Compositions and methods for tissue protection and therapuetic agent delivery

Abstract

New compositions and methods are described herein operable for delivery of therapeutic agents while mitigating damage to healthy tissues. In one aspect, a conjugate comprises a linker joining a pharmaceutical component and a targeting ligand, wherein the linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release of the targeting ligand and / or the pharmaceutical component from the conjugate.

Description

[0001] COMPOSITIONS AND METHODS FOR TISSUE PROTECTION AND THERAPUETIC AGENT DELIVERY

[0002] STATEMENT OF GOVERNMENT RIGHTS

[0003] This invention was made with government support under Grant Numbers HL153744 and HL 176666 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] RELATED APPLICATION DATA

[0005] The present application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to United States Provisional Patent Application Serial Number 63 / 677,804 filed July 31, 2024 which is incorporated herein by reference in its entirety.

[0006] FIELD

[0007] The present invention relates to compositions and methods for the delivery of therapeutic agents to diseased tissue in a patient and, in particular, to compositions operable for delivery of therapeutic agents while mitigating damage to healthy tissues.

[0008] BACKGROUND

[0009] Various compositions have been employed for the delivery of therapeutic agents to diseased tissue and / or diseased sites in a patient. Significant efforts have been undertaken to design therapeutic agents having selectively for the diseased tissue to be treated. Various targeting ligands and associated strategies, for example, have been employed to bind or locate therapeutic agents to the diseased tissue. Once bound to or located in the diseased tissue, the therapeutic agents are locally activated, thereby preserving healthy tissue and / or mitigating damage to healthy tissue.

[0010] Problems with the foregoing targeting strategies arise when the targeting ligand cannot effectively differentiate between healthy and diseased cells and / or when the location of the diseased tissue is unknown or generally widespread, such as metastatic situations. In several cases, healthy cells express receptors being targeted on the diseased cells. For example, widely overexpressed on prostate cancer tissues, Prostate Specific Membrane Antigen (PSMA) has become an attractive target for prostate cancer management. Despite exciting progress, PSMA targeted therapeutic agents could pose high radiation exposure towards normal organs that express the receptor. In fact, salivary glands (SGs) are often the dose-limiting organ that determines how much radioactivity can be administered, patient eligibility and the treatment outcome thereafter. A number of targeting moieties suffer from this same problem.

[0011] SUMMARY

[0012] In view of the foregoing disadvantages, new compositions and methods are needed that are operable for delivery of therapeutic agents while mitigating damage to healthy tissues. In one aspect, a conjugate comprises a linker joining a pharmaceutical component and a targeting ligand, wherein the linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release of the targeting ligand from the conjugate. In another aspect, a conjugate comprises a linker joining pharmaceutical component and a targeting ligand, wherein the linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release of the pharmaceutical component from the conjugate. In another aspect, a conjugate comprises a linker joining a pharmaceutical component and a targeting ligand, wherein the linker is at least one of radiolabile photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release of the targeting ligand or the pharmaceutical component, wherein the targeting ligand is operable to bind with receptors of diseased cells and receptors of non-diseased cells. Pharmaceutical components of conjugates can comprise a therapeutic agent and / or an imaging agent.

[0013] As described further herein, use of a linker operable for degradation in response to the application of external energy (e.g. radiolabile, photolabile, thermolabile, magnetic field, or acoustolabile) can permit release of the targeting ligand prior to or after the conjugate binds to healthy tissue. Additionally, use of a linker operable for degradation in response to reaction with a releasing agent can permit release of the targeting ligand prior to or after the conjugate binds to healthy tissue. In this way, the pharmaceutical component is precluded from binding to or remaining bound to healthy cells in regions where the external energy is applied. Alternatively, use of the degradable linker can permit release of the pharmaceutical component or imaging agent from the conjugate prior to or after binding of the targeting ligand to cells or tissue. In such embodiments, external energy (e.g. radiolabile, photolabile, thermolabile, magnetic field, or acoustolabile) or a releasing agent can be applied to regions of healthy tissue to separate the pharmaceutical component from the conjugate prior to the pharmaceutical component damaging the cells to which the conjugate is bound.

[0014] In another aspect, methods of detecting and / or treating diseased tissue are provided. In some embodiments, a method of treating diseased tissue comprises administering to a patient in need thereof a conjugate comprising a linker joining a pharmaceutical component and a targeting ligand, and locating the pharmaceutical component at one or more regions of the diseased tissue via binding the conjugate to diseased cells with the targeting ligand. The diseased tissue is imaged or treated with the pharmaceutical component. Radiological, photonic, thermal, magnetic field, and / or acoustic energy is applied to one or more locations of non- diseased / healthy tissue in the patient to release the targeting ligand from the conjugate. Alternatively, a releasing agent is introduced at one or more locations of non-diseased / healthy tissue in the patient. The releasing agent, for example, can be locally / directly introduced at the one or more locations of non-diseased / healthy tissue in the patient. The releasing agent chemically reacts with the conjugate to release the targeting ligand from the conjugate. Release of the targeting ligand precludes binding of the conjugate to cells of the non-diseased tissue. Moreover, in some embodiments, release of the targeting ligand separates the linker and pharmaceutical component from cells of non-diseased / healthy tissue bound to the targeting ligand.

[0015] In another aspect, a method of detecting and / or treating diseased tissue comprises administering to a patient in need thereof a conjugate comprising a linker joining pharmaceutical component and a targeting ligand, and locating the pharmaceutical component at one or more regions of the diseased tissue via binding the conjugate to diseased cells with the targeting ligand. The diseased tissue is imaged or treated with the pharmaceutical component. Radiological, photonic, thermal, magnetic field, and / or acoustic energy is applied to one or more locations of non-diseased / healthy tissue to release the pharmaceutical component from the conjugate. Alternatively, a releasing agent is introduced at one or more locations of non- diseased / healthy tissue in the patient. The releasing agent chemically reacts with the conjugate to release the pharmaceutical component from the conjugate. The releasing agent, for example, can be locally / directly introduced at the one or more locations of non-diseased / healthy tissue in the patient. The releasing agent, for example, can be injected into the one or more locations of non-diseased / healthy tissue, such as healthy organs. Release of the pharmaceutical component can occur prior to binding of the targeting ligand to cells of the non-diseased / healthy tissue. Moreover, release of the pharmaceutical component can separate the pharmaceutical component from cells of the non-diseased / healthy tissue bound to the targeting ligand.

[0016] These and other embodiments are further described in the following detailed description. BRIEF DESCRIPTION OF THE FIGURES

[0017] FIGS. 1A-1C illustrate various conjugate constructions employing a cobalamin linker, PSMA targeting ligand, and chelator for binding a metallic radioisotope for imaging or radiotherapy.

[0018] FIG. 2A is a fluorescent dye-cobalamin scaffold coupled to a PSMA ligand via a photolabile bond.

[0019] FIG. 2B illustrates uptake of the scaffold of FIG. 2A by PSMA positive cells.

[0020] FIG. 2C are liquid chromatography-mass spectrometry results confirming cleavage of the PSMA ligand from the cobalamin scaffold of FIG. 2A.

[0021] FIG. 3 are optical images showing reduction of uptake of the cobalamin scaffold of FIG. 2A in salivary gland areas of mice.

[0022] FIG. 4 illustrates various cobalamin scaffolds operable to serve as linkers in conjugates according to some embodiments described herein.

[0023] FIG. 5 A and FIG. 5B are axial PET images prior and post irradiation of a test subject, respectively.

[0024] FIG. 6A and FIG. 6B are coronol PET images prior and post irradiation of a test subject, respectively.

[0025] FIG. 7 provides results of a tumor model, wherein the B12 conjugates slowed and / or reduced tumor size and growth, according to some embodiments.

[0026] FIG. 8A and FIG. 8B provide PET tumor imaging results with a conjugate described herein pre- and post-irradiation, respectively.

[0027] DETIALED DESCRIPTION

[0028] Embodiments described herein can be understood more readily by reference to the following detailed description and examples and their previous and following descriptions. Elements, apparatus and methods described herein, however, are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

[0029] I. Conjugates

[0030] As described herein, a conjugate comprises a linker joining a pharmaceutical component and a targeting ligand, wherein the linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release of the targeting ligand or the pharmaceutical component. A targeting ligand includes molecules with an affinity to bind, or be bound by, a specific cell type, tissue type, lesion, and / or tumor type. In some embodiments, the affinity is due to the presence of a specific receptor protein or other molecule on the surface of cells in the tissue, lesion, and / or tumor. In some embodiments, the targeting ligand forms a covalent or noncovalent attachment to the receptor protein or other molecule. Targeting ligands include, but are not limited to, those molecular species operable to bind to receptors on diseased cells as well as healthy cells. In some embodiments, for example, a targeting ligand comprises a prostate-specific membrane antigen (PSMA), or derivatives thereof. Targeting ligands also include small molecules, peptides, proteins, carbohydrates, aptamers, antibodies, antibody fragment, antibodies or an antigen binding fragment or derivatives thereof, oligonucleotides, and other organic targeting molecules. Specific identity of targeting ligands can be selected according to the disease or indication to be treated in the patient. In some embodiments, targeting ligands include folic acid, mammalian bombesin (BBN), fibroblast activation protein (FAP), netrin-4 (Net4), octreotide, neurotensin receptor 1 (NTSR), choline, RGD ligands, polysaccharides, oncogene trophoblast cell surface antigen 2 (Trop2), epidermal growth factor receptor (EGFR) ligands (including Her2 and Her3), Nectin-4, transferrin, and poly(ADP-ribose) polymerase (PARP) inhibitors.

[0031] A pharmaceutical component associated with the targeting ligand via the linker can be a therapeutic agent (including therapeutic radioisotope), an imaging agent, or a combination thereof. A therapeutic agent is intended to mean a molecule that will, when provided to a subject in a therapeutically effective amount, provide some improvement or benefit to the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. In some embodiments, the therapeutic agent may be a chemotherapeutic agent. Examples of a therapeutic agent include, but are not limited to, a PARP inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a tropomyosin receptor kinase (Trk) inhibitor, a human carbonic anhydrase IX (hCA) inhibitor, ado-trastuzumab emtansine (T-DM1), Paclitaxel (PTX), Doxorubicin (Dx), oleuropein, arsenic trioxide, plumbagin, resveratrol, chlorogenic acid, polypeptides, siRNAs, and oligonucleotides. In some embodiments, the therapeutic agent may be an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent may be a stimulator of interferon genes protein (STING) agonist, a programmed cell death protein 1 (PD1) or PD-L1 ligand, a cytotoxic T-lymphocyte associated antigen 4 (CTLA4) ligand, a T-cell immunoglobulin domain and mucin domain-3 (Tim-3) ligand, a T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain protein (TIGIT) ligand, a lymphocyte activation gene-3 (LAG-3) ligand, a nuclear receptor subfamily 2 group F member 6 (NR2F6) ligand, a V-set immunoregulatory receptor (VISTA) ligand, or a B and T lymphocyte attenuator (BTLA) ligand.

[0032] In some embodiments, a therapeutic agent comprises a radioisotope for radiotherapy. Radiotherapy can be employed to treat cancer whereby one or more cancer cells or tumors are destroyed using the radiation emitted from a radioisotope. Suitable radioisotopes for radiotherapy can include177Lu,90Y,125I,131I,225Ac,211At,212Pb,55Co, and38mCo, in addition to the radioisotopes listed below. Examples of radiation used in radiotherapy include alpha rays, beta rays, and Auger electrons.

[0033] “Radioisotope” or “radionuclide”, as used herein, refers to synthetic and / or naturally occurring atoms that have excess nuclear energy and where this excess energy is emitted as radiation. Examples of this type of radiation energy are alpha rays, beta rays, and gamma rays. Examples of radioisotopes includeUC,13N,150,18F,43Sc,44gSc,47Sc,45Ti,52Mn,55Co,58mCo,61Cu,64Cu,67Cu,67Ga,68Ga,76Br,77Br,82Br,89Zr,87Y,89Sr,90Y, "mTc,mIn,123I,124I,125I,131I,134Ce,134La,149Tb,152Tb,153Sm,155Tb,161Tb,177Lu,186 / 188Re,2O1T1,211At,203Pb,212Pb,212Bi,213Bi,223Ra,225Ac,226Th,227Th, and / or230U.

[0034] Additionally, an imaging agent of the pharmaceutical component comprises a radiolabel or radiotracer for imaging the diseased tissue. The terms “radioactive label”, “radiolabel”, or “radiotracer” are used herein to refer to a radioisotope that is used to generate an image that is detectable often using an appropriate instrument. Examples of techniques that use radiotracers include positon emission tomography (PET) and single photon emission computed tomography (SPECT). Examples of radiation emitted by radiotracers include gamma rays and X-rays. Suitable radiolabels includenC,18F,64Cu,89Zr,124I, and90Sr.

[0035] In some embodiments, the therapeutic agent or imaging agent comprises a chelator for binding a metallic radioisotope. The terms “chelate”, “chelating agent”, or “chelator” are intended to mean a molecule with two or more functional groups that are able to donate at least two electron pairs and so bind a metal ion. It is common for the chelating agent to be an organic molecule. One chelating agent will often use its electron pairs to form a coordinate bond with one metal ion, though it is possible for a chelating agent to bind more than one metal ion. Examples of chelating compounds include, but are not limited to, dimercaptopropanol, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), octadentate macrocyclic bifunctional l,4,7,10-tetraazacyclododacane-l,4,7,10-tetraacetic acid (DOTA), hexadentate macrocyclic bifunctional l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), hydroxyethylidene diphosphonic acid (HEDP), ethylenediamine-N,N,N',N'- tetrakis(methylenephosphonic acid) (EDTMP), 1,4, 7,10-tetraazacyclododecane- 1,4, 7,10- tetraaminomethylenephosphonic acid (DOTMP), mercaptoacetyltriglycine (MAG3), Sar cage, DiAm-Sarcage, l,10-diaza-18-crown-6, Cross Bridge-Cyclam, Cross Bridge-TE2A, DF chelators, salicylic acid, triethanolamine, ferri oxamines, macropa or derivatives thereof, and ionophores.

[0036] Alternatively, one or more radioisotopes of therapeutic agent or imaging agent is bound to an organic moiety, such as an aryl or heteroaryl group.

[0037] As described herein, the pharmaceutical component and targeting ligand are joined by a linker. The linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release targeting ligand from the conjugate. Alternatively, the linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release of the pharmaceutical component from the conjugate. In some embodiments, the linker comprises a transition metal complex. For example, the linker can be selected from various cobalamin (Cbl / Vitamin B12) scaffolds. The Coni-C bond of alkyl cobalamins is operable to undergo homolytic cleavage upon absorption of light within the 330-575 range. In such embodiments, this homolytic cleavage can be employed to separate the targeting ligand from the cobalamin linker. The pharmaceutical component, whether therapeutic agent or imaging agent, can remain bound to the cobalamin. The pharmaceutical component, for example, can be part of a substitution on the corrin ring. In some embodiments, the pharmaceutical component is bound to cobalt of the transition metal complex linker via a nitrogen.

[0038] In other embodiments, the pharmaceutical component is associated with photolytic cleavage of the Coni-C bond, and the targeting ligand remains bound to cobalamin. The targeting ligand, for example, can be bound to cobalt of the transition metal complex linker via a nitrogen.

[0039] The Coni-C chain attaching the targeting ligand or pharmaceutical component to the cobalamin complex can have any desired moieties or character. In some embodiments, the chain is aliphatic and hydrophobic in character. In other embodiments, the chain can be hydrophilic, including one or more hydrophilic moieties, such as alkylene oxide moieties (e.g. polyethylene glycol), urea moieties, and / or thiourea moieties. Metal complexes in addition to cobalamin scaffolds for use as a linker can include ruthenium bipyridyl complexes and silicon phthalocyanine scaffolds.

[0040] In some embodiments, the optical window of photocleavage of metal complex linkers, including cobalamin linkers, can be red shifted or extended by modifying the metal complex linker with one or more conjugated light absorbing species including, but not limited to, one or more dyes or fluorophores. The optical window, for example, can extend from 600 nm to 800 nm by appending fluorophores such as sulfoCy5, Bodipy650m, Alexa700, Atto725 and Dylight800. Other fluorophores are also available for modifying the metal complex linker. In some embodiments, the one or more conjugated light absorbing species are attached to the corrin ring and are independent of the targeting ligand and pharmaceutical component. Alternatively, the one or more conjugated light absorbing species can reside on the ligand structure binding the targeting ligand or pharmaceutical component to cobalt of the transition metal complex linker. FIGS. 1 A-1C illustrate various conjugate constructions employing a cobalamin linker, PSMA targeting ligand, and chelator for binding a metallic radioisotope for imaging or radiotherapy.

[0041] In some embodiments wherein transition metal complexes are used as a linker, the transition metal itself can be a radioisotope for imaging and / or radiotherapy. For example, cobalt of cobalamins described herein can be55Co or58mCo for imaging and radiotherapy applications. In such embodiments, the transition metal complex linker can serve both as a linker for the targeting ligand and a pharmaceutical component. Additionally, one or more additional pharmaceutical components can be attached to the radioisotope transition metal complex as described herein.

[0042] In some embodiments, a conjugate comprising a transition metal complex linker is of Formula I: wherein the corrin ring, R’-R3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, and TL is a targeting ligand. In some embodiments, Ei and E2 independently comprise one or more moieties selected from the group consisting of alkyl, alkylamine, urea, thiourea, amide, alkylene oxide, aryl, and heterocyclyl . In some embodiments, the moiety of R* comprises a chelator. Alternatively, the at least one radionuclide of R* can be bound to an organic moiety, such as an aryl or heteroaryl group.

[0043] In some embodiments, the conjugate is of Formula la: wherein the corrin ring, RJ-R3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, TL is a targeting ligand, and Sc is a conjugated light absorbing species. In some embodiments, Sc is any of the conjugated species described herein, including dyes and fluorophores. In some embodiments, the moiety of R* comprises a chelator. Alternatively, the at least one radionuclide of R* can be bound to an organic moiety, such as an aryl or heteroaryl group. In some embodiments, a transition metal complex of Formula Ila is provided in FIG. 1C.

[0044] A conjugate comprising a transition metal complex, in some embodiments, is of Formula II: wherein the corrin ring, RJ-R3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, and TL is a targeting ligand. In some embodiments, Ei and E2 independently comprise one or more moieties selected from the group consisting of alkyl, alkylamine, urea, thiourea, amide, alkylene oxide, aryl, and heterocyclyl. In some embodiments, the moiety of R* comprises a chelator. Alternatively, the at least one radionuclide of R* can be bound to an organic moiety, such as an aryl or heteroaryl group.

[0045] In some embodiments, the conjugate is of Formula Ila:

[0046] wherein the corrin ring, RJ-R3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, TL is a targeting ligand, and Sc is conjugated light absorbing species. In some embodiments, Sc is any of the conjugated species described herein, including dyes and fluorophores. In some embodiments, the moiety of R* comprises a chelator. Alternatively, the at least one radionuclide of R* can be bound to an organic moiety, such as an aryl or heteroaryl group. In some embodiments, a transition metal complex of Formula Ila is provided in FIGS. 1A and IB.

[0047] In addition to transition metal complexes, suitable linkers of conjugates can comprise small molecules. In some embodiments, the linker is selected from BODIPY caged compounds, cyanine scaffolds, aminobenzoquinone scaffolds, and coumarin-based caging groups.

[0048] The linker, in some embodiments, is chemically labile. The linker, for example, can chemically react with one or more species to release the targeting ligand and / or pharmaceutical component. In some embodiments, a releasing agent is introduced into the environment of the conjugate, wherein the releasing agent reacts with the linker to release the targeting ligand or the pharmaceutical component from the conjugate. The releasing agent can be introduced into healthy tissue or organs according to methods described herein to preclude binding of the conjugate to the healthy tissue or release the pharmaceutical component for clearing once the conjugate is bound to the healthy tissue. In some embodiments, the linker comprises one or more click chemistry moieties. When the complimentary click chemistry moiety is introduced, the linker undergoes chemical reaction with the complimentary moiety and the targeting ligand or pharmaceutical component is released from the conjugate. For example, the linker can comprise a tetrazine moiety that reacts with a TCO releasing agent introduced into the environment of the linker. The TCO and tetrazine react to release the targeting ligand or the pharmaceutical component.

[0049] In some embodiments, the targeting ligand and / or pharmaceutical component can be released from the linker via a pH change. The pH of the local environment can be altered to induce release of the targeting ligand and / or pharmaceutical component. In some embodiments, a change in pH can result in hydrolysis or other chemical reaction for release of the targeting ligand and / or pharmaceutical component from the conjugate.

[0050] The linker employed in conjugates described herein, in some embodiments, is magnetically labile. The linker, for example, can be responsive to changes or alterations in applied magnetic fields resulting in release of the targeting ligand and / or pharmaceutical component. In some embodiments, for example, alterations in magnetic fields provided by magnetic resonance imaging apparatus or other apparatus can induce release of the targeting ligand and / or pharmaceutical component from the conjugate.

[0051] The linker, pharmaceutical component and / or targeting ligand, in some embodiments, can be functionalized / derivatized with click chemistry moieties for facile synthesis of conjugates. Suitable click chemistry moieties can be selected from the group consisting of dibenzocyclooctane (DBCO), bicycle[6.1.0]nonyne (BCN), transcyclooctane (TCO), tetrazine, alkyne and azide. In some embodiments, for example, the cobalamin scaffold can be functionalized with one or more click chemistry moieties. The click chemistry moieties can be associated with the cobalt metal center and / or the macrocyclic ring. Such click chemistry moieties can react with complimentary click chemistry moieties on the targeting ligand, pharmaceutical component (including chelator), and / or other functional structures such as an antenna for altering the light absorption profile of the conjugate. In this way, conjugate construction / synthesis can be modular, permitting a number of different conjugate structures to be readily available.

[0052] Conjugates, in some embodiments, employ a pharmaceutical component comprising a nanoparticle architecture. For example, the therapeutic agent and / or imaging agent can be contained within a nanoparticle or associated with a surface of the nanoparticle. The linker couples the nanoparticle with the targeting ligand. The linker can reside on the nanoparticle surface or proximate the nanoparticle surface for binding with the targeting ligand. Conjugates comprising a nanoparticle pharmaceutical component can operate according to methods described herein where the targeting ligand or the pharmaceutical component is released form the conjugate upon application of radiological, photonic, thermal, and / or acoustic energy. Application of radiological, photonic, thermal, and / or acoustic energy can cleave the targeting ligand or the pharmaceutical component comprising the nanoparticle from the linker, for example.

[0053] Alternatively, photoisomerization can be employed with conjugates comprising nanoparticles to control release of therapeutic agents and / or imaging agents. In some embodiments, the nanoparticle containing a therapeutic agent and / or imaging agent also comprises molecular species operable to undergo isomerization when exposed to light. For example, a molecular species undergoes a conformational change from trans- to cis- or vice versa upon light exposure. This conformational change can increase permeability of the nanoparticle, thereby permitting release of the therapeutic agent and / or imaging agent. The photoisomeric species, for example, can reside in pores of the nanoparticle whereby the conformational change expands the pores and / or increases pore volume for release of the pharmaceutical component. In some embodiments, light is applied to areas of healthy or nondiseased tissue to release the therapeutic agent and / or imaging agent from the nanoparticle of the conjugate prior to entry of the nanoparticle into healthy cells via endocytosis, membrane fusion or diffusion. Such release via photoisomerization can permit clearance of therapeutic agent and / or imaging agent prior to any damage to healthy tissues. In some embodiments, liposomes are nanoparticles that can be paired with photoisomeric compounds to effectuate the foregoing release mechanism. In some embodiments, photoisomeric species comprise azobenzene, spiropyran, and diarylethene.

[0054] Photoisomerization may also play a role in the targeting ligand. In some embodiments, the targeting ligand exhibits photoisomerization. Irradiating the targeting ligand can induce a conformational change whereby the targeting ligand becomes inactive for interaction with the desired cellular environment. In this way, the binding activity of the targeting ligand can be negated, even if the targeting ligand is not cleaved from the conjugate. Accordingly, regions of healthy or non-diseased tissue can be irradiated to inactivate the targeting ligand of any conjugate present in the irradiated regions. Inactivation of the targeting ligand can facilitate clearance of the conjugate prior to any damage to healthy or non-diseased tissue. Photoisomerization of the targeting ligand can be applied with any conjugate architecture described herein.

[0055] II. Methods of Treating and Diagnosing Diseases

[0056] In another aspect, methods of detecting and / or treating diseased tissue are provided. In some embodiments, a method of treating diseased tissue comprises administering to a patient in need thereof a conjugate comprising a linker joining a pharmaceutical component and a targeting ligand, and locating the pharmaceutical component at one or more regions of the diseased tissue via binding the conjugate to diseased cells with the targeting ligand. The diseased tissue is imaged or treated with the pharmaceutical component. Radiological, photonic, thermal, magnetic field, and / or acoustic energy is applied to one or more locations of non-diseased tissue to release the targeting ligand from the conjugate. Alternatively, a releasing agent is introduced at one or more locations of non-diseased / healthy tissue in the patient. The releasing agent, for example, can be locally / directly introduced at the one or more locations of non-diseased / healthy tissue in the patient. In some embodiments, the releasing agent is injected into the non- diseased / healthy tissue. The releasing agent chemically reacts with the conjugate to release the targeting ligand from the conjugate.

[0057] Release of the targeting ligand precludes binding of the conjugate to cells of the nondiseased tissue. Moreover, in some embodiments, release of the targeting ligand separates the linker and pharmaceutical component from cells of the non-diseased tissue bound to the targeting ligand. The ability to release the targeting ligand from the conjugate via selective application of external energy can facilitate or enable the preservation of healthy tissue when diagnosing and treating metastatic cancers. In some embodiments, the pharmaceutical component bound to the linker is not in a form for cellular uptake.

[0058] In another aspect, a method of detecting and / or treating diseased tissue comprises administering to a patient in need thereof a conjugate comprising a linker joining pharmaceutical component and a targeting ligand, and locating the pharmaceutical component at one or more regions of the diseased tissue via binding the conjugate to diseased cells with the targeting ligand. The diseased tissue is imaged or treated with the pharmaceutical component. Radiological, photonic, thermal, magnetic field and / or acoustic energy is applied to one or more locations of non-diseased tissue to release the pharmaceutical component from the conjugate. Release of the pharmaceutical component can occur prior to binding of the targeting ligand to cells of the non-diseased tissue. Alternatively, release of the pharmaceutical component separates the pharmaceutical component from cells of the non-diseased tissue bound to the targeting ligand.

[0059] In other embodiments, a releasing agent is introduced at one or more locations of non- diseased / healthy tissue in the patient. The releasing agent chemically reacts with the conjugate to release the pharmaceutical component from the conjugate. The releasing agent, for example, can be locally / direclty introduced at the one or more locations of non-diseased / healthy tissue in the patient. The releasing agent, for example, can be injected into the one or more locations of non-diseased / healthy tissue, such as healthy organs.

[0060] Use of a linker operable for degradation in response to the application of external energy (e g. radiolabile, photolabile, thermolabile, magnetic field, or acoustolabile) can permit release of the targeting ligand prior to the conjugate binding to healthy tissue. Additionally, use of a linker operable for degradation in response to reaction with a releasing agent can permit release of the targeting ligand prior to or after the conjugate binds to healthy tissue. In this way, the pharmaceutical component or imaging agent is precluded from binding to healthy cells in regions where the external energy is application. Alternatively, use of the degradable linker can permit release of the pharmaceutical component from the conjugate after binding of the targeting ligand to cells or tissue. In such embodiments, external energy or a releasing agent can be applied to regions of healthy tissue to separate the pharmaceutical component from the conjugate prior to the pharmaceutical component damaging the cells to which the conjugate is bound.

[0061] In view of the foregoing capabilities, conjugates described herein find application when diagnosing and treating metastatic cancers and / or other diseases affecting widespread areas of the body. Moreover, the foregoing capabilities can also greatly expand the library of targeting ligands employed for disease diagnosis and treatment by permitting use of ligands binding to both diseased and healthy tissues. Conjugates employed in methods described herein can have any composition and / or properties described in Section I above.

[0062] These and other embodiments are further illustrated in the following non-limiting examples. EXAMPLE 1 - Photolytic Cobalamin Scaffold

[0063] A PSMA targeted cobalamin complex (5) was synthesized having structure in FIG. 2A. Confocal fluorescent microscopy demonstrated that transfected PC3-PSMA (PSMA positive) cells have significant uptake of 5, compared with background signal for native PC3 cells (PSMA negative) (FIG. 2B). Fluorescence-activated cell sorting (FACS) analysis further confirmed the selectivity of 5 toward PSMA positive cells. Furthermore, liquid chromatography-mass spectrometry (LC-MS) confirmed the cleavage of Co-C bond in 5 upon 3 s of 650 nm red light illumination (FIG. 2C), which demonstrates the “un-locking” capability of the constructed complex.

[0064] Fluorescence imaging of PSMA positive tumors in vivo was employed to determine whether methods described herein can reduce salivary gland (SG) uptake of 5. Compound 5 (2.5 nmol) was injected to each animal. In Group 1, the syringe and catheter were illuminated with red light, which cleaves the PSMA motif from B12-Cy5 motif during injection. Indeed, under these conditions, there is minimal uptake of Cy5 fluorescence by the PSMA positive tumor (PC3-PSMA) and SG due to the lack of specific binding to PSMA by the cleaved Bl 2-Cy 5. In Group 2, injections were performed in dark to minimize un-wanted cleavage. As expected, both SG and PC3-PSMA tumor display apparent uptake of 5. Animals in Group 3 the SGs were illuminated using 650 nm light. Other parts of the body were wrapped with black plastic sheets. As shown in FIG. 3, it was notable to observe dramatically reduced SG uptake of Cy5 fluorescence while the tumor uptake is largely maintained. We have previously demonstrated that the mild illumination conditions required to trigger photolysis does not compromise fluorescence integrity, thereby ruling out the possibility of photobleaching.

[0065] EXAMPLE 2 - PSMA-targeted Cobalamin Conjugates for Radioimaging or Radiotherapy

[0066] In the present example, B12 conjugates were constructed by appending the PSMA ligand to the Co of cobalamin. Upon illumination, the PSMA targeting ligand was cleaved, thereby generating B12-radiotherapeutic unable to be internalized by PSMA positive tissue (i.e., light- exposed salivary glands, SG).

[0067] A wide variety of PSMA ligands have are available. One of these ligands (Lys-NH-CO- NH-Glu, highlighted in blue in 5 of Example 1) has found clinical utility as the molecular basis for the creation of prostate cancer radiotracers. Derivatives of this ligand display a high affinity for PSMA (< 1 nM) and are efficiently internalized into PCa cells. In particular, PSMA-617 is rapidly cleared from kidney, which reduces potential side-effects to this healthy organ. PSMA- 617 was selected as the ligand in this example. Structure activity relationships revealed that the lysine amine can accommodate an array of substituents without loss of affinity for PSMA, but that the free carboxylates on the Glu residue are essential for activity. Consequently, the structure of compound 5 is retained while exploring sites elsewhere on B12 for appending metal ion chelators. Two structural archetypes in 6 and 7 are outlined in FIG. 4. The former contains two attachment sites (in the form of amines) off the ribose moiety. The Fmoc and tBoc amines can be selectively deprotected and consequently selectively modified with a fluorophore and subsequently with the metal chelator (8).

[0068] As highlighted in complex 5, Cy5 has been successfully employed to capture red photons (XeX= 650 nm) for drug release. In addition, it has been demonstrated that a variety of fluorophores can be appended to the ribose ring and thereby used to extend photon capture out to the near IR [Alexa700 (700 nm), Atto725 (725 nm), and Dylight800 (780 nm)]. Alternatively, the light capturing fluorophore can be positioned on the corrin ring (compound 7). Following modification of the primary amine with a fluorophore (highlighted in red; step i) a second amine containing ligand is introduced at the ribose (step it) and used as an attachment site for the metal chelator (step Hi). The final synthetic step in both structural archetypes is the covalent attachment of the PSMA ligand to the Co (step iv in compound 7).

[0069] All compounds are assessed for their stability, sensitivity towards light, and ability to bind to PSMA. In brief, the glutamate carb oxy peptidase activity of PSMA is monitored using a standard fluorescent assay and established inhibitors [2-(phosphonomethyl)pentanedioic acid, Ki = 0.3 nM; HO2C-G1U-C(O)-G1U-CO2H, K = 8 nM] are used as controls. Lead compounds (i.e., sub nanomolar) from the library of PSMA-B12-fluorophore(Chelator) derivatives are further examined in cell based assays as follows: competitive binding studies utilize PSMA-expressing cells (PC3-PSMA) and an established PSMA imaging agent68Ga-PSMA(617) as the probe. Non- PSMA-expressing cells (Native PC3) are used to assess any non-specific binding. After in vitro evaluation, the agents are labeled with64Cu followed by stability evaluation of the labeled conjugates. EXAMPLE 3 - PSMA-targeted Cobalamin Conjugates for Radioimaging or Radiotherapy

[0070] In the present example, a B12 conjugate having the structure provided in FIG. 1C was synthesized, wherein64Cu was incorporated into the NOTA chelator. A non-human primate was injected with a composition comprising the B 12 conjugate. The left salivary gland of the test subject was irradiated with 650 nm light provided by a light emitting diode (LED) source. The power of the LED source was 13 mW / cm2providing an energy density of 23.4 J / cm2. Irradiation was administered on the left salivary gland for a time period of 30 minutes.

[0071] Positron emission tomography (PET) was conducted on the test subject at 15 minutes post injection. FIGS. 5A and 5B are axial PET images prior and post irradiation of the test subject, respectively. FIGS. 6A and 6B are coronal PET images prior and post irradiation of the test subject, respectively. As illustrated in FIGS. 5A to 6B, irradiation of the left salivary gland cleaved the pharmaceutical component of the B12 conjugate comprising the64Cu, thereby permitting enhanced clearance of the64Cu from the left salivary gland relative to the nonirradiated right salivary gland.

[0072] EXAMPLE 4 - PSMA-targeted Cobalamin Conjugates for Radioimaging or Radiotherapy

[0073] In the present example, a B12 conjugate having the structure provided in FIG. 1C was synthesized, wherein67Cu was incorporated into the NOTA chelator. A PC3-PIP mouse tumor model was administered to evaluate radiotherapy efficacy at various radiation doses. FIG. 7 provides results of the tumor model, wherein the B 12 conjugates slowed and / or reduced tumor growth and size, thereby confirming therapeutic effects of conjugates described herein.

[0074] EXAMPLE 5 - PSMA-targeted Cobalamin Conjugates for Radioimaging or Radiotherapy

[0075] In the present example, a B12 conjugate having the structure provided in FIG. 1C was synthesized, wherein64Cu was incorporated into the NOTA chelator. The conjugate was injected to mice bearing PSMA-positive PC3 prostate cancer tumors. The right kidney of the mice were irradiated with 650 nm light. PET imaging was conducted pre- and post-irradiation. FIGS. 8A and 8B provide PET imaging results of a mouse subject pre- and post-irradiation respectively. As illustrated in the PET imaging, enhanced clearance of64Cu was observed for the irradiated right kidney relative to the non-irradiated left kidney. Various embodiments of the invention have been described in fulfillment of the various objects of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

Claims

CLAIMS1. A conjugate comprising: a linker joining a pharmaceutical component and a targeting ligand, wherein the linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile, or acoustolabile for release of the targeting ligand.

2. The conjugate of claim 1, wherein the pharmaceutical component comprises a therapeutic agent or an imaging agent.

3. The conjugate of claim 2, wherein the therapeutic agent comprises a radioisotope for radiotherapy.

4. The conjugate of claim 3, wherein the radioisotope is selected from the group consisting of177LU,90Y,131I,131I,225AC,211At,212Pb,55Co, and58mCo.

5. The conjugate of claim 2, wherein the imaging agent is a radiotracer.

6. The conjugate of claim 5, wherein the radiotracer is selected from the group consisting ofnC,18F,64CU,89Zr,124I, and90Sr.

7. The conjugate of claim 1, wherein the targeting ligand binds to cancerous cells or tissue.

8. The conjugate of claim 7, wherein the targeting ligand binds to non-diseased cells or nondiseased tissue.

9. The conjugate of claim 1, wherein the linker comprises a transition metal complex.

10. The conjugate of claim 9, wherein the linker is a cobalamin scaffold.

11. The conjugate of claim 10, wherein the conjugate is of Formula II:whereina corrin ring, R1-]?3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, and TL is a targeting ligand.

12. The conjugates of claim 11, wherein Ei and E2 independently comprise one or more moieties selected from the group consisting of alkyl, alkyl-amine, urea, thiourea, amide, alkylene oxide, aryl, and heterocyclyl.

13. The conjugate of any of claims 9-11, wherein light absorption by the conjugate is red shifted by a conjugated light absorbing species associated with the transition metal complex.

14. The conjugate of claim 13, wherein the conjugated light absorbing species is a fluorophore.

15. The conjugate of claim 13, wherein the conjugate is of Formula Ila:whereinthe corrin ring, RJ-R3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, TL is a targeting ligand, and Sc is the conjugated species.

16. The conjugate of claim 1, wherein the linker is selected from the group consisting ofBODIPY caged compounds, cyanine scaffolds, aminobenzoquinone scaffolds, and coumarin- based caging compounds.

17. The conjugate of any of claims 9 to 16, wherein the linker, targeting ligand, and / or pharmaceutical component is derivatized with click chemistry moieties.

18. The conjugate of claim 17, wherein the click chemistry moieties are selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne and azide.

19. A conjugate comprising: a linker joining a pharmaceutical component and a targeting ligand, wherein the linker is at least one of radiolabile, photolabile, thermolabile, chemically labile, magnetically labile or acoustolabile for release of the pharmaceutical component.

20. The conjugate of claim 19, wherein the pharmaceutical component comprises a therapeutic agent or an imaging agent.

21. The conjugate of claim 20, wherein the therapeutic agent comprises a radioisotope for radiotherapy.

22. The conjugate of claim 21, wherein the radioisotope is selected from the group consisting of177LU,90Y,131I,131I,225AC,211At,212Pb,55Co, and58mCo.

23. The conjugate of claim 20, wherein the imaging agent is a radiotracer.

24. The conjugate of claim 23, wherein the radiotracer is selected from the group consisting of nC, 18FJ64CU, 89Zr, 124I; and90gr25. The conjugate of claim 18, wherein the targeting ligand binds to cancerous cells or tissue.

26. The conjugate of claim 25, wherein the targeting ligand binds to non-diseased cells or non-diseased tissue.

27. The conjugate of claim 19, wherein the linker comprises a transition metal complex.

28. The conjugate of claim 24, wherein the linker is a cobalamin scaffold.

29. The conjugate of claim 28, wherein the conjugate is of Formula I:whereina corrin ring, RJ-R3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, and TL is a targeting ligand.

30. The conjugate of claim 29, wherein Ei and E2 independently comprise one or more moieties selected from the group consisting of alkyl, alkyl-amine, urea, thiourea, amide, alkylene oxide, aryl, and heterocyclyl.

31. The conjugate of any of claims 27-30, wherein light absorption by the conjugate is red shifted by a conjugated light absorbing species associated with the transition metal complex.

32. The conjugate of claim 26, wherein the dye is a fluorophore.

33. The conjugate of claim 31, wherein the conjugate is of Formula la:whereinthe corrin ring, R R3are independently selected from the group consisting of hydrogen and alkyl, Ei and E2 are independently chain extenders, R* is a moiety comprising at least one radionuclide, TL is a targeting ligand, and Sc is the conjugated light absorbing species.

34. The conjugate of claim 19, wherein the linker is selected from the group consisting of BODIPY caged compounds, cyanine scaffolds, aminobenzoquinone scaffolds, and coumarin- based caging compounds.

35. The conjugate of any of claims 27 to 33, wherein the linker, targeting ligand, and / or pharmaceutical component is derivatized with click chemistry moieties.

36. The conjugate of claim 35, wherein the click chemistry moieties are selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne and azide.

37. A method of treating diseased tissue of a patient comprising: administering to the patient a conjugate comprising a linker joining a pharmaceutical component and a targeting ligand; locating the pharmaceutical component at one or more regions of the diseased tissue via binding the conjugate to diseased cells with the targeting ligand; imaging and / or treating the diseased tissue with the pharmaceutical component; and applying radiological, photonic, thermal, magnetic field, and / or acoustic energy to one or more locations of non-diseased tissue of the patient to release the targeting ligand from the conjugate.

38. The method of claim 37, wherein the release of the targeting ligand precludes binding of the conjugate to cells of the non-diseased tissue.

39. The method of claim 37, wherein release of the targeting ligand separates the linker and pharmaceutical component from cells of the non-diseased tissue bound to the targeting ligand.

40. The method of any of claims 37 to 39, wherein the wherein the pharmaceutical component comprises a therapeutic agent or an imaging agent.

41. The method of claim 40, wherein the therapeutic agent comprises a radioisotope for radiotherapy.

42. The method of claim 41, wherein the radioisotope is selected from the group consisting of177LU,90Y,131I,131I,225AC,211At,212Pb,55Co, and58mCo.

43. The method of claim 40, wherein the imaging agent is a radiotracer.

44. The method of claim 43, wherein the radiotracer is selected from the group consisting ofnC,18F,64CU,89Zr,124I, and90Sr.

45. The method of any of claims 37 to 39, wherein the targeting ligand binds to cancerous cells or tissue.

46. The method of any of claims 37 to 39, wherein the linker comprises a transition metal complex.

47. The method of claim 46, wherein the linker is a cobalamin scaffold.

48. The method of claim 46, wherein light absorption by the conjugate is red shifted by a dye associated with the transition metal complex.

49. The method of any of claims 37 to 39, wherein the linker is selected from the group consisting of BODIPY caged compounds, cyanine scaffolds, aminobenzoquinone scaffolds, and coumarin-based caging groups.

50. A method of treating diseased tissue of a patient comprising: administering to the patient a conjugate comprising a linker joining a pharmaceutical component and a targeting ligand; locating the pharmaceutical component at one or more regions of the diseased tissue via binding the conjugate to diseased cells with the targeting ligand; imaging or treating the diseased tissue with the pharmaceutical component; andapplying radiological, photonic, thermal, magnetic field and / or acoustic energy to one or more locations of non-diseased tissue of the patient to release the pharmaceutical component from the conjugate.

51. The method of claim 50, wherein release of the pharmaceutical component occurs prior to binding of the targeting ligand to cells of the non-diseased tissue.

52. The method of claim 50, wherein release of the pharmaceutical component separates the pharmaceutical component from cells of the non-diseased tissue bound to the targeting ligand.

53. The method of any of claims 50 to 52, wherein the wherein the pharmaceutical component comprises a therapeutic agent or an imaging agent.

54. The method of claim 53, wherein the therapeutic agent comprises a radioisotope for radiotherapy.

55. The method of claim 54, wherein the radioisotope is selected from the group consisting of177LU,90Y,131I,131I,225AC,211At,212Pb,55Co, and58mCo.

56. The method of claim 43, wherein the imaging agent is a radiotracer.

57. The method of claim 56, wherein the radiotracer is selected from the group consisting ofnC,18F,64CU,89Zr,124I, and90Sr.

58. The method of any of claims 50 to 52, wherein the targeting ligand binds to cancerous cells or tissue.

59. The method of any of claims 50 to 52, wherein the linker comprises a transition metal complex.

60. The method of claim 59, wherein the linker is a cobalamin scaffold.

61. The method of claim 59, wherein light absorption by the conjugate is red shifted by a dye associated with the transition metal complex.

62. The method of any of claims 50 to 52, wherein the linker is selected from the group consisting of BODIPY caged compounds, cyanine scaffolds, aminobenzoquinone scaffolds, and coumarin-based caging groups.

63. A method of treating diseased tissue of a patient comprising: administering to the patient a conjugate comprising a linker joining a pharmaceutical component and a targeting ligand; locating the pharmaceutical component at one or more regions of the diseased tissue via binding the conjugate to diseased cells with the targeting ligand; imaging or treating the diseased tissue with the pharmaceutical component; and applying a releasing agent to one or more locations of non-diseased tissue of the patient to chemically react with the linker to release the pharmaceutical component and / or targeting ligand from the conjugate.

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