IMAGING AND THERAPEUTICS

Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Application Serial No. 62/196,434 filed on July 24, 2015, and U.S. Application Serial No. 62/213,410 filed on September 2, 2015, the disclosures of which are hereby incorporated by reference in their entireties.

Government Support

[0002] This invention was made with government support under Grant Number

EB014944 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

[0003] This invention relates generally to compositions and methods involving Cerenkov targeted and activated imaging and therapeutics. In particular embodiments, the invention relates to Cerenkov activated photochemistry using (i) azides leading to targeted agents and (ii) photocleavable linkers leading to activatable agents (e.g., contrast agents, photodynamic therapy (PDT) agents, cytotoxic drugs or inhibitors).

Background

[0004] Systemic administration of cytotoxic drugs (e.g., doxorubicin, paclitaxel) is a prevalent treatment strategy. However, clinical application of cytotoxic drugs is limited by its non-targeted nature that affects both normal and cancer tissue. For non-metastasized primary tumors, the drug toxicity is intended to be solely at the tumor site. However, only a fraction of the injected dose is localized in the tumor causing unwanted damage to healthy tissue.

[0005] Various approaches such as chemical modification of chemotherapy agents (e.g., doxorubicin) have attempted to reduce cytotoxicity of chemotherapeutics. For example, one approach administers pro-drugs that are to be transformed into pharmacologically active agents when exposed to changes in endogenous physiological environments specific to a tumor region (e.g., reduction, hydrolytic enzymes, hypoxia or low pH). However, these inherent physiological environments vary between tumor cells.

[0006] Moreover, pro-drugs have been designed to specifically bind to tumor-specific antigens that are expressed on the surface of tumor cells (e.g., by attaching antibodies to the chemotherapy agents that are specific for the tumor-specific antigens). However, tumor cells do not homogeneously express surface markers, they feature endogenous physiological factors (e.g., reduction, hydrolytic enzymes, hypoxia or low pH), and they are subject to poor circulation networks within the tissue. Thus, the chemotherapy drug cannot be reliably and effectively distributed or delivered to each tumor cell.

[0007] In contrast to relying on inherent differences between the tumor cell and the healthy tissue to activate pro-dugs, some approaches focus on inducing artificial changes to the tumor environment in order to distinguish tumor cells from the rest of the body. Examples of these methods include (i) administering ionizing radiation in combination with a small amount of reducing chemical species, (ii) using non-human enzymes to activate the pro-dug, and (iii) covalently attaching photocleavable blocking groups (e.g., cleavable in the UV or near infrared

(NIR) range) to the chemotherapeutic (Ibsen, et al. "A Novel Doxorubicin Prodrug with

Controllable Photolysis Activation for Cancer Chemotherapy," Pharm Pes (2010) 27: 1848-1860; Choi, et al. "Light-controlled release of caged doxorubicin from folate receptor-targeting

PAMAM dendrimer nanoconjugate," Chem Commun (Camb). (2010) 46(15): 2632-2634).

However, these alternative methods require efficient delivery of additional enzymes to the tumor, and use of a trigger source which is external to the body.

[0008] There remains a need for a drug delivery strategy that can be activated by an internal source that creates an environment that distinguishes between tumor and healthy tissues.

Summary of invention

[0009] Described herein are Cerenkov activated tagging agents comprising cargo (e.g., a fluorophore, a therapeutic, a contrast agent) and a covalent binding unit, e.g., an azide. Without wishing to be bound to any particular theory, in certain embodiments, when the tagging agent reaches a site of high Cerenkov luminescence, the cargo-azide undergoes photolysis to form nitrene. The nitrene has a short lifetime and immediately inserts itself into a C-H or N-H bond. In certain embodiments, a photocleavable blocking agent for a fluorophore, drug, or contrast agent that is activated in the presence of Cerenkov luminescence can be used. In preferred embodiments, the cargo blocked by the photocleavable agent has much lower activity than the unblocked cargo thereby generating contrast and/or efficacy.

[0010] In certain embodiments, as described above, the cargo comprises a fluorophore.

Without wishing to be bound to any particular theory, in certain embodiments, the covalent nature of the attachment via nitrene insertion allows the fluorophore to emit light once activated.

Overtime there is fluorophore accumulation in a targeted site (e.g., tumor tissue), and contrast is developed between sites with and without Cerenkov luminescence. The presence of the fluorophore enables reduced times for fluorescence imaging compared to the longer times requires for luminescence imaging. In certain embodiments, a snapshot of the radioisotope distribution can be taken without performing PET imaging due to co-localization of the radioisotope and the fluorophore. This creates a "fluorescent footprint" of the distribution of the radioactive tracer and converts radioactivity into a fluorescent signal, which can be used for facile intraoperative fluorescent imaging.

[0011] Cerenkov luminescence occurs when the velocity of a charged particle exceeds the phase velocity of a medium. The phase velocity is governed in general by Einstein's relativistic kinetic energy equation where phase velocity v, and the speed of light c can be related by the refractive index n such as c/n=v. By increasing the refractive index (RI) of the material and/or environment of a radionuclide, the phase velocity is lowered and charged particles emitted by the radionuclide emit Cerenkov light.

[0012] In a preclinical and/or clinical setting, radionuclides are surrounded by tissues and water which typically have a RI of -1.33-1.41. However, the RI of the environment

surrounding the radionuclide must be greater than about 1.41 in order to increase the yield of emitted particles that produce Cerenkov light. The material properties that govern Cerenkov enhancement remain unknown. Cerenkov enhancement may be produced by altering the RI of the suspension. However, this is not feasible for preclinical and clinical models because serum and tissue become the dominant RI materials, thereby limiting the yield of emitted particles.

[0013] Light generated by radioisotopes (e.g., ¹⁸ F-FDG ) in vivo can be used in photoinitiated chemistry for biomedical imaging and therapy. A variety of isotopes have been clinically translated for positron emission tomography (PET) imaging and radiation therapy.

Moreover, a radioactive isotope that produces charged particles and thus Cerenkov light can be used as in internal light source to photoactivate caged fluorescent compounds in vivo as described by Ran et al., "/« vivo Photoactivation Without "Light": Use of Cherenkov Radiation to Overcome the Penetration Limit of Lighf Molecular Imaging and Biology (2012) 14: 156-162, whose contents are hereby incorporated by reference in its entirety.

[0014] In one aspect, the invention is directed to a method of imaging tumor cells in a subject, the method comprising: administering to the subject a radiolabeled biomarker that emits

Cerenkov luminescence; and administering to the subject a Cerenkov activated tagging agent, wherein the Cerenkov activated tagging agent comprises a payload and a covalent binding unit and, wherein the covalent binding unit is chemically modified upon exposure to the emitted

Cerenkov luminescence, thereby activating the payload of the Cerenkov tagging agent.

[0015] In certain embodiments, the radiolabeled biomarker comprises a member selected from the group consisting of ¹⁸ F-FDG (fluorodeoxyglucose) and ¹⁸ F-FLT (fluorothymidine).

[0016] In certain embodiments, the covalent binding unit comprises an azide.

[0017] In certain embodiments, payload comprises an imaging agent and/or a contrast agent.

[0018] In certain embodiments, administering is via intratumor injection. In certain embodiments, administering is via retroorbital injection.

[0019] In certain embodiments, the covalent binding unit is chemically modified to a nitrene.

[0020] In certain embodiments, the tumor cells are selected from the group consisting of breast tumor cells, prostate tumor cells, lung tumor cells, gastrointestinal tumor cells, pancreatic tumor cells, gynecological tumor cells, soft tissue tumor cells, sarcoma tumor cells, lymphoma tumor cells, kidney tumor cells, urothelial tract tumor cells, and bladder tumor cells. [0021] In certain embodiments, Cerenkov luminescence is limited to local illumination no greater than 5 millimeters from the source of Cerenkov luminescence. In certain

embodiments, the Cerenkov luminescence is limited to local illumination no greater than 3 millimeters from the source of Cerenkov luminescence. In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 1 millimeter from the source of Cerenkov luminescence.

[0022] In certain embodiments, the Cerenkov activated tagging agent is selected from the group consisting of fluorescein arylazide, sulpho-Cy7-fluoroarylazide, Cy5.5-fluoroarylazide, and calcein aryl azide.

[0023] In certain embodiments, the Cerenkov activated tagging agent is pegylated.

[0024] In certain embodiments, the imaging agent is hydrophobic.

[0025] In another aspect, the invention is directed to a Cerenkov activated tagging agent comprising a payload and a covalent binding unit, wherein the payload comprises an imaging agent and/or a contrast agent, wherein the covalent binding unit comprises an azide and the azide is chemically modified to a nitrene upon exposure to the emitted Cerenkov luminescence, and wherein the activated nitrene binds to nearby macromolecules and/or tissue, thereby localizing the imaging agent and/or contrast agent.

[0026] In certain embodiments, the activated nitrene covalently binds to nearby macromolecules and/or tissue.

[0027] In certain embodiments, the activated nitrene covalently binds to nearby macromolecules and/or tissue instantaneously.

[0028] In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 5 millimeters from the source of Cerenkov luminescence. [0029] In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 3 millimeters from the source of Cerenkov luminescence.

[0030] In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 1 millimeter from the source of Cerenkov luminescence.

[0031] In certain embodiments, the Cerenkov activated tagging agent is selected from the group consisting of fluorescein arylazide, sulpho-Cy7-fluoroarylazide, Cy5.5-fluoroarylazide, and calcein aryl azide.

[0032] In certain embodiments, the Cerenkov activated tagging agent is pegylated.

[0033] In certain embodiments, the imaging agent is hydrophobic.

[0034] In another aspect, the invention is directed to a method of treating tumor cells, the method comprising: administering to a subject a pro-drug comprising a therapeutic; a blocking agent; and a cleavable linker, wherein the blocking agent and the therapeutic are attached via the cleavable linker; and administering a targeting agent comprising a radioisotope as a source of Cerenkov luminescence at or near the tumor cells, wherein the blocking agent is cleaved upon (or following) exposure to the Cerenkov luminescence.

[0035] In certain embodiments, the therapeutic comprises a chemotherapy drug. In certain embodiments, the chemotherapy drug is a small molecule. In certain embodiments, the chemotherapy drug comprises a member selected from the group consisting of doxorubicin, paclitaxel, cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine (DTIC), epirubicin, idarubicin, mitoxantrone, valrubicin, docetaxel, abraxane, taxotere, vorinostat, epothilone, romidepsin, irinotecan, topotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, 5-fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, tioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, tretinoin, alitretinoin, bexarotene, vinblastine, vincristine, vindesine, and vinorelbine.

[0036] In certain embodiments, the pro-drug comprises cyclic Arginylglycylaspartic acid

(cRGD).

[0037] In certain embodiments, the radioisotope is selected from the group consisting of

¹⁸ F, ⁹⁰ Y, ⁶⁸ Ga, ⁸⁹ Zr, ⁶⁴ Cu, ¹⁷⁷ Lu, and ³² P. In certain embodiments, the radioisotope is chelated and covalently attached to an immunoglobulin specific for the tumor cells. In certain

embodiments, the immunoglobulin is or comprises a member selected from the group consisting of J591, A33, and 5B 1. In certain embodiments, the radioisotope is covalently attached to a metabolite. In certain embodiments, the metabolite is selected from the group consisting of FDG (fluorodeoxyglucose) and FLT (fluorothymidine).

[0038] In certain embodiments, the pro-drug becomes an activated drug upon exposure to the Cerenkov luminescence.

[0039] In certain embodiments, the tumor cells are selected from the group consisting of breast tumor cells, prostate tumor cells, lung tumor cells, gastrointestinal tumor cells, pancreatic tumor cells, gynecological tumor cells, soft tissue tumor cells, sarcoma tumor cells, lymphoma tumor cells, kidney tumor cells, urothelial tract tumor cells, and bladder tumor cells.

[0040] In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 5 millimeters from the source of Cerenkov luminescence. In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 3 millimeters from the source of Cerenkov luminescence. In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 1 millimeter from the source of Cerenkov luminescence. [0041] In certain embodiments, the cleavable linker is selected from the group consisting of arylcarbonylmethyl, nitroaryl, coumarin, arylmethyl, metal containing groups, and a-carboxy- 2-nitrobenzyl.

[0042] In another aspect, the invention is directed to a method of imaging tumor cells, the method comprising: administering to a subject a pro-contrast agent comprising a contrast agent; a blocking agent; and a cleavable linker, wherein the blocking agent and the contrast agent are attached via the cleavable linker; and administering a targeting agent comprising a radioisotope as a source of Cerenkov luminescence at or near the tumor cells, wherein the blocking agent is cleaved upon (or following) exposure to the Cerenkov luminescence.

[0043] In certain embodiments, the contrast agent is selected from the group consisting of ¹⁸ F, ⁹⁰ Y, ⁸⁹ Zr, ⁶⁸ Ga, ⁶⁴ Cu, ¹⁷⁷ Lu, and ³² P. In certain embodiments, the contrast agent comprises ¹⁸ F.

[0044] In certain embodiments, the pro-contrast agent becomes an activated contrast agent upon exposure to the Cerenkov luminescence.

[0045] In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 5 millimeters from the source of Cerenkov luminescence. In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 3 millimeters from the source of Cerenkov luminescence. In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 1 millimeter from the source of Cerenkov luminescence.

[0046] In certain embodiments, the cleavable linker is selected from the group consisting of arylcarbonylmethyl, nitroaryl, coumarin, arylmethyl, metal containing groups, and a-carboxy- 2-nitrobenzyl. [0047] In another aspect, the invention is directed to a pro-drug comprising: a therapeutic; a blocking agent; and a cleavable linker, wherein the blocking agent and the therapeutic are attached via the cleavable linker which is cleavable by exposure to Cerenkov luminescence.

[0048] In certain embodiments, the therapeutic comprises a chemotherapy drug.

[0049] In certain embodiments, the chemotherapy drug comprises a member selected from the group consisting of doxorubicin, paclitaxel, cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine (DTIC), epirubicin, idarubicin, mitoxantrone, valrubicin, docetaxel, abraxane, taxotere, vorinostat, epothilone, romidepsin, irinotecan, topotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, 5- fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, tioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, tretinoin, alitretinoin, bexarotene, vinblastine, vincristine, vindesine, and vinorelbine.

[0050] In certain embodiments, the pro-drug further comprises cRGD.

[0051] In certain embodiments, the pro-drug becomes an activated drug upon exposure to the Cerenkov luminescence.

[0052] In another aspect, the invention is directed to a pro-contrast agent comprising: a contrast agent; a blocking agent; and a cleavable linker, wherein the blocking agent and the contrast agent are attached via the cleavable linker which is cleavable by exposure to Cerenkov luminescence.

[0053] In certain embodiments, the contrast agent comprises a member selected from the group consisting of ¹⁸ F, ⁹⁰ Y, ⁸⁹ Zr, ⁶⁸ Ga, ⁶⁴ Cu, ¹⁷⁷ Lu, and ³² P. [0054] In certain embodiments, the pro-contrast agent becomes an activated contrast agent upon exposure to the Cerenkov luminescence.

[0055] In another aspect, the invention is directed to a method of imaging tumor cells in a subject, the method comprising: administering to the subject a radiolabeled biomarker that emits electromagnetic radiation (emr); and administering to the subject an emr-activated tagging agent, wherein the emr-activated tagging agent comprises a payload and a covalent binding unit, wherein the covalent binding unit is chemically modified upon exposure to the emr, thereby activating the payload of the tagging agent.

[0056] In another aspect, the invention is directed to a method of treating tumor cells, the method comprising: administering to a subject a pro-drug comprising a therapeutic; a blocking agent; and a cleavable linker, wherein the blocking agent and the therapeutic are attached via the cleavable linker; and administering a targeting agent comprising a radioisotope as a source of electromagnetic radiation (emr) at or near the tumor cells, wherein the blocking agent is cleaved upon (or following) exposure to the emr.

[0057] In another aspect, the invention is directed to a method of imaging tumor cells, the method comprising: administering to a subject a pro-contrast agent comprising a contrast agent; a blocking agent; and a cleavable linker, wherein the blocking agent and the contrast agent are attached via the cleavable linker; and administering a targeting agent comprising a radioisotope as a source of electromagnetic radiation (emr) at or near the tumor cells, wherein the blocking agent is cleaved upon (or following) exposure to the emr.

[0058] In certain embodiments, the emr is a member selected from the group consisting of e.g., Cerenkov luminescence, radio luminescence, and X-ray irradiation.

[0059] In another aspect, the invention is directed to a pro-drug comprising a therapeutic; [0060] a blocking agent; and a cleavable linker, wherein the blocking agent and therapeutic are attached via the cleavable linker for use in a method of treating tumor cells in a subject, wherein the treating comprises delivering the pro-drug to the subject; and delivering a targeting agent comprising a radioisotope as a source of luminescence (e.g., Cerenkov, radio luminescence, X-ray irradiation) at or near the tumor cells, wherein the blocking agent is cleaved (or following) exposure to the luminescence.

[0061] In another aspect, the invention is directed to a pro-drug comprising a therapeutic; a blocking agent; and a cleavable linker, wherein the blocking agent and therapeutic are attached via the cleavable linker, and wherein the blocking agent is cleaved (or following) exposure to the luminescence for use in therapy.

[0062] In certain embodiments, the therapeutic comprises a chemotherapy drug. In certain embodiments, the chemotherapy drug is a small molecule. In certain embodiments, the chemotherapy drug comprises a member selected from the group consisting of doxorubicin, paclitaxel, cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine (DTIC), epirubicin, idarubicin, mitoxantrone, valrubicin, docetaxel, abraxane, taxotere, vorinostat, epothilone, romidepsin, irinotecan, topotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, 5-fluorouracil, gemcitabine, hydroxyurea,

mercaptopurine, methotrexate, tioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, tretinoin, alitretinoin, bexarotene, vinblastine, vincristine, vindesine, and vinorelbine.

[0063] In certain embodiments, the pro-drug comprises cyclic Arginylglycylaspartic acid

(cRGD). [0064] In certain embodiments, the radioisotope is selected from the group consisting of

¹⁸ F, ⁹⁰ Y, ⁶⁸ Ga, ⁸⁹ Zr, ⁶⁴ Cu, ¹⁷⁷ Lu, and ³² P.

[0065] In certain embodiments, the radioisotope is chelated and covalently attached to an immunoglobulin specific for the tumor cells. In certain embodiments, the immunoglobulin is or comprises a member selected from the group consisting of J591, A33, and 5B1.

[0066] In certain embodiments, the radioisotope is covalently attached to a metabolite.

In certain embodiments, the metabolite is selected from the group consisting of FDG

(fluorodeoxyglucose) and FLT (fluorothymidine).

[0067] In certain embodiments, the pro-drug becomes an activated drug upon exposure to the Cerenkov luminescence.

[0068] In certain embodiments, the tumor cells are selected from the group consisting of breast tumor cells, prostate tumor cells, lung tumor cells, gastrointestinal tumor cells, pancreatic tumor cells, gynecological tumor cells, soft tissue tumor cells, sarcoma tumor cells, lymphoma tumor cells, kidney tumor cells, urothelial tract tumor cells, and bladder tumor cells.

[0069] In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 5 millimeters from the source of Cerenkov luminescence. In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 3 millimeters from the source of Cerenkov luminescence. In certain embodiments, the Cerenkov luminescence is limited to local illumination no greater than 1 millimeter from the source of Cerenkov luminescence.

In certain embodiments, the cleavable linker is selected from the group consisting of

arylcarbonylmethyl, nitroaryl, coumarin, arylmethyl, metal containing groups, and a-carboxy-2- nitrobenzyl. [0070] Elements of embodiments involving one aspect of the invention (e.g., methods) can be applied in embodiments involving other aspects of the invention (e.g., systems), and vice versa.

Definitions

[0071] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0072] In this application, the use of "or" means "and/or" unless stated otherwise. As used in this application, the term "comprise" and variations of the term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain

embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%), 2%), 1%), or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0073] "Administration ": The term "administration" refers to introducing a substance into a subject. In general, any route of administration may be utilized including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In some embodiments, administration is oral. Additionally or alternatively, in some embodiments, administration is parenteral. In some embodiments, administration is intravenous.

[0074] "Biocompatible": The term "biocompatible", as used herein is intended to describe materials that do not elicit a substantial detrimental response in vivo. In certain embodiments, the materials are "biocompatible" if they are not toxic to cells. In certain embodiments, materials are "biocompatible" if their addition to cells in vitro results in less than or equal to 20% cell death, and/or their administration in vivo does not induce inflammation or other such adverse effects. In certain embodiments, materials are biodegradable.

[0075] "Biodegradable" . As used herein, "biodegradable" materials are those that, when introduced into cells, are broken down by cellular machinery {e.g., enzymatic degradation) or by hydrolysis into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material do not induce inflammation and/or other adverse effects in vivo. In some embodiments, biodegradable materials are enzymatically broken down. Alternatively or additionally, in some embodiments, biodegradable materials are broken down by hydrolysis. In some embodiments, biodegradable polymeric materials break down into their component polymers. In some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymeric materials) includes hydrolysis of ester bonds. In some embodiments, breakdown of materials (including, for example, biodegradable polymeric materials) includes cleavage of urethane linkages.

[0076] "Carrier ^" . As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0077] "Contrast agent". The term "contrast agent," as used herein, refers to an agent used to highlight specific areas so that organs, blood vessels, and/or tissues are more visible. By increasing the visibility of the surfaces being studied, the presence and extent of disease and or injury can be determined.

[0078] "Peptide" or "Polypeptide" . The term "peptide" or "polypeptide" refers to a string of at least two (e.g., at least three) amino acids linked together by peptide bonds. In some embodiments, a polypeptide comprises naturally-occurring amino acids; alternatively or additionally, in some embodiments, a polypeptide comprises one or more non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, http://www.cco.caltech.edu/~dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed). In some embodiments, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.

[0079] "Photocaging": The term "photocaging" refers to the temporary deactivation of a biologically active molecule using a protective photocleavable group. Upon exposure to irradiation of the photocleavable group, the active form of the caged molecule is irreversibly released.

[0080] "Radiolabel" As used herein, "radiolabel" refers to a moiety comprising a radioactive isotope of at least one element. Exemplary suitable radiolabels include but are not limited to those described herein. In some embodiments, a radiolabel is one used in positron emission tomography (PET). In some embodiments, a radiolabel is one used in single-photon emission computed tomography (SPECT). In some embodiments, radioisotopes comprise ^99m Tc, ^m In, ⁶⁴ Cu, ⁶⁷ Ga, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ¹⁷⁷ Lu, ⁶⁷ Cu, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ^U C, ^N, ¹⁵ 0, ¹⁸ F, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁴⁹ Pm, ⁹⁰ Y, ²¹³ Bi, ¹⁰³ Pd, ¹⁰⁹ Pd, ¹⁵⁹ Gd, ¹⁴⁰ La, ¹⁹⁸ Au, ¹⁹⁹ Au, ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁶⁵ Dy, ¹⁶⁶ Dy, ⁶⁷ Cu, ¹⁰⁵ Rh, ^lu Ag, ⁸⁹ Zr, ²²⁵ Ac, and ¹⁹² Ir.

[0081] "Small molecule": As used herein, the term "small molecule" means a low molecular weight organic and/or inorganic compound. In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about

300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about

2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating agent. In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more steroisomeric forms. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some

embodiments, such a small molecule may be utilized in accoradance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms.

In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some

embodiments, reference to a particular compound may relate to a specific form of that compound. In some embodiments, where a compound is one that exists or is found in nature, that compound may be provided and/or utilized in accordance in the present invention in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a compound preparation including a different level, amount, or ratio of one or more individual forms than a reference preparation or source (e.g., a natural source) of the compound may be considered to be a different form of the compound as described herein. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a compound may be considered to be a different form from another salt form of the compound; a preparation containing one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form from one containing the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0082] "Subject". As used herein, the term "subject" includes humans and mammals

(e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are be mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals will be , for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

[0083] "Substantially": As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0084] "Therapeutic agent": As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.

[0085] "Treatment": As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

[0086] Drawings are presented herein for illustration purposes, not for limitation.

Brief description of drawings

[0087] The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by referring to the following description taken in conduction with the accompanying drawings, in which:

[0088] FIG. 1 depicts a Cerenkov activated tagging agent comprising a payload and reactive covalent binding unit comprising a blocking agent. Following exposure to Cerenkov luminescence, the blocking agent is removed and the Cerenkov tagging agent is activated.

[0089] FIG. 2 depicts an in vivo mechanism of a Cerenkov activated tagging agent.

[0090] FIG. 3 depicts UV catalyzed arylazide decomposition monitored by changes in absorption spectra.

[0091] FIG. 4 depicts various uses of nitrene in a biological system.

[0092] FIGS. 5A-5B depict synthesis of fluorescein arylazide. [0093] FIG. 6 depicts Cerenkov targeting for therapy and/or imaging using fluorescein arylazide.

[0094] FIG. 7 depicts the molecular structure of Calcein.

[0095] FIG. 8 depicts synthesis of Calcein conjugated to arylazide.

[0096] FIG. 9 depicts fluorescence characteristics (λ _€χ = 491 nm, _em ⁼ 509 nm) of calcein arylazide.

[0097] FIG. 10 depicts UV activated cell staining of calcein arylazide.

[0098] FIGS. 11 A-l IB depict two exemplary cyanine dye-arylazide conjugates with different solubilities.

[0099] FIG. 12 depicts Cy5.5 arylazide biodistribution at 15 minutes, 45 minutes, 2 hours, 4 hours, 22 hours, and 49 hours in mice.

[0100] FIG. 13 depicts Sulpho-Cy7 arylazide biodistribution at 10 minutes, 30 minutes,

45 minutes, 75 minutes, and 4 hours. No fluorescence was observed after 24 hours.

[0101] FIG. 14 depicts an image of five mice with HT1080 tumors. Mice 1 through 4 were injected with ¹⁸ F-FDG intratumorally and sulpho-Cy7 arylazide injected retroorbitally, Mice 1 and 2 were injected with ¹⁸ F-FDG, and mouse 5 was a control (no injection). The images were acquired at 34 hours (top) and 50 hours (bottom).

[0102] FIGS. 15A - 15B depict Cerenkov targeted sulpho-cy7 arylazide in vivo imaging.

[0103] FIG. 16 depicts an image of five HT1080 tumors from five mice that had been injected with ¹⁸ F-FDG intratumorally and sulpho-Cy7 arylazide retroorbitally.

[0104] FIGS. 17A - 17B depict in vitro photolabeling of sulpho-cy7 arylazide after incubation of ¹⁸ F-FDG. [0105] FIG. 18 depicts an image of five mice with HT 1080 tumors injected with F-

FDG intratumorally and Cy5.5 arylazide injected retroorbitally. The image was acquired at 50 hours.

[0106] FIG. 19 depicts synthesis of an exemplary immunotargeted Cerenkov source.

[0107] FIG. 20 depicts a schematic of synthesizing Cerenkov targeted azides as MRI contrast agents.

[0108] FIG. 21 depicts a schematic of synthesizing Cerenkov targeted azides as therapeutics.

[0109] FIG. 22 depicts Cerenkov luminescence from ¹⁸ F-FDG treated PC3-hsPSMA tumors at 0 and 3 hours.

[0110] FIG. 23 shows sulfoCy7 aryl azide Cerenkov activation in PC3-hPSMA mice at 0,

3, 6, 30, and 55 hours.

[0111] FIG. 24 depicts sulfoCy7 aryl azide FDG activation in PC3-hPSMA mice injected retroorbitally at 0, 3, 6, 30, and 55 hours.

[0112] FIG. 25 depicts an increase in fluorescence radiance for ¹⁸ F-FDG treated versus untreated tumors with time.

[0113] FIG. 26 depicts data measured by instant thin layer chromatography (iTLC) using a radiodetector.

[0114] FIG. 27 depicts the stability of ⁹⁰ Y-DOTA-J591 in serum. In this example, 50 μΐ, of serum was added to 20 μϋϊ ⁹⁰ Y-DOTA-J591 at 37°C. Over the course of 10 days, iTLC was performed on 0.5 μΙ_, of the sample to check integrity of ⁹⁰ Y-DOTA-J591.

[0115] FIGS. 28A-28C show in vivo activation of Cy7-azide using Cerenkov

luminescence. Mice bearing HT1080 tumors were injected retroorbitally with ⁹⁰ Y-DOTA- (cRGD) ₂ or saline followed by Cy7-azide injection 7 hours afterwards in the alternate eye. Mice were imaged at various timepoints up to 156 h.

[0116] FIG. 28 A shows representative Cerenkov Luminescence images showing tumor uptake of ⁹⁰ Y-DOTA-(cRGD) ₂ in the lower left and right flanks.

[0117] FIG. 28B depicts representative fluorescence images that show Cy7 signal at 36 hours is only present in tumors with Cerenkov luminescence. In contrast, no Cy7 is present in control groups.

[0118] FIG. 28C shows a quantitative analysis of fluorescence signals in tumors at different timepoints. The highest contrast enhancement could be detected at 3 hours after Cy7- azide injection. The highest percentage increase in contrast was 65% at 156 h. P<0.01 for all timepoints.

[0119] FIG. 29 shows an in vivo experiment with Cy7 control dye. Mice bearing

HT1080 tumors were injected retroorbitally with ⁹⁰ Y-DOTA-(cRGD) ₂ or saline followed by Cy7 control compound injection 7 hours afterwards in the alternate eye. Mice were imaged at various timepoints up to 156 h. Quantitative analysis of the fluorescence signals in tumors show no statistically significant contrast enhancement between the groups with or without Cerenkov luminescence.

[0120] FIG. 30 shows an exemplary photocaged doxorubicin construct (doxorubicin- photocage(PC)-PEG ₃ -Biotin). Note that this construct is not limited to doxorubicin but can be extended to any chemotherapeutic drug.

[0121] FIG. 31 shows enhancement of cytotoxicity after photocleavage release of an active drug, such as doxorubicin. In this example, when the construct of FIG. 30 is exposed to

Cerenkov luminescence, doxorubicin is released from the construct. Drug delivery is shown to be 200 fold less toxic than doxorubicin alone. In certain embodiments, the structure of doxorubicin-photocage(PC)-PEG ₃ -Biotin is higher than IC50.

Detailed Description

[0122] Throughout the description, where compositions are described as having, including, or comprising specific components, or where methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are

compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0123] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0124] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.

[0125] Described herein are compositions and methods to confine Cerenkov

luminescence to a localized area (e.g., from less than millimeter to a few millimeters for a beta or positron emitter respectively) (e.g., due to its wavelength) for targeted treatment of a tumor cell via a Cerenkov-luminescence-cleavable blocking agent.

[0126] Isotopes used in PET and/or radiation therapy can be used as sources for imaging and supplementary and/or complementary therapy. The light generated by Cerenkov luminescence is of low wavelength, the propagation of the light is weak, and, hence, the generation of activated molecules is local, e.g., confined to the immediate environment of the source.

[0127] Photolabeling, in certain embodiments, refers to the activation of fluoroarylazide to generate nitrene species. As described herein, Cerenkov luminescence was used to activate sulpho-Cy7-fluoroarylazide, for example, and generate nitrene species in vivo and in vitro.

Photocaging refers to the temporary inactivation of a biologically active molecule using a protective photocleavable group. In certain embodiments, Cerenkov luminescence can be used to cleave a photocleavable group or linker. For example, a photocleavable group or linker includes, but is not limited to, arylcarbonylmethyl, nitroaryl, coumarin, arylmethyl, and metal containing groups. A variant of nitroaryl group with a carboxylic acid, a-carboxy-2-nitrobenzyl was found to be more reactive and is used as described herein. In certain embodiments,

Cerenkov-light activated photocleavage can be used to transform a pro-contrast agent to a contrast agent. This functionality can be used in a variety of imaging modalities, for example, optical imaging magnetic resonance imaging MRI), positron emission tomography (PET), and/or magnetic resonance with coregistration using PET. In certain embodiments, a photocleavable group or linker increases the efficacy or contrast of the drug or imaging agent, respectively, upon photoactivation. The payload is not localized to the tissue, and the clearance of the payload is not affected.

[0128] In certain embodiments, nitrenes generated in situ from azides bind to

surrounding molecules irreversibly. Azides can be loaded with any payload, for example, imaging agents and/or drugs. [0129] The azide is activated by Cerenkov luminescence and converts to a nitrene. This promotes accumulation of the payload in the tissue. The azide may or may not change the potency of the drug, and in certain embodiments, it does not change the efficacy of a drug payload. Without wishing to be bound to a particular theory, in certain embodiments, the clearance properties of the payload can be modified because it is believed that the payload is bound to tissue upon activation (conversion to a nitrene).

[0130] In certain embodiments, Cerenkov-light activated photocleavage can be used to transform a pro-drug to a drug. In certain embodiments, caged doxorubicin with photocleavable linkers have been synthesized and optimized to cleave at low light levels.

[0131] In certain embodiments, the drug is a chemotherapeutic. In certain embodiments, the chemotherapeutic is a small molecule. In certain embodiments, the small molecule is selected from the group consisting of alkylating agents, anthracyclines, cytoskeletal disruptors (taxanes), epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents, retinoids, and vinca alkaloids and derivatives. In certain embodiments, the small molecule is described in

https://en.wikipedia.org/wiki/List_of_chemotherapeutic_ag ents, the contents of which are hereby incorporated by reference in its entirety.

[0132] In certain embodiments, Cerenkov light can be used in Photo Dynamic Therapy

(PDT). For example, the generation of singlet oxygen by small molecules can be activated by

Cerenkov light (e.g., radioisotope-generated light). The small molecules (e.g., porphyrin based molecules) are targeted towards a receptor or transporter which promotes accumulation in a tumor. After accumulation of the small molecules in a tumor, a radioisotope (e.g., ¹⁸ F, ⁹⁰ Y) can be administered to the tumor as a source of light. Thus, the treatment regimen is dependent on the tumor accumulation time of the PDT agent and the half-life of the radioisotope being used.

[0133] In certain embodiments, Cerenkov luminescence from the tumor site of ¹⁸ F-FDG

(fluorodeoxyglucose) accumulation can be used for in vivo pro-drug activation leading to theranostic strategies. In certain embodiments, Cerenkov luminescence from the tumor site of ¹⁸ F-FLT (fluorothymidine) can be used for in vivo pro-drug activation leading to theranostic strategies.

Examples

Synthesis and characterization of calcein arylazide, an exemplary Cerenkov activated tagging agent

[0134] FIG. 1 depicts a Cerenkov activated tagging agent comprising a payload and reactive covalent binding unit comprising a blocking agent. In certain embodiments, the

Cerenkov activated tagging agent is exposed to Cerenkov luminescence and the blocking agent is removed from the reactive covalent binding unit.

[0135] FIG. 2 depicts an in vivo mechanism of a Cerenkov activated tagging agent. In certain embodiments, tumor cells preferentially uptake ¹⁸ F-FDG. The higher uptake of ¹⁸ F-FDG generates increased Cerenkov light in tumor cells compared to normal tissue. After

administration and delivery of the Cerenkov activated tagging agent to the tumor tissue, or the tissue with increased uptake of ¹⁸ F-FDG, the Cerenkov activated tagging agent is activated and the payload is delivered specifically to tumor cells.

[0136] FIG. 3 depicts UV catalyzed arylazide decomposition monitored by measuring absorption spectra. As the arylazide is exposed to short bursts of UV radiation, the spectral features of the reaction mixture change as more product is formed. [0137] FIG. 4 depicts various uses of nitrene in a biological system. In certain embodiments, a phenyl azide reagent forms a nitrene after exposure to UV light. The nitrene formed can be used in addition reactions, active hydrogen (C-H) insertions, or active hydrogen (N-H) insertions. The nitrene formed can also undergo ring expansion and become a

dehydroazepine intermediate. This intermediate can become a nucleophile or a reactive hydrogen.

[0138] FIGS. 5A - 5B depict synthesis of fluorescein arylazide. The synthesis of fluorescein arylazide can be performed in two reactions and is shown in FIG. 5A. The first reaction is a two-step substitution of the amine to azide. The second reaction is the coupling of the carboxylic group to the amine of the fluorescein. In certain embodiments,

dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) are coupling agents. FIG. 5B shows fluorescein arylazide purified by UPLC and is confirmed using ESI MS.

[0139] FIG. 6 depicts Cerenkov targeting for therapy and/or imaging using fluorescein arylazide. Fluorescein arylazide is quickly internalized by cells, which increases the background signal and limits the reaction time of azide with the tumor cell as shown in FIG. 6. In certain embodiments, the kinetics of fluorescein arylazide can be improved by using a less permeable dye motif. There is zero signal to noise ratio (S/N) in the presence of UV and in the absence of fluorescein azide (data not shown). This data was collected using UV light because incubation of dye with ¹⁸ F-FDG resulted in cell permeabilization.

[0140] In certain embodiments, calcein, the molecular structure which is shown in FIG.

7, can be used as a fluorophore that can be conjugated to arylazide as shown in FIG. 8. Calcein provides at least the following advantages. First, calcein has four negative charges that prevent cellular internalization and therefore reduce background noise. Second, calcein provides multiple carboxyl groups for functionalization. Third, the absorption range is different from an arylazide moiety. Fourth, the probe is soluble in water. The fluorescence characteristics of calcein arylazide are shown in FIG. 9. Calcein arylazide has an excitation maximum wavelength of about 491 nm and an emission maximum wavelength of about 509 nm.

[0141] FIG. 10 shows UV activated cell staining using calcein aryl azide in vitro. MDA-

MB-231 cells were incubated with calcein-azide and free calcein with and without UV-light. Quantitative signal to noise (S/N) analysis and microscopy images show efficient cell labeling of calcein-azide after UV treatment. *P<0.01. A GFP channel can be used to collect emitted light calcein aryl azide.

[0142] FIG. 10 shows fluorescence images of a monolayer of MDA-MB-231 cells that were incubated either with the free dye calcein ± UV or the calcein functionalized azide ± UV. The cells incubated with calcein-azide + UV had a 4.5 times higher signal to noise (S/N) ratio than cells exposed to no UV and free calcein showing photoactivated binding of calcein-azide on the cells. As shown in FIG. 10, the Signal to Noise ratios of untreated (1), calcein dark (without UV) (2), calcein UV treated (3), and calcein azide dark (without UV) (4) are low compared to calcein azide UV treated (5).

Translation to in vivo systems

[0143] For translation of Cerenkov activated tagging agents to in vivo systems, an infrared (IR) dye is preferred when imaging tissue due to its low scattering and increased tissue penetration. In the examples described herein, Cy7 was used instead of calcein to take advantage of the higher penetration depth of NIR light. First, HT1080 cells were incubated with and without ¹⁸ F-FDG followed by Cy7-azide incubation and fluorescence images of the cell pellets were taken. In certain embodiments, the hydrophobicity of the dye-arylazide before and after photoactivation plays a role in the bioavailability of the azide available to be photoactivated. Moreover, the hydrophobicity can determine if the dye is to be delivered to the lymph or blood system.

[0144] The source of photoactivation is based on accumulation of a radioisotope in a particular tissue. For example, the accumulation can be facilitated via immunotargeting (e.g.,

90 18 18

J591-DOTA- Ύ as described below) or via trapped radiolabeled metabolites (e.g., F-FDG, F- FLT). Each of these accumulation approaches can provide unique accumulation characteristics. For example, the dye-arylazide construct that is injected into the subject (e.g., a human, mice, etc.) depends on the system (e.g., the hydrophobicity of the dye), and the kinetics can be characterized and matched accordingly.

[0145] To this end, cyanine dye-arylazide conjugates with different solubilities were investigated in mice. As shown in FIGS. 12 and 13, Cy5.5 arylazide (e.g., a hydrophobic dye) (FIG. 11 A) and sulpho-Cy7 arylazide (e.g., a hydrophilic dye) (FIG. 1 IB) were tested in mice.

[0146] FIG. 12 depicts Cy5.5 arylazide biodistribution in 5 mice at 15 minutes, 45 minutes, 2 hours, 4 hours, 22 hours, and 49 hours. FIG. 13 depicts sulpho-Cy7 arylazide biodistribution at 10 minutes, 30 minutes, 45 minutes, 75 minutes, and 4 hours. No fluorescence was observed after 24 hours.

¹⁸ F-FDG as a source of Cerenkov luminescence and in vivo experiment design

[0147] ¹⁸ F-FDG is clinically used to diagnose, stage, and evaluate recurrence in oncology. Accumulation of ¹⁸ F-FDG in a tumor is due to higher metabolic activity in a tumor.

[0148] To first test if ¹⁸ F-FDG can be used as a source for Cerenkov luminescence, five mice with HT1080 tumors were injected with ¹⁸ F-FDG or saline intratumorally. Next, sulpho-

Cy7-fluoroarylazide was injected at 100 μΐ., 10 μΜ retroorbitally. The mice were imaged at various time points; the 34 and 50 hours time points are shown in FIG. 14. FIG. 14 shows that mice 1 through 4 were injected with ¹⁸ F-FDG intratumorally and sulpho-Cy7 arylazide injected retroorbitally, Mice 1 and 2 were injected with ¹⁸ F-FDG, and mouse 5 was a control (no injection). FIGS. 15A and 15B show a graph plotting the average fluorescence signal in the circled regions of interest at different time points. FIG. 15A shows the average signal (RFU) of ¹⁸ F-FDG treated and untreated control mice at 20 hours, 34 hours, and 50 hours. FIG. 15B shows that mice treated with ¹⁸ F-FDG have significantly higher accumulation of the Cerenkov targeted sulpho-Cy7-arylazide construct compared to the ¹⁸ F-FDG untreated mice. The mice were euthanized at 70 hours, and the tumors were measured in IVIS as shown in FIG. 16.FIGS. 17A and 17B shows Cerenkov activated labeling of HT 1080 cells with photolabeling of sulpho- Cy7-arylazide using ¹⁸ F-FDG in vitro. Fluorescence images and quantitative analysis of cell pellets (n=3) pretreated or untreated with ¹⁸ F-FDG and post incubated with Cy7-azide show activation and binding of Cy7 only in combination with Cerenkov. ¹⁸ F-FDG uptake was measured to be 117 μθ per pellet in 30 minutes. Next, the cells were incubated for 2 hours with 0.01 nanomoles of sulpho-Cy7-arylazide and washed with PBS and 15% DMSO in PBS. As shown in FIGS. 17A and 17B, sulpho-Cy7-arylazide is activated only in cells that had uptaken ¹⁸ F-FDG. Pre-treated ¹⁸ F-FDG cells had a 95% higher Cy7 compared to untreated cells.

[0149] The above steps were repeated by replacing sulpho-Cy7-arylazide with Cy5.5 aryl azide. As seen in FIG. 18, lower fluorescence signal of Cy5.5 arylazide construct is present in mice at the 50 hour time point compared to sulpho-Cy7-arylazide construct present in mice at the 50 hour time point as shown in FIG. 14.

[0150] In vivo experiments using the Cy7 derivative were performed using male nude mice with PC3-hPSMA and are shown in FIGS. 22-24. Mice 1 and 2 were injected with ¹⁸ F- FDG. The Cerenkov luminescence images FIG. 22 and 24 show the radioactive signal of F. After ¹⁸ F-FDG administration, mice 1 through 4 were injected with 50 nmol of the Cy7 derivative and imaged at various time points as shown in FIG. 23. The Cy7 derivative requires about 50 hours to clear from the non ¹⁸ F-FDG treated tumors and to develop contrast. Contrast enhancement of ⁸ F-FDG tumors can be visualized over time in FIG. 25. Mouse 5 received neither ¹⁸ F-FDG nor the Cy7 derivative.

Immunotargeted Cerenkov source in vivo

[0151] In certain embodiments, ¹⁸ F, which has a short half-life, is attached to metabolites that quickly accumulate in tissues. In certain embodiments, ⁹⁰ Y, which has a longer half-life, is attached to immunoglobulins (e.g., J591) that slowly accumulate in tissues. In addition, ⁹⁰ Y provides more Cerenkov luminescence compared to ¹⁸ F. Therefore, in certain embodiments, a lower concentration of ⁹⁰ Y is required compared to ¹⁸ F.

[0152] In certain embodiments, immunoglobulins can be conjugated to Cerenkov light sources as shown in FIG. 19. For example, J591 is an immunoglobulin that is specific to prostate specific membrane antigen (PSMS) and is clinically used to target PSMA positive tumors. ⁹⁰ Y is a source that generates Cerenkov luminescence. Thus, in certain embodiments, accumulation of ⁹⁰ Y-DOTA-J591 at the tumor site can generate Cerenkov luminescence and be used to activate Cerenkov activated tagging agents.

Other applications of Cerenkov targeted azides

[0153] In certain embodiments, Cerenkov targeted azides can be developed as MRI contrast agents as depicted in FIG. 20 (e.g., pro-contrast agents to be activated to contrast agents upon exposure to Cerenkov luminescence). In certain embodiments, Cerenkov targeted azides can be developed as therapeutics as depicted in FIG. 21 (e.g., pro-drugs activated to drugs upon exposure to Cerenkov luminescence).

Exemplary drug activation by Cerenkov illumination

[0154] In certain embodiments, a pro-drug that can be synthesized by functionalizing doxorubicin or other drugs into a caged analog using a Cerenkov activated tagging agent (FIG. 1). The caged doxorubicin can increase the blood circulation time and lower the toxicity of the pro-drug compared to doxorubicin. Moreover, the caged doxorubicin can maintain metabolic stability. Upon activation of the caged doxorubicin by the Cerenkov enhanced tissue, toxicity is rendered only to the tumor site. This approach is not limited to doxorubicin and can be used with other small molecule chemotherapeutic drugs.

Exemplary imaging of tumors and drug delivery in vivo by Cerenkov illumination

[0155] The present Example further demonstrates that an activated Cerenkov

Luminescence payload delivery system using azides can be used for contrast enhancement or specific drug delivery for cancer therapy.

[0156] In this Example, for instance, mice with two HT1080 xenografts (n=10) were retroorbitally injected with ⁹⁰ Y labeled DOTA-(cRGD) ₂ or saline The beta emitting ⁹⁰ Y radioisotope was chosen due to its higher Cerenkov Luminescence compared to FDG. However, other radioisotopes can be used.

[0157] After 7 h, all mice were injected with the Cy7-azide in the alternate eye.

Fluorescence images of the mice were taken up to 156 hours after injection. As shown in FIGS. 28A-28C, mice injected with ⁹⁰ Y had a higher uptake of the dye in the tumor than mice injected with saline alone, where the highest contrast enhancement occurred at 3 h. [0158] A control compound was synthesized where the azide moiety of the Cy7-azide was replaced by a fluoride to have a Cerenkov Luminescence non-activatable dye. An in vivo experiment was performed as described herein (n=8). As shown in FIG. 29, there was no preferential accumulation of the control dye at the tumor site of the ⁹⁰ Y injected mice. This confirms that the Cy7 azide accumulation at the tumor site is a direct result of the Cerenkov Luminescence activation.