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WO2009038776A1 - Nanoconjugués thérapeutiques - Google Patents

Nanoconjugués thérapeutiques Download PDF

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Publication number
WO2009038776A1
WO2009038776A1 PCT/US2008/010921 US2008010921W WO2009038776A1 WO 2009038776 A1 WO2009038776 A1 WO 2009038776A1 US 2008010921 W US2008010921 W US 2008010921W WO 2009038776 A1 WO2009038776 A1 WO 2009038776A1
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nanoconjugate
therapeutic
molecule
polymer
moiety
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Victor Manneh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6933—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to therapeutic and imaging nanoconjugate molecules.
  • the aim of polymer-drug conjugation is to (a) improve drug targeting and therapeutic index, (b) reduce drug toxicity through limited access to sites of toxicity, and (c) overcome the mechanisms of drug resistance (Duncan, 2006).
  • drug resistance Dermat, 2006
  • polymer-drug conjugates enhance drug targeting and limit toxicity by reducing their cellular uptake while in route to the tumor site.
  • targeting ligands i.e. antibodies, peptides, sugars
  • the present invention provides a method and compositions for controlled release of therapeutic agents (e.g., active therapeutic or prophylactic agents) and/or imaging or other diagnostic agents at a desired site, such as a desired site of action.
  • therapeutic agents e.g., active therapeutic or prophylactic agents
  • imaging or other diagnostic agents at a desired site, such as a desired site of action.
  • a particular advantageous application of this method is for destroying and/or imaging abnormal tissue or cells, e.g., cancer tissue or cells, in a patient, typically at an internal treatment site.
  • the method of the invention commonly involves using a therapeutic and/or diagnostic nanoconjugate, e.g., an anticancer nanoconjugate. (In describing the present invention, emphasis is placed on therapeutics and especially anticancer agents.
  • Such description also applies to the other therapeutic nanoconjugates and applications, as well as imaging and other diagnostic applications unless the context dictates a narrower application.
  • Such nonoconjugates can provide targeted delivery and temporal control of the release of an active agent, which is often a therapeutic agent such as a small molecule drug, e.g., anticancer drug.
  • Particularly useful examples utilize photocleavable linkers to release the active agent, with the cleavage accomplished by reaction with singlet oxygen produced by a photosensitizer in response to an appropriate light exposure.
  • the nanoconjugate can be configured to include multiple releasable moieties (which may be the same or different), which may, for example, be linked on a polymer backbone or scaffold. Such an arrangement allows rapid release of large numbers of therapeutic agent molecules, which can provide locally high concentrations of the agent or agents.
  • the invention also concerns such nanoconjugate molecules.
  • the invention can be applied to assist in imaging or diagnosis, either in conjunction with delivery of therapeutic agents, or separately.
  • the nanoconjugate that includes a therapeutic agent moiety may also include an imaging agent moiety and/or a portion of the nanoconjugate (e.g., a portion exclusive of the therapeutic agent moiety) may function as an imaging moieity.
  • an imaging moiety may, for example, be a contrast agent.
  • a nanoconjugate with an imaging agent but without a therapeutic agent moiety may be used, either in conjunction with a separate nanoconjugate having a therapeutic moiety or alone.
  • a first aspect of the invention concerns a method for destroying and/or imaging abnormal cells in a patient at an internal treatment site by administering to a subject having such abnormal cells a conjugate that includes at least one therapeutic or imaging agent moiety, a photosensitizer, a photocleavable linker, targeting agent, e.g., a specific binding agent targeting the abnormal cells, and a polymer or particle.
  • the method further involves delivering light of a wavelength effective to cause generation of singlet oxygen by the photosensitizer to the internal treatment site.
  • the conjugate is configured such that generation of the singlet oxygen causes release of the therapeutic or imaging agent moiety.
  • the conjugate is linked with a moiety or moieties which decrease immune system response (or enhance immune system tolerance) to the conjugate; such moiety is linked to the polymer or particle, and/or to the linker; such moiety is polyethyleneglycol (PEG) or a derivative thereof such as monomethoxypolyenthylene glycol, e.g., of about 2000-10000, 3000-8000, 3000- 5000, or about 3000 daltons average molecular weight.
  • PEG polyethyleneglycol
  • monomethoxypolyenthylene glycol e.g., of about 2000-10000, 3000-8000, 3000- 5000, or about 3000 daltons average molecular weight.
  • the polymer is biocompatible and/or the polymer is a high molecular weight polymer with a highly flexible main chain; the polymer moiety bears a plurality (e.g., at least 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, 70, 100, 2-10, 10-20, 20-50, 50-100, 100-1000) of therapeutic agent moieties linked to the polymer through photocleavable linkers, where activation of the photosensitizer causes cleavage of a plurality of the photocleavable linkers; at least a portion of the polymer moiety is a polymer selected from the group consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA), polyethyleneglycol (PEG), polysaccharides such as polydextrans, dendrimers, liposomes, micelles, polymeric particles, linear cyclodextrins, polymerized ⁇ /-isopropyl
  • HPMA N-(2-
  • the photosensitizer absorbs light within a defined waveband and generates singlet oxygen in response to absorbing that light; the photosensitizer absorbs light above 450 nm, above 550 nm, above 600 nm, above 700 nm, above 800 nm, above 900 nm, in the range of 450-1000 nm, in the range of 550-900 nm, in the range of 550-700 nm, in the range of 650-800 nm, or in the range of 700-900 nm, and generates singlet oxygen in response to absorbing that light; the photosentizer is selected from the group consisting of pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3- hydroxyphenyl)-21-23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphen
  • the photocleavable linker is hydrolyzed upon interaction with singlet oxygen; the photocleavable linker is selected from the group consisting of oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.
  • the targeting moiety includes a specific binding agent, e.g., a moiety which directly or indirectly specifically binds with one or more accessible components of the target cells; the targeting moiety includes a member of a specific binding pair, e.g., where the other member of the specific binding pair is on the target cells; the specific binding agent is a moiety that specifically binds with one or more molecules at the surface of the abnormal cells; the specific binding agent is a small molecule that specifically binds with a cell surface receptor, e.g., a folate receptor; the specific binding agent is an antibody (which may be a specific binding antibody fragment; the antibody preferentially binds to an antigen which is present substantially only at target cells, e.g., abnormal cells, in the patient; the antibody links with the abnormal cells at the treatment site, and the polymer increases an in vivo residence time of the conjugate proximal to the abnormal cells.
  • a specific binding agent e.g., a moiety which directly or indirectly specifically binds with one or
  • the singlet oxygen is generated in close proximity to the cleavable linker, resulting in hydrolysis of the cleavable linker and release of the therapeutic agent; the therapeutic agent is released quickly and in high concentrations at the treatment site; the therapeutic agent is an anticancer drug, an antiinfective agent, a hormone, a small molecule drug; the at least one therapeutic agent includes a plurality of different therapeutic agents, e.g., 2, 3, 4, 5, or even more; the therapeutic agent is highly toxic; the therapeutic agent is considered too toxic for non-targeted administration to a patient by the majority of medical practitioners.
  • the imaging agent is a contrast agent, a fluorescent moiety, a chemiluminescent moiety.
  • the conjugate includes each discrete combination of polymer, therapeutic agent (or imaging or other diagnostic agent), photosensitizer, cleavable linker, and targeting moiety described for embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, cleavable linker, and targeting moiety described for embodiments above; the conjugate includes each discrete combination of polymer, therapeutic agent, photosensitizer, and cleavable linker, described for embodiments embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, and cleavable linker described for embodiments above.
  • a related aspect provides a method for treating a disease or condition in an animal, by administering to a subject suffering from or at risk of a disease of condition, a pharmacologically effective amount of a nanoconjugate molecule, where the nanoconjugate molecule includes at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially targets said molecule to a biological target (e.g., target cells). Photoactivation of the sensitizer causes cleavage of the photocleavable linker releasing at least one of the therapeutic moieties.
  • the conjugate is as described above for the preceding aspect or otherwise described herein for conjugates that include a therapeutic moiety.
  • a further related aspect of the invention concerns a therapeutic nanoconjugate molecule which includes at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially targets the molecule to a biological target, e.g., target cells, where photoactivation of the sensitizer causes cleavage of the photocleavable linker releasing at least one of the therapeutic moieties.
  • a biological target e.g., target cells
  • the conjugate includes each discrete combination of polymer, therapeutic agent, photosensitizer, cleavable linker, and targeting moiety described for aspects and embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, cleavable linker, and targeting moiety described for aspects and embodiments above; the conjugate includes each discrete combination of polymer, therapeutic agent, photosensitizer, and cleavable linker, described for aspects and embodiments embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, and cleavable linker described for aspects and embodiments above.
  • a therapeutic or diagnostic (e.g., imaging) nanoconjugate molecule which includes at least one therapeutic or diagnostic (e.g., imaging) moiety; at least one exogenously cleavable linker; and at least one targeting moiety that preferentially targets said molecule to a biological target (e.g., target cells), where introduction of an exogenous cleaving agent causes cleavage of the exogenously cleavable linker releasing at least one of the therapeutic moieties.
  • the nanoconjugate molecule also includes a polymer moiety which bears a plurality of therapeutic moieties each linked to said polymer through an exogenously cleavable linker, where activation of the photosensitizer causes cleavage of a plurality of the exogenously cleavable linkers;
  • the therapeutic moieties include at least 2, 3, 4, 5, or more different therapeutic moieties;
  • the exogenously cleavable linker is cleaved in vivo and/or in vitro by an exogeneously introduced agent selected from the group consisting of an enzyme, X-rays, and high energy radiation, the nanoconjugate is as described for the preceding aspect or otherwise described herein for therapeutic conjugates.
  • the invention also provides, in another aspect, a therapeutic kit which includes a packaged, pre-measured quantity of a nanoconjugate that includes at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially or specifically targets the molecule to a biological target (usually target cells), where photoactivation of the sensitizer causes cleavage of the photocleavable linker releasing at least one of the therapeutic moieties.
  • the kit also includes instructions for administering the nanoconjugate to a subject suffering from or at risk of a disease or condition treatable or at least potentially treatable by administration of the therapeutic agent.
  • the nanoconjugate is as described for an aspect above or otherwise described herein.
  • Another aspect of the invention concerns a method for preparing a therapeutic nanoconjugate molecule by covalently linking together a targeting moiety, e.g., a cell-targeting moiety, at least one photosensitizer, a polymer backbone, a plurality of photocleavable linkers, and a plurality of therapeutic agents, where illumination of the conjugate in physiological solution causes the photosensitizer to generate singlet oxygen which causes cleavage of the photocleavable linkers, releasing the therapeutic agents from the polymer backbone.
  • a targeting moiety e.g., a cell-targeting moiety
  • at least one photosensitizer e.g., a cell-targeting moiety
  • a polymer backbone e.g., a cell-targeting moiety
  • a plurality of photocleavable linkers e.g., a plurality of photocleavable linkers
  • therapeutic agents e.g., a cell-targeting moiety
  • the nanoconjugate is as described for an aspect above or otherwise described herein.
  • Yet another aspect concerns a method for therapeutic use of a drug having unacceptably high toxicity when administered systemically, by administering to a subject suffering from or at risk of a disease or condition against which the drug has activity a conjugate as described above or otherwise described herein for therapeutic conjugates, where the conjugate includes that drug.
  • the drug is substantially non-toxic when attached in said conjugate; the method also includes selecting a drug having unacceptably high system toxicity; the conjugate includes a moiety allowing removal of circulating conjugate from the bood of subject, such as by affinity purification.
  • the invention concerns a method for temporally and/or spatially controlled (e.g., at a target site) delivery and release of a release moiety such, as a molecule, conjugate or complex, intended for delivery to a target, e.g., an active moiety such as a therapeutic agent, a prodrug, an imaging agent or other diagnostic agent, in a subject.
  • a release moiety such as a molecule, conjugate or complex
  • the method involves delivering to a target environment, e.g., a cellular environment such as to a subject, in a tissue culture, or in a cell suspension, a conjugate, pair of conjugates or complex which includes a photosensitizer moiety, a photocleavable linker, an active moiety or other release moiety, and usually but not necessarily a target binding moiety.
  • a target environment e.g., a cellular environment such as to a subject, in a tissue culture, or in a cell suspension
  • a conjugate, pair of conjugates or complex which includes a photosensitizer moiety, a photocleavable linker, an active moiety or other release moiety, and usually but not necessarily a target binding moiety.
  • the photocleavable linker is located proximal (often adjacent) to the active moiety or other release moiety such that cleavage of the photocleavable linker releases the release moiety.
  • a single conjugate is used, which is configured such that upon exposure of the photosensitizer to light of an appropriate wavelength, singlet oxygen is produced by the photosensitizer causing cleavage of the photocleavable linker, and release of the active moiety from the conjugate.
  • a pair of conjugates are used, where one conjugate includes a photosensitizer, and the other includes a photocleavable linker and an active moiety. Either or both of the conjugates may include a target binding moiety. In most cases, one of the conjugates will include a target binding moiety, and each of the conjugates will include a member of a specific binding pair which binds to the other, e.g., one conjugate will include streptavidin and the other will include biotin. Thus, the conjugate which includes the target binding moiety will bind to the target, and the two conjugates will bind together, thus bringing the photosensitizer into close proximity to the photocleavable linker.
  • conjugates and complexes may be used which include the photosensitizer and photocleavable linker; highly preferably the conjugate or complex is stable in the delivery environment.
  • the complex includes a small number of non-covalently linked molecules, e.g., 2, 3, 4, 5, 2-5, or 5-10; the complex is or includes a particle, which may be or include a solid phase particle; the complex is or includes a liposome.
  • the conjugate is as described for an aspect above or otherwise described herein for the present invention; the photosensitizer is as described herein for the present invention; the photocleavable linker is as described herein for the present invention; the release moiety is as described herein for the present invention; a polymer bearing photocleavable linkers and release moieties is as described herein for the present invention.
  • a further related aspect concerns a conjugate, set of complementary conjugates, or complex as specified for the preceding aspect, but in which the target binding moiety is optional.
  • the single conjugate, set of linked or linkable conjugates, or complex includes a photosensitizer and a release moiety linked in a molecule or conjugate through a photocleavable linker which is cleaved by reaction with singlet oxygen.
  • the conjugate, set of conjugates, or complex include at least one target binding moiety, e.g., an antibody (which may be an antibody fragment).
  • Another related aspect concerns a method for imaging a tumor or other tissue or selected group of cells.
  • the method involves the targeted delivery of a conjugate, conjugates, or complex as described for an aspect above or otherwise described for the present invention, where the conjugate or complex includes at least one binding moiety targeted to the selected tumor, tissue, or group of cells, and also includes at least one contrast agent or other imaging agent (e.g., fluorescent or chemiluminescent agent).
  • the conjugate or complex is administered to a subject (e.g., a human), tissue culture, or cell suspension.
  • the imaging agent is released by exposing target-bound conjugate or complex to a suitable exteneral agent suitable to cause release of the imaging agent.
  • the conjugate or complex includes a photosensitizer and a photocleavable linker, and the conjugate or complex is exposed to light of a wavelength suitable to cause generation of singlet oxygen by the photosensitizer with cleavage of the photocleavable linker.
  • the invention also provides similar conjugates that include releasable diagnostic moieties, e.g., imaging agents, probes, and the like.
  • the invention also provides diagnostic methods utilizing such diagnostic moieties, diagnostic conjugates, diagnostic kits including such conjugates along with instructions for use, and methods for making diagnostic conjugates in the same manner as for therapeutic conjugates.
  • abnormal cells refers to cells within a subject that are different from normal cells, e.g., cells that are not properly growth-regulated by the subject (for example, neoplastic cells such as cancer cells), virus-infected cells, and cells of pathogenic organisms (e.g., pathogenic bacterial cells, pathogenic fungi, and the like).
  • the term "antigen” means a molecule bearing an epitope recognized by the particular antibody. It does not require that the "antigen” be the particular molecule against which the antibody was raised.
  • Indication that a conjugate or a polymer or particle is "biocompatible" means that the indicated entity does not cause or elicit significant adverse effects when administered in vivo to a selected subject, e.g., a human subject. Examples of possible adverse effects include excessive inflammation and/or an excessive or adverse immune response, as well as toxicity.
  • Indication that singlet oxygen is generated in "close proximity" to a cleavable linker means that the singlet oxygen is generated sufficiently close that the concentration of singlet oxygen is sufficient at the cleavable linker such that the cleavable linker will more likely than not react with a singlet oxygen within one minute.
  • “close proximity” will be within the diffusion distance for the lifetime of the singlet oxygen in the particular intended in vivo environment. In many cases, "close proximity” will be 100 nm or less, and often 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, or 5 nm or less.
  • the term "diameter” refers to the mean linear dimension of the particle for lines passing through the center of mass of the particle. Acceptable approximation of the diameter of non-spherical particles is provided by taking the mean of the thickness of the particle along 3 orthogonal axes of a coordinate system, with one of the axes aligned with the longest dimension of the particle.
  • the term "highly flexible” means that the polymer has a short persistence length, generally less than 100 nm (preferably determined using single molecule measurements, though bulk property measurements can be used if single molecule measurements are not available), usually less than 50 nm, often less than 10, 1 nm, 500 angstroms, 100 angstroms, 50 angstroms, or even 10 angstroms.
  • the term "highly toxic” means that the drug entity exhibits toxicity for cells different from target cells in a subject at a level such that a reasonable medical practitioner would not use the drug in a non-targeted manner.
  • internal treatment site refers to a location in the body of a subject that is at least partially below the outer skin of the subject.
  • polymer refers to a molecule (which may be linear, branched, cyclic, or even a network) which is formed of many covalently linked small molecules.
  • the covalently linked small molecules may be of one type or may be a combination of two or more different types.
  • nanoparticle refers to a particle that is not more than 1000 nm in at least one dimension, often no more than 100 nm, 70 nm, 50 nm, 40 nm, 30 nm, or 20 nm. In many cases, a nanoparticle will have a diameter of a just-specified value (as defined herein for particles).
  • the term "photosensitizer” refers to a molecule or moiety of the conjugate which generates singlet oxygen when exposed to light of a particular wavelength.
  • photocleavable linker refers to a moiety which reacts with singlet oxygen resulting in the cleavage of the moiety.
  • a photocleavable linker links a therapeutic or diagnostic agent moiety with a carrier portion of the molecule, e.g., a polymer or particle.
  • small molecule refers to a molecule or a moiety of a conjugate that has a MW of 1000 daltons or less, usually 600, 500, 400, 300, 200, 100 daltons or less.
  • the term "specific binding agent” is used to refer to a molecule or moiety which binds to a particular other molecule or complex with a significant level of specificity. That is, the specific binding agent binds to the particular other molecule or complex to a substantially greater degree than to other-molecules ro complexes that are normally present in the particular environment. In many case the specific binding agent binds with another particular molecule, and the two binding molecules constitute a specific binding pair. Examples include antibody/antigen pairs (including specific binding antibody fragments), ligand/receptor pairs, and enzyme/substrate pairs (including substrate analogs).
  • a molecule or molecule/complex or complex/complex "specifically binds" with another means that the molecule binds with the other to a substantially greater extent than to other similar molecules in the environment. Highly preferably, the molecule does not bind to a significant extent to any other molecules in the relevant environment.
  • the term "subject” means an individual complex organism, e.g., a person or a non-human animal.
  • patient refers to a human subject, i.e., a person.
  • targeting moiety refers to a portion of a conjugate which causes the conjugate to preferentially locate to a particular target area or tissue of a subject, or to associate with particular types of cells.
  • a target moiety can cause a conjugate to preferentially associate with tumor cells. In many cases the association is mediated by the binding of specific binding pairs.
  • therapeutic agent means that the referenced molecule or conjugate moiety can beneficially affect the initiation, course, and/or one or more symptoms of a disease or condition in a subject.
  • the present invention concerns the preparation and use of advantageous therapeutic and/or diagnostic nanoconjugate molecules (e.g., imaging nanoconjugates) designed to release therapeutic or diagnostic moieties (or other molecular entities for which temporal and/or spatial control is desired), e.g., small molecule drugs, dyes, and the like, with selected timing and/or location.
  • advantageous nanoconjugates include a polymer-drug linker that is stable during its transit to the tumor or other site, specific in their binding, and possessing a novel mechanism for abrupt and rapid drug release at the tumor site.
  • the nanoconjugate molecules include a cleavable linker, where the cleavage is accomplished through the administration of an exogenous agent that is deliverable to the desired site.
  • cleavable linkers the combination of photosensitizers and photocleavable linkers, but it should be understood that other types of cleavable linkers, the cleavage of which can be controlled by an exogenously administered agent, e.g., an electromagnetic radiation, particle radiation, or small molecule, can similarly be used in the present invention with corresponding modification for providing the active cleaving entity to the cleavable linker at the desired location.
  • an exogenously administered agent e.g., an electromagnetic radiation, particle radiation, or small molecule
  • This approach of using cleavable linkers can be advantageously extended by using a polymeric or particulate backbone or carrier to simultaneously target a plurality of therapeutic or diagnostic entities to the target site.
  • a high dose and/or an extended dose of the active entity can be achieved with good localization. This can greatly assist in providing therapies with high therapeutic indices.
  • a particularly advantageous application for the present invention is in cancer treatment, or more generally, the destruction of abnormal tissue. Providing a suitable therapeutic conjugate and using it to destroy the abnormal tissue can be illustrated by the following:
  • (i) Manufacturing -The conjugate can be manufactured by creating a conjugate comprised of (B) x -P-(S-L-D) y (1) a hydrophilic, nontoxic polymer (P), responsible for carrying all active ingredients.
  • a binding agent (B) specific for the cancer (or other target) such as an antibody or a specific molecule that binds to the receptor on the surface of the cancer cell, that is attached (most often covalently) to the polymer and is responsible for directing the conjugate to the tumor.
  • An anticancer drug (or other release moiety) (D) such as paclitaxel, (or other active agent) responsible for destroying abnormal tissue such as cancer.
  • a photosensitizer (S) (also referred to as PS) such as Phthalocyanine that generates singlet oxygen.
  • a cleavable linker (L) such as an oxazole.
  • the bond between the anticancer drug and the polymer has a cleavage-inducing moiety with an effective proximity to the singlet oxygen generating agent.
  • the number (y) of the complex molecules S-L-D which determine the number of drug molecules per conjugate.
  • the irradiation may be provided externally by a lamp or a laser, or internally by an endoscopic laser or the like for deep seated tumors.
  • Application of light activates the S and produces a high level of singlet oxygen which reacts very quickly with the cleavable linker and separates the anticancer drug (or other release moiety) from the polymer.
  • the anticancer drug is then released in close proximity to the tumor and enters inside the cancer cell to produce anticancer activity.
  • the present invention utilizes at least one photosensitizer moiety, often at least one photosensitizer moiety per cleavable linker.
  • Photosensitizers are compounds (attached as moieties in the present conjugates or complexes) that photochemically generate a reactive form of oxygen called singlet oxygen (Haugland et al., 2002).
  • a number of these photosensitizers, such as phthalocyanine, have found utility as anticancer drugs when used as singlet oxygen generators in Photodynamic Therapy (PDT).
  • PDT Photodynamic Therapy
  • Those photosensitizers identified for PDT can be used in the present invention also with corresponding suitable cleavable compounds, although the present invention is not limited to those entities.
  • the photosensitizer is commonly administered intravenously and is allowed to selectively localize and concentrate in the tumor while largely clearing from normal tissue.
  • the drug is then activated by excitation with laser energy delivered to the diseased site, e.g., through a fiber optic device, generating large amounts of highly reactive singlet oxygen.
  • the singlet oxygen destroys the diseased tissue in which the photosensitizer has concentrated with minimal damage to healthy tissue.
  • a single molecule of the photosensitizer can generate half a million molecules of singlet oxygen per second (Youngjae et al., 2003).
  • the higher wavelengths (650 nm and above) of activating laser energy are preferred because they can penetrate up to three centimeters (over 1 inch) deep into human tissues. This means that targeted tissue beneath the skin's surface can be irradiated by light that is simply shined on the skin, avoiding any need for invasive surgical procedures. Alternatively, if deeper tissue illumination is needed, light can be delivered via fiber optic devices through blood vessels, the Gl tract, etc. (Zheng et al., 2001 ).
  • a non-limiting list of examples of useful photosensitizers include the pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3- hydroxyphenyl)-21-23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, photofrin.
  • Other photosensitiers are described in US Patents 5990149, 5880145, 5283255, 5171749, 5095030, 4920143, and 4883790, each of which is incorporated herein by reference in its entirety, including for purposes of their descriptions of photosensitizers and PDT.
  • Other photosensitizers are also known and can be used in this invention.
  • cleavable linkers are known that may serve as linker L.
  • cleavable linkers include photocleavable linkers that react with singlet oxygen in solution, resulting in cleavage of the linker.
  • Such a photocleavable linkage includes an oxidation-labile linkage that is cleaved by singlet oxygen.
  • linkers are heterocyclic compounds, such as diheterocyclopentadienes, as exemplified by substituted imidazoles, thiazoles, oxazoles, etc., where the rings will usually be substituted with at least one aromatic which contains carbon-carbon double bonds.
  • these compounds Upon reaction with singlet oxygen, these compounds form an oxo group which then hydrolyzes into two separate molecules (Ando et al., 1973). For example, see the example of the mechanism of oxazole cleavage below.
  • the cleavable linkage is an oxidation-labile linkage, and preferably it is a linkage cleavable by reaction with singlet oxygen.
  • the linker may, for example, be a thioether or its selenium analog; or an olefin, which contains carbon-carbon double bonds. Cleavage of a double bond to an oxo group releases the active moiety, e.g., an anticancer drug.
  • olefins which may be used include vinyl sulfides, vinyl ethers, enamines, imines substituted at the carbon atoms with an a-methine (CH, a carbon atom having at least one hydrogen atom), where the vinyl group may be in a ring, the heteroatom may be in a ring, or substituted on the cyclicolefinic carbon atom, and there will be at least one and up to four heteroatoms bonded to the olefinic carbon atoms.
  • the resulting dioxetane may decompose spontaneously or, highly preferably, by reaction with singlet oxygen from a photosensitizer. Such reactions are described in the following exemplary references: Adam and Liu, J. Amer.
  • the dioxetane occurs upon reaction of singlet oxygen with an activated olefin substituted with a drug moiety or other active moiety at one carbon atom and the second binding agent at the other carbon atom of the olefin. See, for example, U. S. Patent No. 5,807,675 (incorporated herein by reference in its entirety).
  • Exemplary cleavable linkages include S-3-thiolacrylic acid, -N, N-methyl 4-amino-4butenoicacid,-0, 3-hydroxyacrolein, N- (4-carboxyphenyl) 2-imidazole, oxazole, and thiazole.
  • cleavable linkers include N-alkyl acridinyl derivatives, substituted at the 9 position with a divalent group of the formula: - (CO) X' (A) wherein: X'is a heteroatom selected from the group consisting of O, S, N, and Se, usually one of the first three; and A is a chain of at least 2 carbon atoms and usually not more than 6 carbon atoms substituted with anticancer drug, where preferably the other valences of A are satisfied by hydrogen, although the chain may be substituted with other groups, such as alkyl, aryl, heterocyclic groups, etc., A generally being not more than 10 carbon atoms.
  • cleavable linkers are heterocyclic compounds, such as diheterocyclopentadienes, as exemplified by substituted imidazoles, thiazoles, oxazoles, etc., where the rings will, in some cases, be substituted with at least one aromatic group and in some instances hydrolysis will be necessary to release the drug.
  • Still other cleavable linkers are tellurium (Te) derivatives, where the Te is bonded to an ethylene group having a hydrogen atom beta to the Te atom.
  • the ethylene group is part of an alicyclic or heterocyclic ring that may have an oxo group, preferably fused to an aromatic ring and the other valence of the Te is bonded to the drug.
  • the rings may be, for example, coumarin, benzoxazine, tetralin, etc.
  • R is a group having from 1-8 carbon atoms and from 0 to 4 heteroatoms selected from the group consisting of O, S, and N which enhances cell uptake.
  • Photocleavable linkers suitable for use in this invention which are currently regarded as preferable include, for example, oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.
  • cleavable linkers and corresponding cleaving agents can also be used.
  • enzymes that cleave a corresponding substrate can be used (with the substrate serving as a cleavable linker).
  • enzymes include, for example, alkaline phosphatases, penicillin amidases, arylsulfatases, cytosine deaminases, proteases, D-alanyl carboxypeptidases, carbohydrate-cleaving enzymes, beta-lactamases, DNA nucleases, and RNA nucleases.
  • the present invention provides a new generation of drug delivery systems (as well as diagnostic agent delivery systems) made of a synthetic nanovector to achieve high specificity to a target site (e.g., a tumor), which utilizes a cleavable release mechanism, e.g., using photocleavable linkers and photosensitizer agents.
  • a cleavable release mechanism e.g., using photocleavable linkers and photosensitizer agents.
  • cleavage of the linker(s) releases the therapeutic or diagnostic agent.
  • This system can, for example, readily deliver therapeutic concentrations of free anti-cancer drugs to a tumor.
  • a photosensitizer is introduced in close proximity (typically less than 1 nm) to a photocleavable linker to release an active entity, e.g., a long acting lipophilic anticancer drug such as paclitaxel, thereby avoiding the limitations of singlet oxygen's extremely short diffusion distance.
  • an active entity e.g., a long acting lipophilic anticancer drug such as paclitaxel
  • the components of the delivery system can advantageously be loaded onto a polymer backbone (as described below) functionalized with a specific binding agent (e.g., an antibody) to achieve the desired concentration of drug at the tumor site.
  • the resulting nanomedicine can overcome the limitations of current nanomedicine and PDT approaches, and be of great use in the treatment of common solid tumors such as breast, prostate, lung and gastrointestinal cancers.
  • the present invention provides advantageous treatment for a varieity of conditions.
  • Particulary advantageous is the use of the invention against solid tumors
  • light can penetrate from outside to the tumor site, thereby effecting cleavage of the linker and release of the active agent.
  • light can be provided by inserting an endoscopic laser to the tumor site, operated using remote control from outside.
  • the present conjugates include a nontoxic polymer to which multiple delivery/targeting moieties and/or therapeutic moieties are attached.
  • the polymer itself, as well as degradation products, if any, should be suitable for in vivo applications.
  • the polymer should have or be able to be modified to have suitable functional groups for attaching cleavable linker and active agent combinations. Selection of appropriate functional groups and the corresponding reaction conditions and any protective or blocking groups useful for the reactions are well within the skill of synthetic chemists and can be utilized in preparation of the present conjugates.
  • the polymer can advantageously have the ability to deliver a wide range of therapeutic payloads, ranging from small molecules to proteins and peptides.
  • the polymer and resulting conjugate should be useful in a variety of diseases such as cancer, infectious diseases, and the like.
  • Polymers such as N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA) 1 polyethyleneglycol (PEG), and polysaccharides such as polydextrans, and dendrimers have been used for polymer-drug conjugates and can be utilized in the present invention.
  • HPMA N-(2-hydroxypropyl)methacrylamide
  • PGA polyglutamic acid
  • PEG polyethyleneglycol
  • dendrimers polysaccharides
  • dendrimers dendrimers
  • other biocompatible polymers can also be utilized, including but not limited to liposomes, micelles, polymeric particles, linear cyclodextrins, polymerized N- isopropylacrylamide, polyglutamic acid, polylysine, and polyaspartic acid.
  • Selection of appropriate polymer backbone candidates may be guided by criteria such as water solubility, size and/or size distribution, density of chemical functional groups (usually a high density is desirable), and capability of delivering a wide range of therapeutic payloads, e.g., ranging from small molecules to proteins and peptides.
  • Dendrimers in particular are a useful class of polymeric nanoparticles that have found use in biological applications ranging from drug delivery to tissue repair.
  • Dendrimers are polymers that are branched (as opposed to conventional linear polymers) where the branches radiate in a symmetric fashion from a central core.
  • dendrimers found in biological applications are usually based on polyamidoamines, polyamides, carbohydrates or polypeptides.
  • Dendrimers are commercially available in a variety of sizes and chemistries.
  • nanogels Another type of useful polymer materials are nanogels.
  • the DeSimone group utilized inverse microemulsion polymerization techniques to synthesize stable, biocompatible polymeric nanogels less than 200 nm in size, for antisense and gene delivery to HeLa cells via the exploitation of charge. (McAllister et al., 2002)
  • Ppy poly(pyrrole)
  • targeting moieties have been utilized in therapeutic applications. Perhaps the most common is the use of site or target specific antibodies.
  • Other targeting moieties include, for example, targeting peptides, nucleic acid molecules, ligand analogs, oligosaccharides, and the like. Such targeting moieties have been used, for example, to target molecules to tumors.
  • antibodies including antibody fragments
  • the use of antibodies has been well documented, and serves as an exemplary targeting method herein.
  • the antibody is a monoclonal antibody or a fragment thereof, which can be produced by conventional methods.
  • the antibody can be selected to recognize and bind to a tumor marker.
  • the marker will be a protein accessible on the outside of tumor cells, e.g., a tumor-specific receptor or the like.
  • binding pairs may be used, such as receptors with their specific binding partner, e.g., folate and folate receptor.
  • Folate receptors are known to be overexpressed in many different types of tumor cells.
  • the present invention is adapted to delivery of a variety of different active agents, especially therapeutic agents, including many different small molecule drugs. WhIe the present conjugates are advantageous for therapeutic agents, they are also useful for localized delivery of certain diagnostic agents also.
  • the therapeutic moiety can be selected to have desirable properties for the particular application.
  • Non-limiting examples of therapeutic moieties or agents include paclitaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxandrone, chloroambucil, melphalan, 5-fluorouracyl, 5'-desoxy-5-fluorouridine, thioguanine, methotrexate, docetaxel, topotecan, 9-aminocamptothecin, mitopodoside, vinblastine, vincristine, vindesine, vinorelbine, etoposide, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, cis-platinum and cis- platinum analogues, bleomycins, esperamicins, melphalan and other nitrogen mustards.
  • a therapeutic agent may be selected that facilitate its entry into target cells, e.g., tumor cells, once the agent is freed from the conjugate.
  • the agent may be recognized and internalized through particular receptors, or may have such properties as to enable it to pass through the cellular membranes passively.
  • the therapeutic agent and the other components of the conjugate are selected so that they have little or no effect while not associated with the target cells.
  • the linkage of the therapeutic agent can be selected so that the agent is essentially inactive until it is released by cleavage of the cleavable linker.
  • the present conjugates are also useful for imaging and other diagnostic applications in which it is advantageous to spatially and/or temporally control release of a particular agent.
  • agents may include, for example, contrast agents and the like.
  • the present conjugates are designed such that the attached therapeutic moiety is preferentially delivered to a desired site or sites before being released from the conjugate.
  • the release is accomplished using a cleavable linker that can be temporally controlled using an exogenous agent, e.g., light, penetrating radiation, or other type of electromagnetic radiation that is sufficiently energetic to directly or indirectly cause cleavage of a selected cleavable linker, exogenously administered chemical moiety, and the like.
  • an exogenous agent e.g., light, penetrating radiation, or other type of electromagnetic radiation that is sufficiently energetic to directly or indirectly cause cleavage of a selected cleavable linker, exogenously administered chemical moiety, and the like.
  • the present therapeutic conjugate molecules are adaptable to a variety of different therapeutic, prophylactic, and/or diagnostic applications.
  • a particularly appropriate and important application is in treatment of cancers or other localized cells or groups of cells that may be targeted with physical targeting (e.g., by localized injection) or preferably using a targeting moiety that specifically or preferentially causes the conjugate molecule to locate to or in the desired cells.
  • present therapeutic conjugates can be administered by methods suitable for administering other molecules of similar size.
  • the present cleavable conjugates may be administered by any route appropriate to the condition to be treated.
  • the conjugates will usually be administered parenterally, e.g., by injection or infusion, such as subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.
  • pharmaceutical formulations of the present conjugates are typically prepared for parenteral administration with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form.
  • a conjugate having the desired degree of purity can also be optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).
  • the pharmaceutical formulation can be prepared, for example, as a lyophilized formulation or an aqueous solution.
  • Pharmaceutically acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydro
  • the active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules
  • sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the conjugates, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and gamma-ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • the formulations to be used for in vivo administration should be sterile. Sterilization can be accomplished by any of a variety of methods compatible with the components present, e.g., by filtration through sterile filtration membranes.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients may include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbid
  • the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • a sterile injectable preparation such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents, e.g., as mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1 ,3-butane-diol or prepared as a lyophilized powder.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may likewise be used in the preparation of injectables.
  • an aqueous solution intended for intravenous infusion may contain from about 3 to 500 microgram of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • formulations of the present conjugates suitable for oral administration may be prepared as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the conjugate.
  • the formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials.
  • the formulations may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use.
  • sterile liquid carrier for example water
  • Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.
  • Exemplary unit dosage formulations include a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.
  • the invention further provides veterinary compositions comprising at least one conjugate together with a veterinary carrier therefore.
  • Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.
  • the present conjugates may be used to treat various diseases or conditions, e.g., diseases or conditions characterized by the overexpression of a tumor antigen.
  • diseases or conditions include benign or malignant tumors; leukemia and lymphoid malignancies; as well as other disorders such as neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic disorders.
  • the disease or condition to be treated is cancer.
  • cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,
  • the cancer may include HER2-expressing cells, such that the conjugates are able to bind to the cancer cells.
  • HER2-expressing cells such that the conjugates are able to bind to the cancer cells.
  • various diagnostic/prognostic assays are available.
  • ErbB2 overexpression may be analyzed by IHC, e.g. using the HERCEPTEST (Dako).
  • Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a ErbB2 protein staining intensity criteria as follows: Score 0, no staining is observed or membrane staining is observed in less than 10% of tumor cells; Score 1+, a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells, the cells are only stained in part of their membrane; Score 2+, a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells; Score 3+, a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells. Those tumors with 0 or 1+ scores for ErbB2 overexpression assessment may be characterized as not overexpressing ErbB2, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing ErbB2.
  • FISH assays such as the INFORMTM (Ventana Co., Ariz.) or PATHVISIONTM (Vysis, III.) may be carried out on formalin- fixed, paraffin-embedded tumor tissue to determine the extent (if any) of ErbB2 overexpression in the tumor.
  • the cancer to be treated herein may be one characterized by excessive activation of an ErbB receptor, e.g. HER2. Such excessive activation may be attributable to overexpression or increased production of the ErbB receptor or an ErbB ligand.
  • a diagnostic or prognostic assay will be performed to determine whether the patient's cancer is characterized by excessive activation of an ErbB receptor. For example, ErbB gene amplification and/or overexpression of an ErbB receptor in the cancer may be determined.
  • Assays for determining such amplification/overexpression are available in the art and include the IHC, FISH and shed antigen assays described above.
  • levels of an ErbB ligand, such as TGF-alpha., in or associated with the tumor may be determined according to known procedures. Such assays may detect protein and/or nucleic acid encoding it in the sample to be tested. In one embodiment, ErbB ligand levels in the tumor may be determined using immunohistochemistry (IHC); see, for example, Scher et al. (1995) Clin. Cancer Research 1 :545-550. Alternatively, or additionally, one may evaluate levels of ErbB ligand-encoding nucleic acid in the sample to be tested; e.g. via FISH, southern blotting, or PCR techniques.
  • IHC immunohistochemistry
  • ErbB receptor or ErbB ligand overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label (e.g. a radioactive isotope) and externally scanning the patient for localization of the label.
  • a detectable label e.g. a radioactive isotope
  • the appropriate dosage of a conjugate will depend, for example, on the type of disease to be treated, the severity and course of the disease, whether the conjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the conjugate is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of conjugate is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • An exemplary dosage of conjugate to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight.
  • the present conjugates may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second active compound, e.g., a compound also having anti-cancer properties.
  • a second active compound e.g., a compound also having anti-cancer properties.
  • the second compound of the pharmaceutical combination formulation or dosing regimen may have complementary activities to the present conjugates such that they do not adversely affect each other.
  • the second compound may, for example, be a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant.
  • a chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.
  • Other therapeutic regimens may be combined with the administration of an anticancer conjugate as described herein.
  • the combination therapy may be administered as a simultaneous or sequential regimen.
  • the combination When administered sequentially, the combination may be administered in two or more administrations.
  • the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • treatment with the present conjugates involves the combined administration of an anticancer agent, e.g., as identified herein, and one or more chemotherapeutic agents or growth inhibitory agents, including coadministration of cocktails of different chemotherapeutic agents.
  • Chemotherapeutic agents include, for example, taxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers 1 instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).
  • the anticancer agent may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti- progesterone such as onapristone (EP 616812); or an anti-androgen such as flutamide, in dosages known for such molecules.
  • an anti-hormonal compound e.g., an anti-estrogen compound such as tamoxifen; an anti- progesterone such as onapristone (EP 616812); or an anti-androgen such as flutamide
  • an anti-hormonal compound such as tamoxifen
  • an anti- progesterone such as onapristone (EP 616812)
  • an anti-androgen such as flutamide
  • Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the newly identified agent and other chemotherapeutic agents or treatments.
  • the combination therapy may provide "synergy” and prove “synergistic", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect may be attained when the active ingredients are: (1 ) co- formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
  • Combination therapy may be accomplished in a variety of ways.
  • the present conjugates may be provided containing two or more different therapeutic agent moieties, which may be releasable under the same or different conditions.
  • a polymer may be used to which at least two different types of therapeutic agents are linked through photocleavable linkers. Activation of attached photosensitizers will essentially simultaneously release the different therapeutic agents. Similar results can be obtained by using separate, concurrently administered conjugates, where the different conjugates include different therapeutic agents linked through photocleavable linkers.
  • EXAMPLE 1 Demonstration of release of selected moiety using a photosensitizer and a photocleavable linkage.
  • a photosensitizer (Phthalocyanine) was incorporated into white, carboxy modified, polystyrene nanoparticles. These particles were then aminated by coating them with amino-dextran polymer in the presence of EDC. After extensive washing, the amine-modified nanoconjugate was resuspended, characterized for the presence of amines and monodispersity, and stored in double-deionized water for later use (see Construct 1).
  • Construct 1 Amine-modified nanoconjugate.
  • the photocleavabe linker 2-Amino-4-thiazoleacetic acid (Alpha-Aesar) was modified with amine reactive, N-hydroxysuccinimide activated Alexa-546 dye (NHS-Alexa-546, Invitrogen) as a drug surrogate to create the following subcomponent (see Construct 2).
  • Alexa-546 dye NHS-Alexa-546, Invitrogen
  • each nanoconjugate suspension was diluted 1 :10 in double-deionized water and applied to a glass slide for analysis. Each suspension was then centrifuged at 13,000 x g for five minutes and the resulting supernatants applied to glass slides for analysis.
  • Each of the nanoconjugate suspension samples were analyzed microscopically to confirm the presence and release of the Alexa-546 fluorophore (drug surrogate).
  • the analysis included imaging of the photosensitizer nanoconjugate using the excitation/emission wavelengths of the phthalocyanine dye, as well as imaging of the location of signal from the drug surrogate using excitation/emission wavelengths for Alexa-546 (Excitation at 556 nm / Emission at 570 nm).
  • the fluorescence intensity measured in the final nanoconjugate post-laser activation's supernatant exhibits the successful cleavage of the photocleavable linker holding the Alexa-546 fluorophore to the nanoconjugate. It is clear from the lack of fluorescence measured in the pre-laser activated and amino-modified nanoconjugate supernatants that it is the activation of the photosensitizer dye that has caused this cleavage.
  • EXAMPLE 2 Preparation of Exemplary Nanovector Conjugate.
  • An example of a group of related nanovectors containing a drug moiety, a photocleavable linker, a photosensitizer moiety, and a targeting moiety is shown below.
  • This resulting compound is a novel conjugate with a stable, covalent attachment of its drug to a polymer backbone.
  • the conjugate binds specifically to tumor cells, such as breast cancer cell line MDA-MB-453, by way of its tumor specific antibody - Anti-HER2.
  • the drug is released by activating the phthalocyanine with 680 nm light, causing this photosensitizer to rapidly produce singlet oxygen.
  • the singlet oxygen reacts with the cleavable linker that is situated in close proximity, breaking the bond between the polymer and Paclitaxel to release the Paclitaxel at the tumor site. Since free Paclitaxel is very hydrophobic, it will diffuse inside the tumor cell at high concentration for efficient cell killing.
  • Photosensitizer compound is in extremely close proximity to the cleavable linker (an oxazole) ensuring highly efficient cleavage by the generated singlet oxygen (diminished half-life of singlet oxygen in biologic systems no longer a limiting factor) • Highly loaded conjugate ensures delivery of high concentrations of drug to the tumor site (dendrimers, polysaccharides, and other polymers can have thousands of sites for drug loading)
  • a hydrophobic drug molecule that diffuses passively inside the cell and has a relatively long half life around the tumor could be used
  • the lmmunopolymer part of the conjugate can be constructed utilizing many different antibodies, e.g., different monoclonal antibody (MAb) fragments.
  • a demonstration conjugate can include rhuMAb HER2-Fab' (Genentech, Inc.).
  • the antibody can be modified for immunopolymer conjugation by addition of a cysteine residue near the COOH terminus of the recombinant sequence and reacting this with a maleimide activated polymer (Sigma-Aldrich, Inc. or Molecular Probes, Inc.).
  • a negative control consisting of inactivated rhuMAb HER2-Fab', in which the antibody is exposed to a specific inactivating agent resulting in disruption of the antigen-binding domain essential for binding activity can also be prepared.
  • the Phthalocyanine-cleavable linker-Paclitaxel (PCT) portion of the conjugate can be prepared by conjugation of amine-modified Paclitaxel (Sigma- Aldrich, Inc.) to a COOH group on the cleavable linker (Alfa-Aesar, Inc) in the presence of EDC. The remaining amino group on the cleavable linker can then be complexed with Phthalocyanine (Sigma-Aldrich, Inc).
  • the final conjugate can then be prepared by combining the two subcomponents (immunopolymer with remaining amine groups and PCT conjugate).
  • the immunopolymer can be conjugated to the PCT at a ratio of 1000 PCT's per 1 immunopolymer.
  • Assays to measure binding specificity can be used to demonstrate specific conjugate binding to tumor cells.
  • anti-HER2 antibody conjugated to the nanovector will provide specific binding to MDA-MB-453, a cell line expressing this known tumor antigen.
  • Direct measurement of the conjugate's fluorescence (Excitation 680 nm / Emission 700 nm) on a fluorescence microscope will allow its level of specific binding to a cancer cells to be assessed.
  • MDA-MB-453 cells will be incubated with the nanovector at various concentrations and centrifuged to separate cells from unbound nanovector. The cells will then be applied to glass microscope slides and measured fluorometrically to determine the level of nanovector binding.
  • a separate assay measuring the degree of drug release from the conjugate can also advantageously be used.
  • the levels of free drug relative to conjugated drug can be quantified by homogeneous Taxol immunoassay (ALPHAscreen, Perkin-Elmer). Briefly, a sample of nanovector can be activated with a 680 nm laser. This released drug and conjugate can then be separated via dialysis, and the level of drug remaining on the conjugate can be measured and compared to the amount on the conjugate initially.
  • cell line MDA-MB-453 can be obtained from American Type Culture Collection. This cell line has been extensively characterized for HER2 expression by flow cytometry, homogeneous immunoassay, ELISA, and immunohistochemistry (Lewis et a!.. 1993). lmmunohistochemistry (IHC) scoring of HER2 expression on a semiquantitative scale (0-3+) was first developed for trastuzumab clinical trials and is now routinely used clinically (Park. 2000; Mass et al., 2001) .
  • nanoconjugate It can also be beneficial to conduct pharmacokinetic studies using a selected therapeutic nanoconjugate.
  • healthy, adult, Sprague Dawley rats can be used which will receive single or multiple intravenous injections of the complete nanovector conjugate, polymer-phthalocyanine, or free drug via indwelling jugular venous catheters. After the injection, blood will be serially sampled via catheter up to 48 h post injection, and plasma will be assayed for nanovector and Paclitaxel. Paclitaxel will be measured by homogeneous immunoassay method while nanovector or polymer-phthalocyanine concentration will be determined by flourometric analysis after extraction from plasma.
  • tumor cells can be implanted subcutaneously with or without Matrigel in the dorsum of athymic nude mice or SCID mice.
  • mice will be randomly assigned to different treatment groups (5-15 mice/group per condition). All intravenous treatments will be performed via tail vein injection.
  • Anti-HER2 nanovector will be administered intravenously at 20 mg Paclitaxel/kg/dose every week for 3 weeks, for a total dose of 60 mg/kg. Additional treatment groups will be included as in the pharmacokinetic studies. Saline (PBS) will be administered intravenously at the same injection volume and schedule as the nanovector. Free Paclitaxel will also be administered intravenously at its MTD of 25 mg/kg on the same schedule as the nanovector.
  • Negative control nanovector will be prepared identically as described above with the omission of the MAb conjugation step or the photosensitizer. All preparations of the negative control conjugate will be administered intravenously at the same dose and schedule as the complete nanovector.
  • mice will receive illumination of the tumor site with a 6.2 mm diameter, 670 nm, 300 mW PDT laser (Edmund Optics) following standard PDT administration energies (i.e. 25 J/cm 2 ) and techniques (Kolarova et al., 1999).
  • Tumors will be measured twice a week by caliper, and tumor volumes will be calculated using the equation: length x width x depth x 0.5. Mice with complete tumor regressions by measurement will be necropsied at the end of the study and classified as "cured” if no residual tumor cells are detected on histopathological examination of the tumor injection site.
  • Noise in an assay is defined as the standard deviation from the mean of a zero input (negative control).
  • Assay sensitivity is defined as the input resulting in a signal-to-noise ratio of three.
  • Assay Dynamic Range will be calculated by dividing the highest input yielding a change in signal by the sensitivity of the assay.

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Abstract

La présente invention concerne la construction et l'utilisation de molécules de nanoconjugués qui incorporent un lieur, clivable par un agent exogène, en même temps qu'un groupement thérapeutique ou diagnostique. Ces nouveaux nanoconjugués utilisent un mécanisme de libération médicamenteuse qui peut incorporer des médicaments de thérapie photodynamique, ou des composés similaires, et un lieur apparié clivable pour cibler, imager et/ou administrer des concentrations thérapeutiques de médicament libre à des cellules cibles telles que des cellules cancéreuses. Par conséquent, cette nanomédicine procure un système d'administration de médicament hautement spécifique et puissant, présentant un indice thérapeutique hautement amélioré du médicament libre.
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WO2014004278A1 (fr) * 2012-06-26 2014-01-03 The Curators Of The University Of Missouri Conjugués de médicament photoclivables
EP3079664A4 (fr) * 2013-12-10 2017-06-28 The Regents of The University of California Nanoparticules pour administration de médicament activé dans certaines régions
WO2018156815A1 (fr) * 2017-02-23 2018-08-30 Aspyrian Therapeutics, Inc. Compositions thérapeutiques et méthodes associées pour la photoimmunothérapie
WO2019109126A1 (fr) * 2017-12-06 2019-06-13 Newsouth Innovations Pty Limited Système liposomal pour l'administration de médicaments
US10538590B2 (en) 2010-07-09 2020-01-21 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Photosensitizing antibody-fluorophore conjugates
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US11013803B2 (en) 2015-08-07 2021-05-25 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Near infrared photoimmunotherapy (NIR-PIT) of suppressor cells to treat cancer
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CN115926177A (zh) * 2022-11-11 2023-04-07 四川大学 一种聚合物、聚合物胶束及其制备方法和应用
CN116963778A (zh) * 2021-01-29 2023-10-27 波尔多大学 包含光敏剂单元的生物缀合物、其制备方法及其用途

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JP2012525409A (ja) * 2009-04-28 2012-10-22 セラムオプテック ゲーエムベーハー テトラピロール誘導体のための新規の経口製剤
US11364298B2 (en) 2010-07-09 2022-06-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Photosensitizing antibody-fluorophore conjugates
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US12296013B2 (en) 2010-07-09 2025-05-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Photosensitizing antibody-fluorophore conjugates
EP2438908A1 (fr) 2010-10-11 2012-04-11 Vectum Pharma, S.L. Compositions d'ancrage pour applications topiques
WO2012049453A2 (fr) 2010-10-11 2012-04-19 Vectum Pharma S.L. Compositions destinées à des applications topiques
US20170014510A1 (en) * 2012-04-24 2017-01-19 The Board Of Regents Of The University Of Oklahoma Singlet Oxygen-Labile Linkers and Methods of Production and Use Thereof
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US20150165026A1 (en) * 2012-04-24 2015-06-18 The Board Of Regents Of The University Of Oklahoma Singlet oxygen-labile linkers and methods of production and use thereof
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US10159735B2 (en) 2012-06-26 2018-12-25 The Curators Of The University Of Missouri Photocleavable drug conjugates
US20150328314A1 (en) * 2012-06-26 2015-11-19 The Curators Of The University Of Missouri Photocleavable drug conjugates
WO2014004278A1 (fr) * 2012-06-26 2014-01-03 The Curators Of The University Of Missouri Conjugués de médicament photoclivables
EP3079664A4 (fr) * 2013-12-10 2017-06-28 The Regents of The University of California Nanoparticules pour administration de médicament activé dans certaines régions
US10830678B2 (en) 2014-08-08 2020-11-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Photo-controlled removal of targets in vitro and in vivo
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US11147875B2 (en) 2015-08-18 2021-10-19 Rakuten Medical, Inc. Compositions, combinations and related methods for photoimmunotherapy
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