
Kevin Neumann
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The University Court Of The University Of Edinburgh
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Publication of WO2017046602A1 publication Critical patent/WO2017046602A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Tetrazine as a Trigger to Release Caged Cargo
  • the invention relates to the field of small molecule chemical triggers and masked active agents and peptides, specifically, chemical triggers for specifically transforming a masked active agent into an active agent, or selectively unprotecting a peptide which work under physiological conditions.
  • inactive agents that can be activated at a specific site
  • a drug or therapeutic active agent in an inactive form such as a prodrug
  • prodrug forms are used to enable an active agent to pass through the digestive system of a subject without degradation of the active agent, and to then be transformed in the gut or blood stream of the subject into the active form.
  • Prodrug forms of therapeutic active agents have been activated in a targeted manner, by conjugating an activating enzyme to an antibody that targets tissue in the site of interest. Once the prodrug arrives at the target tissue, it is activated into the active form by the activating enzyme. 1
  • the activating enzyme must not be an endogenous enzyme, which would lead to non-specific activation of the prodrug, and as a result the activating enzyme is often highly immunogenic, making repeat administration impossible. 2
  • the therapeutic active agent is coupled to a targeting moiety such as a monoclonal antibody.
  • a targeting moiety such as a monoclonal antibody.
  • this approach has been used to deliver anticancer therapeutics specifically to tumour cells in an attempt to mitigate toxicity of the therapeutic active agent to other cells of the body. Whilst this approach should reduce the side effects of such conjugated therapeutic active agents, it has met limited success as the coupled therapeutic active agents are often inactive or significantly less active in the coupled form, resulting in a loss of efficacy of the treatment, while the dosing levels are stoichiometric.
  • WO2012/156920 in the name of Koninklijke Philips Electronics N.V., discloses the use of tetrazines as chemical triggers to activate bio-orthogonal drugs comprising a trans-cyclooctene derivative.
  • the strain in trans-cyclooctene ring is released, thereby driving the reaction forward.
  • coupling of the desired active agent to a trans-cyclooctene ring is synthetically complex, the resulting conjugated form of the active agent undergoes a significant background cleavage rate.
  • further trans-cis isomerizaton of trans-cyclooctene has been shown to occur in the presence of catalysts such as free thiols or transition metals bound to serum proteins in vivo. 2.
  • one object of the present invention is the provision of an improved conjugated active agent and an improved method of activating the improved conjugated active agent at a specific site of interest.
  • kits for the administration and activation of a masked active agent comprising:
  • the trigger is configured to release the active agent from the masking moiety or the cage moiety, and wherein the trigger is a tetrazine, and the masking moiety comprises a monovinyl ether and the cage moiety comprise a monovinyl ether or an allyl group.
  • the masked active agent comprises an active agent connected to a masking moiety, and the trigger is configured to initiate the controlled removal of the masking moiety from the active agent, wherein the masking moiety comprises a monovinyl ether and the trigger comprises a tetrazine.
  • the masked active agent comprises an active agent enclosed within a cage moiety, and the trigger is configured to release the active agent from the cage moiety, wherein the trigger is a tetrazine and the cage moiety comprises a monovinyl ether or an allyl group.
  • the allyl group may be an allyl ether, allyl ester or other activated allyl group.
  • the masking moiety or the cage moiety comprise a phenolic monovinyl ether.
  • the kit of the invention allows selective release of an active agent from a masked active agent in a versatile way.
  • the use of a masking moiety that comprises a monovinyl ether or cage moiety that comprises monovinyl ether or an allyl group allows a wide range of active agents to be converted to the masked form easily.
  • the masked active agent comprises a masking moiety
  • a hydroxyl group in the active agent may be converted to the monovinyl ether group.
  • the masked active agent is not significantly more bulky that the active agent itself, and may be formed readily from the active agent itself.
  • the tetrazine has the general structure:
  • the reaction between the masked active agent and the tetrazine is an inverse electron demand Diels-Alder reaction, where the monovinyl ether group or allyl group acts as the dienophile, and the tetrazine acts as the diene.
  • Diels-Alder reaction where the monovinyl ether group or allyl group acts as the dienophile, and the tetrazine acts as the diene.
  • Scheme 1 Example reaction scheme of release of a drug (an example of an active agent) an inverse electron demand Diels-Alder reaction.
  • the use of a tetrazine with a trans-cyclooctene moiety is known in the art.
  • the masked active agent of the kit of the invention comprises a monovinyl ether or allyl group, rather than a trans-cyclooctene ring moiety, which is typically simpler to synthesise from the active agent of interest, and results in the dienophile (the vinyl/ethylene group that reacts with the tetrazine) being more readily available for reaction.
  • dienophile the vinyl/ethylene group that reacts with the tetrazine
  • the rate of reaction of a tetrazine with a vinyl group will typically depend in part on the identity of Ri and R2.
  • Ri and R2 are selected to be strong electron withdrawing groups, the electron density within the central tetrazine ring will be reduced, and the rate of reaction with a vinyl group may consequently increase.
  • Ri and R2 are selected to be weak electron withdrawing groups, the electron density within the central tetrazine ring will be relatively higher, and the rate of reaction with a vinyl group may be consequently decreased.
  • the reactivity of the tetrazine with the masked active agent can be tailored by changing the identity of Ri and/or R2.
  • Ri and/or R2 may be chosen to be weaker electron withdrawing groups, as defined below.
  • Ri and/or R2 may be selected to be stronger electron withdrawing groups.
  • Ri and R2 are typically selected to ensure that the resulting tetrazine is stable in aqueous solution.
  • the tetrazine has the general structure (1) and one of Ri and R2 are chosen to be a polymer, and the remaining group Ri and/or R2 IS chosen to be an electron withdrawing group or simple alkyl group.
  • the polymer may act as a tethering group to bind the tetrazine to a specific area of interest or target tissue, for example, such that activation of the masked active agent predominantly occurs in the area of interest or in the target tissue.
  • N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates and polyglutamate-paclitaxel display significant passive tumor targeting (>70-fold). This targeting may be due to the enhanced permeability and retention (EPR) effect. 4 Ri and R2 may be weak electron withdrawing groups.
  • Ri and R2 may be weak electron withdrawing groups, and may be chosen from pyridines, diazines, aryl halides, aryl ketones, aryl aldehydes or aryl esters, such as methyl benzoate, for example.
  • Ri and R2 are selected from pyridines, and diazines. Typically, Ri and R2 are independently selected from
  • Ri and R2 are both (2) and the tetrazine has the structure (8):
  • Ri is (5) and R2 is (3) and the tetrazine has the structure (9):
  • Ri is (6) and R2 is (3) and the tetrazine has the structure (10): NH 3
  • Ri is (6) and R2 is (6) and the tetrazine has the structure (11):
  • Tetrazines having the structures (8), (9), (10) and (11) have been found to be particularly effective as triggers for releasing active agents from masked active agents comprising monvinyl ether moieties or a!lyl group moieties under physiological conditions.
  • the masked active agents are not triggered by endogenous species found in the environment within which the masked active agent is to be administered.
  • the masked active agent is typically not triggered to release the active agent by enzymes, proteins or chemical species that are present in the living subject.
  • the active agent is substantially only released when the trigger is administered and the trigger reacts with the masked active agent, thereby providing specific and controllable release of the active agent into the environment into which it has been administered.
  • the masked active agent comprises an active agent connected to a masking moiety
  • the active agent is directly linked to the masking moiety.
  • the active agent comprises a hydroxyl group and the oxygen of the hydroxy! group of the active agent is connected to a vinyl group to thereby form the monovinyi ether masking moiety.
  • the monovinyi ether is converted by reaction with the tetrazine to a hydroxyl group, thereby reforming the original active agent.
  • the masked active agent comprises an active agent connected to a masking moiety
  • the active agent is indirectly linked to the masking moiety via a linker.
  • This combination is known as a "safety catch" linker or protecting group which is tetrazine cleavabie.
  • the linker comprises a hydroxyl group and the oxygen of the hydroxyl group of the linker is chemically bonded to a vinyl group to thereby form the monovinyi ether masking moiety.
  • the linker is also chemically bonded to the oxygen of the hydroxy! group of the active agent.
  • the structure of the linker is chosen such that when the monovinyi ether is converted into a hydroxy! group on the linker, the newly formed hydroxy! group of the linker initiates a structural rearrangement reaction of the linker, resulting in the release of the active agent.
  • the structural rearrangement reaction may be a 1 ,4- and/or a 1 ,6-elimination reaction. 5 ' 6
  • the linker may comprise a polymer.
  • the polymer may be configured as a tether for the masked active agent via the linker, acting as an anchor for the masked active agent during use to fix the masked active agent to a specified location.
  • the linker may comprise a bridge and a polymer extending away from the bridge. The bridge may connect the active agent to the masking moiety.
  • the polymer may act as a targeting moiety such that the masked active agent preferentiaiiy targets or binds to a specific target, e.g. target tissue within which it is desired to activate the active agent.
  • Multiple bridges may be connected to the polymer such that multiple masked active agents are connected to a common polymer.
  • the linker is selected from the group of linkers comprising:
  • X is a monovinyl ether masking moiety
  • R is selected from H, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, a polymer such as polyethylene glycol, poly(methyl methacrylate), poly(oxazoline), C1-C20 hydroxyl, halide (e.g. F, CI, Br, I), C1-C20 ether, C1-C20 carboxylic acid, C1-C20 amide, C1-C20 amine, C1-C20 aldehyde, C1-C20 ketone.
  • a polymer such as polyethylene glycol, poly(methyl methacrylate), poly(oxazoline), C1-C20 hydroxyl, halide (e.g. F, CI, Br, I), C1-C20 ether, C1-C20 carboxylic acid, C1-C20 amide, C1-C20 amine, C1-C20 aldehyde, C1-C20 ketone.
  • R may be selected from H, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alky a polymer such as polyethylene glycol, poly(methyl methacrylate), and poly(oxazoline).
  • the active agent is connected to the carbamate group in structures (12), (13) and (15), to the carbonate group in structures (14) and (16), and to the carboxyl group in structure (17).
  • the active agent may be released from the masking moiety linked to the active agent via the linker (12) to (17) via a 1 ,4- and/or a 1 ,6-elimination reaction, respectively, when X is converted from a monovinyl ether to a hydroxyl group.
  • R is a polymer
  • the remainder of the linker may act as a bridge between the masking moiety and the active agent.
  • R comprises a moiety that is not H
  • R is connected to the benzene ring in (12) to (17) at the 3- or 5- position (meta position). Accordingly, the identity of R is less likely to interfere with the intended 1 ,4- and/or 1 ,6-elimination reaction that results in the release of the active agent.
  • R may be a polymer.
  • the polymer is a straight chain polymer and the active agent may be connected to a first end of the polymer and the masking moiety may be connected to the opposed second end of the polymer.
  • the polymer may be a branched polymer and the active agent may be connected to a first end of the polymer and the masking moiety may be connected to the end of a branch of the polymer, or at the opposed end of the polymer chain.
  • the polymer may comprise multiple copies of an active agent.
  • the multiple copies of the active agent may be pendent from the polymer.
  • the multiple copies of the active agent may be connected to the polymer periodically along the chain of the polymer. For example, a copy of the active agent may be connected to the polymer at every monomer subunit of the polymer, or at every other monomer subunit, or one active agent per three monomer subunits of the polymer etc.
  • the polymer is a branched polymer
  • the multiple copies of the active agent may be located on one or more branches. Fragmentation of the polymer may release the multiple copies of the active agent from the polymer.
  • the polymer chain may comprise multiple copies of the active agent. Accordingly, when the polymer fragments upon activation by a tetrazine, the fragments of the polymer may correspond to individual or multiple copies of the active agent. The fragments of the polymer may comprise individual or multiple copies of the active agent.
  • the polymer is configured to fragment when the monovinyl ether moiety is removed from the polymer, to thereby release the active agent.
  • the polymer may be configured to fragment via a cascade reaction when the monovinyl ether moiety is removed from the polymer.
  • the polymer may have the structure:
  • Scheme 2 fragmentation of an example polymer upon removal of the masking moiety.
  • the polymer may act as a spacer between the active agent and the monovinyl ether, or may act as a targeting moiety to direct binding of the masked active agent to a specifc target site or target tissue, for example, such that when the masked active agent is triggered by a tetrazine trigger, the active agent is substantially only released in the target site of target tissue.
  • the monovinyl ether is removed from the masked active agent at room temperature.
  • the monovinyl ether is removed from the masked active agent in physiological conditions.
  • the kit is typically used in aqueous solutions at 37°C, conditions that are compatible with biological applications.
  • the kit may be used to administer a masked active agent to a subject and subsequent delivery of a tetrazine trigger may allow controlled release of the active agent within the subject.
  • the masked active agent may be administered to a specific tissue or area of the subject and the trigger may be delivered generally to the subject, activating the localised masked active agent in the specific tissue or area of interest.
  • the masked active agent may be administered to a subject generally and the trigger may be administered to a specific tissue or target area of the subject such that only active agent in the specific tissue or target area of the subject are released by the trigger.
  • Some known methods of tetrazine triggered release of agents typically require elevated temperatures, and/or organic solvents that are not compatible with biological applications.
  • kits that allows tetrazine triggered release of active agents at room temperature, and/or in physiological conditions, allows masked active agents to be used with biological samples in vitro, or in vivo within a subject.
  • the masked active agent may comprise a targeting moiety.
  • the targeting moiety may preferentially bind to a specific target such that generally administered masked active agent may tend to accumulate in or bind to the specific target. Accordingly, general or targeted administration of the trigger to the subject may predominantly release the active agent in the specific target area only. Therefore, the kit of the invention may allow specific delivery of the active agent to a specific target within the body of a subject.
  • the masked active agent comprises a masking moiety and the masking moiety comprises a polymer
  • the polymer may be configured to act as a targeting moiety.
  • the masked active agent may be a masked fluorophore.
  • the masked fluorophore may be substantially non-fluorescent, and the fluorophore may be fluorescent when released from the masking moiety or cage moiety by the trigger.
  • the masked fluorophore may be fluorescent and the released fluorophore may be more fluorescent than the masked fluorophore. Accordingly, the release of fluorophore from the masking moiety or cage moiety may be accompanied by an increase in fluorescence.
  • the masked fluorophore comprises a masking moiety
  • the masked fluorophore is a masked conjugated fluorophore.
  • conjugated fluorophore we refer to a fluorophore comprising a conjugated pi system through which electrons are delocalised.
  • the conjugated fluorophore comprises a hydroxyl or carbonyl group adjacent to the conjugated pi system, such that the masking moiety is directly bonded to the oxygen of the hydroxyl or carbonyl group of the conjugated fluorophore.
  • masked conjugated fluorophore we refer to a conjugated fluorophore having a hydroxyl group that has been converted to a monovinyl ether.
  • a masked conjugated fluorophore is substantially non-fluorescent or the level of fluorescence is significantly reduced compared to the fluorescence of the released conjugated fluorophore.
  • the inventors suggest that the reduction or inhibition of fluorescence may be due to the reduction in the extent of conjugation of the masked fluorophore due to the conversion of the hydroxyl group or carbonyl group to the monovinyl ether masking moiety.
  • the masked fluorophore may be a masked fluorescein, a masked naphthofluorescein, a masked resorufin, a masked 5(6)-carboxyfluorescein or a masked coumarin.
  • the active agent may be a therapeutic active agent and the masked active agent may be a prodrug of that therapeutic active agent.
  • the prodrug may be a monovinyl florouracil, a monovinyl doxorubicin, a monovinyl camptothecin, a monovinyl clofarabine, a monovinyl fludarabine, a monovinyl cladribine, a monovinyl etoposide, a monovinyl daunorubicin, or a monovinyl cis-platin.
  • the prodrug of the therapeutic active agent may comprise a single masking moiety.
  • the prodrug of the therapeutic active agent may comprise two masking moieties.
  • the therapeutic active agent may be cytotoxic.
  • the prodrug of the cytotoxic therapeutic active agent may have reduced cytotoxicity.
  • the prodrug of the cytotoxic therapeutic active agent may have substantially no cytotoxicity. Accordingly, targeted release of the active agent by a tetrazine trigger in situ may allow the active agent to by cytotoxic at the target tissue or area of interest and not cytotoxic, or less cytotoxic in other tissues. Therefore, the kit of the invention may at least reduce the side effects associated with cytotoxicity affecting healthy cells.
  • a cytotoxic therapeutic active agent may target rapidly multiplying cells, and therefore, may target red blood cells in bone marrow and cells in the digestive tract, in addition to its intended target of tumour cells. Reducing the cytotoxicity of the therapeutic active agent by converting the therapeutic active agent to the prodrug form comprising a monovinyl ether, and activating the prodrug form with a tetrazine trigger at the site of the tumour or tumours to be treated may result in a reduction of side effects experienced by the subject, without compromising the potency of the therapeutic active agent.
  • the kit of the invention allows a prodrug form of a therapeutic active agent to be activated by release of the therapeutic active agent from the prodrug form of the active agent in the presence of tetrazine at the site of interest only.
  • the active agent may be a protein.
  • the active agent may be a peptide or polypeptide.
  • the masking moiety of the masked active agent may be bonded to the C- terminal of the peptide or protein.
  • the masking moiety may be bonded to the N-terminal of the peptide or protein.
  • the masking moiety may be bonded to an amino group of the peptide or protein.
  • a masked peptide may have the general structure:
  • AA n is the nth amino acid in the sequence.
  • the masked active agent may be a masked protein or a masked peptide.
  • the monovinyl ether of the masking moiety may prevent a masked protein from folding correctly and thereby prevent or inhibit the functionality of the mis-folded protein. Removal of the masking moiety may allow the mis-folded protein of the masked protein to refold correctly and thereby restore the functionality of the protein.
  • the masked peptide may be more resistant to degradation of the peptide by enzymes in biological environments, and thereby allow the peptide to be released at the target where it is required, even in the presence of protease or the like.
  • the masked peptide may allow selective peptide conjugation.
  • the masked active agent comprises a cage moiety
  • the cage moiety may encapsulate a protein, thereby sterically hindering the functionality of the encapsulated protein. Release of the protein from the cage moiety may therefore remove the steric hindrance to the protein and thereby restore the functionality of the protein.
  • the masked active agent comprises an active agent connected to a masking moiety
  • the masked active agent comprises at least one masking moiety.
  • the masked active agent may comprise at least two masking moieties.
  • the masked active agent may be linked to a further active agent.
  • the further active agent may be active whilst linked to the masked active agent.
  • the further active agent may be a fluorophore that is fluorescent when linked to the masked active agent. Accordingly, masked active agent may be visualised during delivery to a target site during use.
  • the further active agent may be a further masked active agent.
  • the masked active agent may be linked to the further masked active agent via a common masking moiety, such that removal of the monovinyl ether of the masking moiety results in the release of the active agent and/or the further masked active agent.
  • the common masking moiety may have the structure:
  • the common masking moiety may have the structure:
  • Xi is the active agent and X2 is the further active agent.
  • Xi may be a therapeutic active agent and X2 may be a fluorophore.
  • the masked active agent may allow delivery and activation of a therapeutic active agent to a target site and at the same time allow visualisation of activation via fluorescence of the released fluorophore.
  • Xi and X2 may be therapeutic active agents.
  • Xi and X2 may be different therapeutic active agents. Therefore, the masked active agent may deliver two different therapeutic active agents to a target tissue or area of interest.
  • Xi and X2 may be the same therapeutic active agent.
  • the masked active agent may deliver a higher concentration of the therapeutic active agent to the target tissue or area of interest.
  • the cage moiety may comprise a plurality of branch elements extending from a core.
  • the core comprises a monovinyl ether, the removal of which results in the release of the active agent from the cage moiety.
  • the core may comprise a benzene ring and the oxygen of the monovinyl ether may be directly connected to the benzene ring, such that the trigger converts the aryl monovinyl ether to a phenol group that may then further react to release the active agent from the cage moiety.
  • the phenol may allow one or more of the branch elements within the plurality of branch elements to be eliminated from the benzene ring via a 1 ,6- and/or 1 ,4-elmination pathway.
  • the elimination of the one or more branch elements may allow the active agent to be released from the cage moiety.
  • the core may comprise an allyl group and modification of the allyl group may result in the release of the active agent from the cage moiety.
  • At least some of the branch elements within the plurality of branch elements may be cross- linked such that the active agent is restrained between the plurality of branch elements, and therefore constitutes the masked active agent.
  • Cleaved crosslinks Scheme 3 cross-linking of cage moieities comprising monovinyl ether followed by reaction with a tetrazine trigger and subsequent release of the active agent (trapped cargo).
  • the masked active agent may comprise a plurality of cage moieties.
  • the plurality of cage moieties may be linked.
  • the plurality of linked cage moieties may form a superstructure that may encapsulate the active agent.
  • the application of the trigger to the superstructure may initiate modification of the superstructure due to the reaction of the tetrazine with the monovinyl ether or allyl group of the plurality of cage moieties.
  • the superstructure may be modified sufficiently to release the active agent when the monovinyl ether or allyl group of at least one of the cage moieties react with the trigger.
  • the superstructure may be modified sufficiently to release the active agent when the monovinyl ether or allyl group of the majority of the cage moieties react with the trigger.
  • the superstructure may be modified sufficiently to release the active agent when the monovinyl ether or allyl group of substantially all of the cage moieties react with the trigger.
  • the plurality of cage moieties may comprise polymers.
  • the polymers may form aggregates in aqueous solution.
  • the aggregates may be nanoparticles in aqueous solution, such as micelles, or vesicles. Accordingly, an active agent may be encapsulated within the polymer nanoparticles in aqueous solution, for example.
  • the polymers may comprise one or more allyl groups. Accordingly, reaction of the allyl groups with a tetrazine may change the electrostatic charge on the polymer, thereby changing polymer's interaction with the aqueous solution. As a result, the shape or morphology of the polymer aggregates may change. For example, the size of the polymer aggregates may be reduced, thereby releasing at least a majority of an active agent retained therein (see Scheme 4 below).
  • the morphology of the polymer aggregate may change such that the average spacing between the polymers making up the polymer aggregate increases, thereby allowing an active agent encapsulated within the polymer aggregate to be released.
  • the superstructure may switch from hydrophobic to hydrophilic to release the active agent upon application of a tetrazine trigger.
  • the plurality of cage moieties may comprise one or more amino acids.
  • the one or more amino acids of the plurality of cage moieties may form a shell around the active agent enclosed within the superstructure.
  • the superstructure may be a dendrimer structure.
  • the dendrimer structure may comprise a plurality of generations terminating in cage moieties. Accordingly, the monovinyl ether or allyl groups of the cage moieties may be located at the perimeter of the dendrimer.
  • the superstructure may be a 5 th generation poly(propyleneimine) dendrimer modified to include cage moieties at the terminus of each dendron. 8
  • the superstructure may be in the form of nanoparticles, a hydrogel, fibres, vesicles or microcapsules.
  • the active agent may be enclosed within the superstructure. Accordingly, the plurality of cage moieties may form a shell around the active agent. The plurality of cage moieties may form a superstructure around the active agent.
  • Release of the active agent from a masked active agent comprising a plurality of cage moieties forming a shell around the active agent may be triggered by reaction of the monovinyl ether moieties at the end of each cage moiety to result in primary amine groups, or by reaction of allyl group moieties to covalently attach a modifying group to the superstructure.
  • tetrazine triggered materials can be termed 'smart materials'
  • the invention extends in a second aspect to a masked active agent comprising an active agent connected to a masking moiety, or a masked active agent comprising an active agent enclosed within a cage moiety; wherein the masking moiety comprises a monovinyl ether and the cage moiety comprise a monovinyl ether or allyl group moiety.
  • the masked active agent is configured to release the active agent from the masking moiety or the cage moiety in the presence of a trigger.
  • the trigger is a tetrazine.
  • the reaction of the tetrazine with the monovinyl ether of the masking moiety or the monovinyl ether or allyl ether of the cage moiety releases the active agent from the masking moiety or the cage moiety.
  • the masked active agent is substantially less active than the released active agent. In some embodiments, the masked active agent is substantially inactive and the released active agent is active.
  • the active agent is directly linked to the monovinyl ether moiety.
  • the active agent comprises a hydroxy! group and the oxygen of the hydroxyl group of the active agent is chemically bonded to a vinyl group to thereby form the monovinyl ether masking moiety. Accordingly, when the masked active agent is triggered by the application of a tetrazine, the monovinyl ether is converted by reaction with the tetrazine to a hydroxyl group, thereby reforming the original active agent.
  • the active agent is indirectly linked to the masking moiety via a linker.
  • the linker comprises a hydroxyl group and the oxygen of the hydroxy! group of the linker is connected to a vinyl group to thereby form the monovinyl ether masking moiety.
  • the linker may be connected to an oxygen of a hydroxyl group of the active agent.
  • the structure of the linker is chosen such that when the monovinyl ether is converted into a hydroxy! group on the linker, the newly formed hydroxyl group of the linker initiates a structural rearrangement reaction of the linker, resulting in the release of the active agent.
  • the structural rearrangement reaction may be a 1 ,4- or a 1 ,6-elimination reaction.
  • the linker may comprise a polymer.
  • the polymer may be configured as a tether for the masked active agent via the linker, acting as an anchor for the masked active agent during use to fix the masked active agent to a specified location.
  • the linker may comprise a bridge and a polymer extending away from the bridge. The bridge may connect the active agent to the masking moiety.
  • the polymer may act as a targeting moiety such that the masked active agent preferentially targets or binds to a specific target, e.g. target tissue within which it is desired to activate the active agent.
  • the linker is selected from the group of linkers comprising structures (12) to (16) as defined in the first aspect.
  • the active agent is connected to the carbamate group in structures (12) to (16).
  • the active agent may be released from the masking moiety linked to the active agent via the linker (12) to (16) via a 1 ,4- and/or a 1 ,6-elimination reaction, respectively.
  • the remainder of the linker may act as a bridge between the masking moiety and the active agent.
  • Multiple bridges may be connected to the polymer such that multiple masked active agents are connected to a common polymer.
  • R comprises a moiety that is not H
  • R is connected to the benzene ring in (12) to (17) at the 3- or 5- position (meta position). Accordingly, the identity of R is less likely to interfere with the intended 1 ,4- and/or 1 ,6-elimiation reaction that results in the release of the active agent.
  • the linker may be a polymer.
  • the polymer is a straight chain polymer and the active agent may be connected to a first end of the polymer and the masking moiety may be connected to the opposed second end of the polymer.
  • the polymer may be a branched polymer and the active agent may be connected to a first end of the polymer and the masking moiety may be connected to the end of a branch of the polymer, or at the opposed end of the polymer chain.
  • the polymer is configured to fragment when the monovinyl ether moiety is removed from the polymer, to thereby release the active agent.
  • the polymer may have the structure (18) above, and fragment following removal of the monovinyl ether moiety via the general reaction as shown in scheme 2.
  • the polymer may act as a spacer between the active agent and the monovinyl ether, or may act as a targeting moiety to direct binding of the masked active agent to a specific target site or target tissue, for example, such that when the masked active agent is triggered by a tetrazine trigger during use, the active agent is substantially only released in the target site or target tissue.
  • the monovinyl ether is removed from the masked active agent at room temperature.
  • the monovinyl ether is removed from the masked active agent in physiological conditions. Accordingly, the masked active agent may be triggered to release the active agent in aqueous solutions at 37°C, conditions that are compatible with biological applications.
  • the masked active agent may be administered to a subject and subsequent delivery of a tetrazine trigger may allow controlled release of the active agent within the subject.
  • the masked active agent may be administered to a specific tissue or area of the subject and the trigger may be delivered generally to the subject, activating the localised masked active agent in the specific tissue or area of interest.
  • the masked active agent may be administered to a subject generally and the trigger may be administered to a specific tissue or target area of the subject such that only active agent in the specific tissue or target area of the subject are released by the trigger.
  • Some known methods of tetrazine triggered release of agents typically require elevated temperatures, and/or inorganic solvents that are not compatible with biological applications.
  • a masked active agent that allows tetrazine triggered release of an active agent from the masked active agent at room temperature, and/or in physiological conditions, allows masked active agents to be used with biological samples in vitro, or within a subject in vivo.
  • the masked active agent may comprise a targeting moiety.
  • the targeting moiety may preferentially bind to a specific target such that generally administered masked active agent may tend to accumulate in or bind to the specific target. Accordingly, general or targeted administration of the trigger to the subject may predominantly release the active agent in the specific target area only. Therefore, during use, the masked active agent may allow specific delivery of the active agent to a specific target within the body of a subject.
  • the polymer may be configured to act as a targeting moiety.
  • the masked active agent may be a masked fluorophore.
  • the masked fluorophore may be substantially non-fluorescent, and the fluorophore may be fluorescent when the masked fluorophore is released from the masking moiety or cage moiety by the trigger.
  • the masked fluorophore may be fluorescent and the released fluorophore may be more fluorescent than the masked fluorophore.
  • the release of fluorophore from the masking moiety or cage moiety may be accompanied by an increase in fluorescence.
  • the masked fluorophore comprises a masking moiety
  • the masked fluorophore is a masked conjugated fluorophore.
  • the conjugated fluorophore comprises a hydroxyl or carbonyl group adjacent to the conjugated pi system, such that the masking moiety is directly bonded to the oxygen of the hydroxyl or carbonyl group of the conjugated fluorophore.
  • a masked conjugated fluorophore is substantially non-fluorescent or the level of fluorescence is significantly reduced compared to the fluorescence of the released conjugated fluorophore.
  • the masked fluorophore may be a masked fluorescein, a masked naphthofluorescein, a masked resorufin, a masked 5(6)-carboxyfluorescein or a masked coumarin.
  • the active agent may be a therapeutic active agent and the masked active agent may be a prodrug of that therapeutic active agent.
  • the prodrug may be a monovinyl florouracil, a monovinyl doxorubicin, a monovinyl clofarabine, a monovinyl fludarabine, a monovinyl cladribine, a monovinyl Etoposide, a monovinyl daunorubicin or a monovinyl camptothecin.
  • the prodrug of the therapeutic active agent may comprise a single masking moiety.
  • the prodrug of the therapeutic active agent may comprise two masking moieties.
  • Therapeutic active agents may be cytotoxic.
  • the prodrug of the cytotoxic therapeutic active agent may have reduced cytotoxicity.
  • the prodrug of the cytotoxic therapeutic active agent may have substantially no cytotoxicity. Accordingly, targeted release of the active agent by a tetrazine trigger in situ may allow the active agent to by cytotoxic at the target tissue or area of interest, and may therefore at least reduce the side effects associated with cytotoxicity affecting healthy cells.
  • a cytotoxic therapeutic active agent may target rapidly multiplying cells, and therefore, may target red blood cells in bone marrow and cells in the digestive tract, in addition to its intended target of tumour cells.
  • Reducing the cytotoxicity of the therapeutic active agent by converting the therapeutic active agent to the prodrug form comprising a monovinyl ether, and activating the prodrug form with a tertazine trigger at the site of the tumour or tumours to be treated may result in a reduction of side effects experienced by the subject, without compromising the potency of the therapeutic active agent.
  • the invention allows a prodrug form of a therapeutic active agent to be activated by release of the therapeutic active agent from the prodrug form of the active agent in the presence of tetrazine at the site of interest only.
  • the active agent may be a protein.
  • the active agent may be a peptide or polypeptide.
  • the masking moiety of the masked active agent may be bonded to the C- terminal of the peptide or protein.
  • the masking moiety may be bonded to the N-terminal of the peptide or protein.
  • a masked peptide may have the general structure (19).
  • the masked active agent comprises a masking moiety
  • the masked active agent may be a masked protein or a masked peptide.
  • the monovinyl ether of the masking moiety may prevent a masked protein from folding correctly and thereby prevent or inhibit the functionality of the mis-folded protein. Removal of the masking moiety may allow the mis-folded protein of the masked protein to refold correctly and thereby restore the functionality of the protein.
  • the masked peptide may prevent degradation of the peptide by enzymes in biological environments, and allow the peptide to be released at the target where it is required.
  • the cage moiety may encapsulate the protein, thereby sterically hindering the functionality of the encapsulated protein. Release of the protein from the cage moiety may therefore remove the steric hindrance to the protein and thereby restore the functionality of the protein.
  • the masked active agent comprises a masking moiety
  • the masked active agent comprises at least one masking moiety.
  • the masked active agent may comprise at least two masking moieties.
  • the masked active agent may be linked to a further active agent.
  • the further active agent may be a further masked active agent.
  • the masked active agent may be linked to the further masked active agent via a common masking moiety, such that removal of the monovinyl ether of the masking moiety results in the release of the active agent and/or the further masked active agent.
  • the further active agent is a further masked active agent and the further masked active agent is inactive or less active when linked to the masked active agent via a common masking moiety.
  • the common masking moiety may have the structure (20) or (21).
  • the common masking moiety may have the structure (22) to (24).
  • Xi may be a therapeutic active agent and X2 may be a fluorophore.
  • the masked active agent may allow delivery and activation of a therapeutic active agent to a target site and at the same time allow visualisation of activation via fluorescence of the released fluorophore.
  • Xi and X2 may be therapeutic active agents. Xi and X2 may be different therapeutic active agents. Therefore, the masked active agent may deliver two different therapeutic active agents to a target tissue or area of interest. Xi and X2 may be the same therapeutic active agent. Therefore, the masked active agent may deliver a higher concentration of the therapeutic active agent to the target tissue or area of interest.
  • the further active agent may be active whilst linked to the masked active agent.
  • the further active agent may be a fluorophore that is fluorescent when linked to the masked active agent. Accordingly, masked active agent may be visualised during delivery to a target site during use.
  • the cage moiety may comprise a plurality of branch elements extending from a core.
  • the core comprises a monovinyl ether or allyl group, the modification of which results in the release of the active agent from the cage moiety.
  • the core may comprise an aryl monovinyl ether and the oxygen of the monovinyl ether may be directly connected to the benzene ring, such that the trigger converts the aryl monovinyl ether group to a phenol group that may then further react to release the active agent from the cage moiety.
  • the phenol may allow one or more of the branch elements within the plurality of branch elements to be eliminated from the benzene ring of the phenol group via a 1 ,6- or 1 ,4-elimination pathway.
  • the elimination of the one or more branch elements may allow the active agent to be released from the cage moiety.
  • the core may comprise an allyl group, modification of which converts the allyl group to a dihydropyridazine. Accordingly, the chemical and physical properties of the superstructure may be modified to allow release of the active agent from the masked active agent.
  • At least some of the branch elements within the plurality of branch elements may be cross- linked such that the active agent is restrained between the plurality of branch elements, and therefore, constitutes the masked active agent.
  • the masked active agent may comprise a plurality of cage moieties.
  • the plurality of cage moieties may be linked.
  • the plurality of linked cage moieties may form a superstructure that may encapsulate the active agent.
  • the application of the trigger to the superstructure may initiate fragmentation of the superstructure due to the reaction of the tetrazine with the monovinyl ether of the plurality of cage moieties.
  • the superstructure may fragment sufficiently to release the active agent when the monovinyl ether of at least one of the cage moieties react with the trigger.
  • the superstructure may fragment sufficiently to release the active agent when the monovinyl ether of the majority of the cage moieties react with the trigger.
  • the superstructure may fragment sufficiently to release the active agent when the monovinyl ether of substantially all of the cage moieties react with the trigger.
  • the core may comprise an allyl group, modification of which converts the allyl group to a hydro pyridazine. Accordingly, the chemical and physical properties of the superstructure may be modified to allow release of the active agent from the masked active agent.
  • An example reaction scheme for the cross-linking polymerisation and subsequent release of the retained active agent via reaction with a tetrazine trigger is shown in scheme 3 above.
  • the plurality of cage moieties may comprise one or more amino acids.
  • the one or more amino acids of the plurality of cage moieties may form a shell around the active agent enclosed within the superstructure.
  • the superstructure may form a dendrimer structure.
  • the dendrimer structure may comprise a plurality of generations terminating in cage moieties. Accordingly, the monovinyl ether or allyl groups of the cage moieties may be located at the perimeter of the dendrimer.
  • the superstructure may be a 5 th generation poly(propyleneimine) dendrimer modified to include cage moieties at the terminus of each dendron.
  • the active agent may be enclosed within the superstructure. Accordingly, the plurality of cage moieties may form a shell around the active agent. The plurality of cage moieties may form a superstructure around the active agent.
  • Release of the active agent from a masked active agent comprising a plurality of cage moieties forming a shell around the active agent may be triggered by reaction of the monovinyl ether or allyl group moieties at the end of each cage moiety.
  • Preferred and optional features of the masked active agent of the first aspect are preferred and optional features of the second aspect.
  • a pharmaceutical composition comprising a prodrug form of a therapeutic active agent, wherein the prodrug form of the therapeutic active agent comprises the therapeutic active agent and a monovinyl ether moiety.
  • the pharmaceutical composition may comprise a diluent.
  • the pharmaceutical composition may comprise a filler.
  • the pharmaceutical composition may comprise a carrier.
  • the pharmaceutical composition may comprise an excipient.
  • the pharmaceutical composition may comprise a lubricant or lubricating agent.
  • the pharmaceutical composition may comprise a dispersing agent.
  • the prodrug may be configured to release the therapeutic active agent upon reaction of the prodrug with a tetrazine trigger.
  • the prodrug may be a prodrug according to any of the first and second aspects.
  • a method of modifying an active agent into a masked active agent comprising the steps of:
  • the monovinyl ether is configured to be converted to a hydroxyl group by a tetrazine trigger.
  • the resulting masked active agent may be a masked active agent according to the second aspect of the invention, and may be provided in the kit of the first aspect of the invention.
  • a method of modifying an active agent into a masked active agent comprising the steps of:
  • the caging moiety comprises a monovinyl ether configured to be removed from the caging moiety by a tetrazine trigger, or
  • caging moiety comprises an allyl group configured to be modified by a tetrazine trigger
  • the active agent may be released form the masked active agent upon application of a tetrazine trigger.
  • the resulting masked active agent may be a masked active agent according to the second aspect of the invention, and may be provided in the kit of the first aspect of the invention.
  • the invention extends in a sixth aspect to the use of a tetrazine as a trigger for the release, in a physiological environment, of an active agent, wherein the active agent is connected to one or more monovinyl ether moieties before release to form a masked active agent, and wherein the one or more monovinyl ether moieties are removed from the active agent by the tetrazine during release.
  • the active agent may be directly connected to the one or more monovinyl ether moieties.
  • the active agent may be indirectly connected to the one or more monovinyl ether moieties via a linker.
  • the masked active agent may be a masked active agent according to the second aspect of the invention.
  • a tetrazine to remove one or more monovinyl ether groups during active agent synthesis, wherein the one or more monovinyl ether groups prevent hydroxyl groups on the substrate from reacting in one or more earlier steps of the synthesis.
  • the one or more monovinyl ether groups may be acting as a protecting group during at least one step of the synthesis.
  • the invention extends to the use of a tetrazine to selectively remove one or more monovinyl ether groups from a peptide comprising one or more monovinyl ether groups.
  • the monovinyl ether group may act as a cleavable linker during synthesis of a peptide.
  • An example of using a tetrazine trigger to cleave a linker comprising a monovinyl ether connecting a peptide to a support during synthesis eptide is shown in scheme 6 below.
  • the polymer may comprise an active agent.
  • the polymer may have a linear structure.
  • the active agent may be linked to the polymer at a first end of the polymer and the monovinyl ether may be linked to a second opposed end of the polymer.
  • the polymer may have a branched structure.
  • the active agent may be linked to one of the branches of the polymer and the monovinyl ether may be linked to another branch of the polymer.
  • the polymer may encapsulate an active agent and fragmentation of the polymer activated by the reaction of the tetrazine with the monovinyl ether may release the active agent.
  • the polymer may form aggregates in aqueous media and the active agent may be encapsulated within the aggregates. Tetrazine triggered fragmentation of the polymers that make up the aggregates may release the active agent therein.
  • the polymer comprising the active agent may correspond to a masked active agent in accordance with the first and second aspects of the invention. Accordingly, the polymer may have the general structure (18).
  • Preferred and optional features of the polymer and the fragmentation of the polymer of the first aspect are preferred and optional features of the ninth aspect.
  • the invention extends to the use of a tetrazine as an activator for modifying the hydrophobicity of a polymer comprising one or more allyl groups.
  • the polymer may be hydrophobic, or substantially hydrophobic, and the reaction of a tetrazine with the one or more allyl groups of the polymer may increase the hydrophilicity of the polymer, thereby modifying the hydrophobicity of the polymer.
  • at least one of the monomers of the polymer comprises one or more allyl groups.
  • the tetrazine comprises one or more hydrophilic groups.
  • the polymer may form aggregates in aqueous solution and modification of the hydrophobicity of the polymer may result in a change in the morphology of the aggregates such that active agents encapsulated within the aggregates before activation by the tetrazine are released.
  • Preferred and optional features of the polymer and reaction of tetrazines with allyl groups of the first aspect are preferred and optional features of the present aspect.
  • Figure 1 a). Structures of bis-O-vinylfluorescein and fluorescein, b). Fluorescence intensity of Fluorescein and bis-O-Vinylfluorescein 8 ⁇ 2 ⁇ ;
  • Figure 2 Graph showing percentage of bis-O-vinylfluorescein remaining and converting to mono-vinylfluorescein before converting to fluoresein. Calculated by using 1 H NMR via decrease in integration of vinyl protons in comparison to standard (1 ,2,4,5 Tetramethylbenzene);
  • Figure 7. Graph showing 20 fold increase in fluorescence in comparison to bis-O- vinylfluorescein over time indicating release of fluorescein;
  • Figure 8. Graph showing increase in fluorescence in comparison to bis-O-vinylfluorescein over time indicating release of fluorescein;
  • Figure 9 Graph showing 123 fold increase in fluorescence in comparison to bis-O- vinylfluorescein over time indicating release of fluorescein;
  • Figure 10 a) Structures of vinylresorufin and resorufin. b) Fluorescence intensity of resorufin and vinylresorufin at 2 ⁇ ;
  • Figure 11 Graph showing percentage of vinylresorufin remaining and converting to resorufin. Calculated by using 1 H NMR via decrease in integration of vinyl protons in comparison to standard (1 ,2,4,5 Tetramethylbenzene);
  • Figure 17 Graph showing 32.6 fold increase in fluorescence in comparison to vinylresorufin over time indicating release of resorufin (TZ1);
  • FIG. 19 Graph showing 10.8 fold increase in fluorescence in comparison to vinylresorufin over time indicating release of resorufin (TZ3);
  • Figure 20 Graph showing cell viability of TZ1 and TZ3;
  • FIG. 21 Graph showing cell viability of Vinylresorufin (VR), Vinylfluorescein (VF), Fluorescein (F) and Resorufin (R);
  • Figure 22 Graph showing percentage of vinyl groups of Divinyl-5, Fluorouracil remaining. Calculated by using 1 H NMR via decrease in integration of vinyl protons in comparison to standard (1 ,2,4,5 Tetramethylbenzene). c). 1 H NMR spectra of vinyl protons decreasing over time;
  • Figure 23 Graph showing percentage of vinyl group of polymer P1 reacted with tetrazine TZ1 at 37 °C. Calculated by using 1 H NMR via decrease in integration of vinyl protons in comparison to standard (1 ,2,4,5 Tetramethylbenzene);
  • Figure 24 1 H-NMR of PAGE homopolymer P3 (9k Da) and tetrazine (9) in d 6 -DMSO at 60°C. The NMR was measured every 30 min;
  • FIG. 28 Release profile of encapsulated Rhodamine B in nanoparticles formed by block PEG-co-PAGE (P6) in water.
  • the experiment was carried out in water at 37°C and followed for 72 hours by fluorescence detection. Tetrazine 1 1 (TZ37 was added after 210 min.
  • the black line shows the control experiment where no tetrazine was added whereas the red line shows the experiment which includes the addition of tetrazine;
  • FIG. 29 Synthesis and characterisation of Doxorubicin conjugated nanoparticles (PEG-/ Dox).
  • FIG. 30 Tetrazine triggered release of Doxorubicin.
  • HEK273T cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS. Nanoparticles, tetrazine 5 and/or free Doxorubicin were incubated with cells at 37°C with 5% CO2 for 48 h.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Nanoparticles, tetrazine 5 and/or free Doxorubicin were incubated with cells at 37°C with 5% CO2 for 48 h.
  • a) Control just cells
  • PEG- Dox 15 nanoparticles (1 ⁇ equiv. of Dox
  • the samples were stained with propidium iodide (2 ⁇ ) and analysed by flow cytometry (AE x 488 nm with 500- 554 nm broad pass filter). Forward versus side scatter (SSC-A) profiles were used to gate intact cellular materials and determine membrane integrity (PI );
  • FIG. 33 PC3 cells were stained with CellTrackerTM Green prior to incubation with NPs which were loaded with DOX (at 1 ⁇ , 6 ⁇ and 18 ⁇ ) at 37 °C in the presence of 5% C0 2 for 72 hours,
  • a-b Cells were harvested, stained with propidium iodide PI (1 ⁇ g ml-1) and examined by flow cytometry.
  • the x-axis measures the fluorescein intensity of cells (FITC) due to CellTrackerTM staining, (cell viability) and the y-axis corresponds to the PI fluorescence intensity due to the dead cell staining.
  • Electrospray ionization mass spectrometry (ESI-MS) analyses were carried out on an Agilent Technologies LC/MSD Series 1100 quadrupole mass spectrometer (QMS) in an ESI mode.
  • MALDI spectra were acquired on a Bruker Ultraflextreme MALDI TOF/TOF with a matrix solution of sinapic acid (10 mg/mL) in H2O/CH3CN/TFA (50/50/0.1). Florescence monitoring experiments were done on BioTek SIAFRT.
  • Tetrazines TZ1 to TZ6 (shown below) were synthesized to perform de-caging reactions. These tetrazines were used in further reactions.
  • TZ2 was synthesised from a modified procedure. 9 To a stirred solution of compound 4 (600 mg, 1.46 mmol) in 1 , 2-Dicholoroethane (35 ml) Isopentyl nitrite (0.39 ml, 2.9 mmol) was added dropwise at room temperature. The reaction was completed in 2 hours and the solvents were evaporated to afford TZ2 as a pink solid (525 mg, 88%).
  • Novel de-caging of vinyl ether was performed to release free hydroxyl groups.
  • Phenylvinylether was used as model compound.
  • Various tetrazines were used to de-cage the phenylvinylether (Scheme 2a-e). The de-caging was successful with three tetrazines with different percentage of de-caging.
  • TZ6 mediated de-caging was monitored by 1 H NMR.
  • TZ6 only de-caged 19% of the vinyl ether ( Figure 2). All the reactions were performed at physiological temperature.
  • TZ1 , TZ2 and TZ3 were considered as ideal candidates for the novel de-caging of various caged fluorophores.
  • Phenylvinylether and tetrazine are mixed in MeOH. Solution was heated to 37 °C and stirred till the reaction was complete. Reaction was monitored by TLC. Further column chromatography was performed to yield pyridazine 1 (4 mg, 81 %) and pyridazine 2 (19 mg, 77%). Pyridazine 1 1 H NMR (500 MHz, Chloroform-d) ⁇ 8.82 - 8.74 (m, 4H), 8.72 (s, 2H), 7.94 (dt, 2H), 7.44 (m, 2H).
  • Non-Fluorescent Fluorescent Scheme 10 Structures of vinylfluorescein and fluorescein
  • Vinylfluorescein was de-caged by TZ2 in DMSO-cfe at 37° C. 100% of vinylfluorescein reacted with TZ1 after 44 hrs giving 97% of de-caged fluorescein with 3% of mono-vinylfluorescein.
  • the reaction was monitored by 1 H NMR at intervals of 1 hr ( Figure 16). This confirms the de- caging of vinyl ether by tetrazine.
  • Fluorescent Scheme 13 Reaction between a) vinylfluorescein (35 ⁇ ) and TZ3 (500 ⁇ ) in PBS at 37°C and b) between vinylfluorescein (35 ⁇ ) and TZ1 (200 ⁇ ) in PBS at 37 °C.
  • TZ1 Scheme 15 Reaction between vinylfluorescein (1 eq, 2mg) and TZ2 (5 eq, 14.5 mg) in 0.6 ml in DMSO-de at 37°C.
  • TZ3 was used for further experiments as its solubility in aqueous media was found to be 1 mM. Vinylresorufin was de-caged by TZ3 in water at 37° C (Scheme 16). The reaction was monitored by HPLC ( Figure 14). After 24 hrs 3.7%, 51.9% and 44.3% of TZ3, vinylresorufin and resorufin was found ( Figure 15 and Table 2). This further confirms the de-caging of vinyl ether by tetrazine.
  • TZ3 Scheme 17 Reaction between vinylresorufin (125 ⁇ ) and TZ3 (1 mM) in water at 37 °C.
  • the de-caging of resorufin was monitored by increase in fluorescence in comparison to vinylresorufin.
  • the novel de-caging reaction was performed at 37°C and in phosphate saline buffer (PBS) or DM EM with 10% FBS (Dulbecco's Modified Eagle Medium with 10% Fetal bovine serum).
  • TZ1 200 ⁇
  • vinylfluorescein 10 ⁇
  • DMEM Dulbecco's Modified Eagle Medium with 10% Fetal bovine serum
  • TZ1 200 ⁇
  • vinylfluorescein (10 ⁇ ) in PBS to give 32.6 fold increase in fluorescence after 40 hrs (Scheme 18, Figures 16 and 17).
  • TZ3 500 ⁇
  • vinylresorufin 100 ⁇
  • vinylresorufin was found to be stable, showing stability of vinyl ether in PBS and DMEM with 10% FBS at physiological temperature. This technique confirms the novel de-caging of vinyl ether in real time.
  • MTT assay which determines the cell viability was performed to determine the toxicity of the vinylfluorescein, fluorescein, vinylresorufin, resorufin, TZ1 and TZ3. The various concentrations were tested to find the optimum safe concentration to be used in further cell assays. TZ1 and TZ3 showed no toxicity in comparison to control cells at concentration of 10 ⁇ and 100 ⁇ respectively. Vinyl ether caged fluorescein showed no toxicity even at concentrations of 200 ⁇ ( Figure 20 and 21).
  • Divinyl-5, Fluorouracil was synthesised as a masked drug of 5-Fluorouracil.
  • 5-Fluorouracil kills the cells through irreversible inhibition of thymidylate synthase. Synthesis of Divinyl-5-Fluorouracil
  • a pro-drug form of 5-fluorouracil, the monovinyl-form was made using the following synthesis.
  • 5-Flourouracil 25 mg, 0.2 mmol, 1 equiv.
  • 2,4,6-Trivinylcyclotriboroxane-pyridine 48 mg, 0.2 mmol, 1 equiv.
  • caesium carbonate 65 mg, 0.2 mmol, 1 equiv.
  • copper (II) acetate 36 mg, 0.2 mmol, 1 equiv.
  • TZ1 was used to de-cage the Divinyl-5, Fluorouracil.
  • the two vinyl group (1 & 2) were de- caged 20% and 18% respectively ( Figure 22).
  • reaction solution was quenched with cooled (10 ml) water and extracted with DCM (30 ml) twice. After drying over sodium sulphate and evaporation of the solvent the product obtained in quantitative yield as a white solid (97 mg, quant.).
  • PEG Mn 480 (400 mg, 0.83 mmol) and the monomer 9 (71 mg, 0.24 mmol) were given together and dissolved in THF (0.6 mL). The solution was degassed via nitrogen flow. DMA (6.9 ⁇ _, 1 equiv.), BPO (13 mg, lequiv.) and 2-Cyano-2-propyl dodecyl trithiocarbonate (19 mg, 1 equiv.) was added. The solution was degassed again. After degassing the solution was stirred at room temperature for 24 h.
  • GPC (DMF): M n 6020 g mor 1 , PDI 1.32.
  • Caging moieties comprising polymers may allow triggered release of active agents in situ.
  • PAGE Poly (allyl glycidyl ether)
  • Scheme 21 An example is the use of Poly (allyl glycidyl ether) (PAGE) polymers that react with tetrazine via the inverse Diels Alder reaction.
  • PAGE is synthesized by an anionic ring opening polymerization of the glycidyl group (28) to form a polymer comprising multiple allyl ether moieties (Scheme 21).
  • Nanoparticle were prepared by dissolving block PEG-co-PAGE (P6) in DMF and followed by precipitation in water. (Figure 27) The hydrophobic part of the polymer becomes more hydrophilic after reacting with tetrazine
  • the release profile confirms the trigger to release the cargo encapsulated by PAGE nanoparticles with tetrazine.
  • the methacrylate moiety of the Doxorubicin monomer was polymerized with the RAFT (Reversible Addition-Fragmentation chain Transfer) reagent PEG CTA (Poly(ethylene glycol) 4-cyano-4-(phenylcarbonothioylthio)pentanoate, M n 10,000 g mol "1 ) using APS/TMEDA as the redox initiator to give the amphiphilic PEG-/ Dox co-polymer 15 of Figure 29a (13,000 g mol "1 ).
  • RAFT Reversible Addition-Fragmentation chain Transfer
  • PEG CTA Poly(ethylene glycol) 4-cyano-4-(phenylcarbonothioylthio)pentanoate, M n 10,000 g mol "1 ) using APS/TMEDA as the redox initiator to give the amphiphilic PEG-/ Dox co-polymer 15 of Figure 29a (13,000 g mol "1 ).
  • the PEG-/ Dox co-polymer 15 formed nanoparticles with a diameter of 35 nm ( Figure 29c), with the hydrophobic core of each particle, consisting of the four Doxorubicin units (determined by 1 H NMR), linked to the methacrylate backbone surrounded by a hydrophilic PEG shell.
  • Tetrazine mediated controlled drug release was explored using HEK273T cells.
  • the PEG-/ Dox nanoparticles (loading equivalent to 8 ⁇ of Dox) showed no cytotoxicity (MTT assay) after 48 h, whereas 1 ⁇ "free" Doxorubicin resulted in complete cell death.
  • Tetrazine TZ1 showed no toxicity at a concentration of 35 ⁇ .
  • the addition of tetrazine TZ1 (35 ⁇ ) triggered cytotoxicity with 73 % cell death after 48 h incubation (Figure 29). This indicates that the nanoparticles underwent efficient tetrazine triggering, even in a complex cellular environment, leading to a controlled switch-on of cytotoxicity.
  • Nanoparticles containing multiple covalently attached Doxorubicins (attached via a 4-hydroxymethyl phenyl vinyl ether linker) demonstrated efficient tetrazine mediated switch-on of cytotoxicity via a 1 ,6-elimination driven release.
  • the PEG- b-Dox nanoparticles display low cytotoxicity and inherently have EPR targeting abilities.
  • TEM measurement confirmed that a population of micelles was generated with a uniform diameter of 30 ⁇ 5 nm (Fig. 31 C, and 31 D). These changes take place due to the modification of the hydrophobic moieties, arising from the conversing of the hydrophobic moieties into hydrophilic side chains (due to the nature of the tetrazine used and the high level of polymer modification). In support of these observation, the zeta-potential values of the NP exhibited a dramatic change from 0.12 mV to 24.10 mV following tetrazine modification.
  • IC50 values of the NPs and tetrazine against the PC3 cells were determined as 5 mg ml "1 and 100 ⁇ respectively, indicating relatively low cytotoxicity.
  • FITC labelled PEG-b-PAGE (FITC-PEG-/ PAGE) was prepared.
  • the self- assembled NPs showed a time-dependent increase in fluorescence intensity in cells with an enhanced internalisation capacity in the present of tetrazine as evidenced by flow cytometry and cell imaging.
  • DOX Doxorubicin
  • the triggered nanoparticles exhibited full cargo release, with the release profile mirroring the reduction in particle diameter (see Fig. 32b.
  • the amount of liberated DOX was negligible over 48 h, while in the in the presence of tetrazine, the release was rapid, with almost quantitative release over 10 hours.
  • the trigger release of DOX was attributed to the gross morphological changes occurring to the polymer. Hydrophobic DOX is encapsulated in the membrane of the vesicles. The reaction of tetrazine with the allyl group will cause vesicles collapse rapidly as the global properties change quickly, but small hydrophobic pockets were still binding DOX release.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne un kit comprenant un agent actif masqué et de la tétrazine à rôle de déclencheur, l'agent actif masqué comprenant un agent actif lié à un élément à rôle de masquage comprenant un éther de monovinyle ou un agent actif enfermé à l'intérieur d'un élément de type cage comprenant un éther monovinylique ou un groupe allyle, la tétrazine à rôle de déclencheur étant conçue pour libérer l'agent actif depuis l'élément à rôle de masquage ou l'élément de type cage. L'invention concerne également des méthodes de préparation des agents actifs masqués, et des utilisations du kit.
PCT/GB2016/052895 2015-09-17 2016-09-16 Tétrazine comme déclencheur afin de libérer une charge enfermée dans une cage WO2017046602A1 (fr)

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GBGB1516480.9A GB201516480D0 (en) 2015-09-17 2015-09-17 Tetrazine as a trigger to release caged cargo

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US10662274B2 (en) 2016-12-02 2020-05-26 Georgia Tech Research Corporation Self-immolative polymers, articles thereof, and methods of making and using same
US11607458B2 (en) 2017-10-25 2023-03-21 Georgia State University Research Foundation, Inc. Enrichment-triggered chemical delivery system

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10662274B2 (en) 2016-12-02 2020-05-26 Georgia Tech Research Corporation Self-immolative polymers, articles thereof, and methods of making and using same
US11607458B2 (en) 2017-10-25 2023-03-21 Georgia State University Research Foundation, Inc. Enrichment-triggered chemical delivery system

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