N-oxide-based zwitterionic near-infrared fluorophore imaging agents and methods of use thereof

Abstract

According to at least one aspect of the present disclosure, there is provided an imaging agent comprising a dye compound conjugated to an antigen-specific targeting vehicle, which can be particularly advantageous as its behavior in vivo can contribute to superior optical imaging properties, for example by significantly increasing the solubility of the dye and / or the targeting vehicle, increasing the target-to-background ratio of the imaged tissue, yielding higher resolution imaging, and ultimately providing better identification of malignant tissue for resection and margin assessment, as well as improved visualization, for example during minimally invasive laparoscopic surgery. A method of imaging tumor cells in a subject can comprise: administering an imaging effective amount of an imaging agent according to at least one embodiment of the present application; irradiating a region in the subject where tumor cells are expected to be found with a wavelength absorbed by the imaging agent; and detecting a signal from the imaging agent.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 538,038, filed September 12, 2023; U.S. Provisional Patent Application Serial No. 63 / 648,302, filed May 16, 2024; and U.S. Provisional Patent Application Serial No. 63 / 656,858, filed June 6, 2024. The disclosure of each of these applications is incorporated herein by reference.

[0003] This application also relates to U.S. Utility Patent Application Serial No. 18 / 640,169, filed April 19, 2024; U.S. Utility Patent Application Serial No. 18 / 732,982, filed June 4, 2024; U.S. Utility Patent Application Serial No. 18 / 641,097, filed April 19, 2024; U.S. Utility Patent Application Serial No. 18 / 641,149, filed April 19, 2024; and U.S. Utility Patent Application Serial No. 18 / 641,620, filed April 19, 2024, the disclosures of each of which are incorporated herein by reference.

[0004] sequence list

[0005] The sequence list conforming to WIPO standard ST.26 is incorporated herein by reference. The sequence list was submitted as an electronic document via PatentCenter as UTF-8 text encoded as XML. This electronic document was created on September 7, 2024, with the title “1515138_110WO2_SL.xml” and a size of 6,253 bytes. Technical Field

[0006] This invention relates to a method for optical imaging of tissues or cells using zwitterionic imaging agents having compact N-oxide-based zwitterions, resulting in improved stability and solubility compared to other near-infrared (NIR) fluorophore contrast agents. Background Technology

[0007] Near-infrared (NIR) fluorescence holds potential importance in the medical field, particularly in diagnostics and image-guided surgery. However, the availability of suitable fluorophores as imaging agents has been a major obstacle. For clinical feasibility, an ideal NIR fluorophore should possess both good optical properties and excellent in vivo properties in terms of solubility, metabolism, biodistribution, and clearance. Known fluorophores tend to be cleared via the liver, resulting in undesirable fluorescence throughout the gastrointestinal tract. Furthermore, in some cases, known fluorophores have insufficient solubility, particularly in saline solutions, leading to more difficult and expensive administration methods.

[0008] In recent years, more advanced fluorophores have been developed, producing improved target-background interactions. See, for example, U.S. Patent Nos. 11077210, 9687567, 10493169, 10201621, and 10478512. While these advanced fluorophores reduce non-specific uptake in non-target tissues, they are relatively unstable in the blood and are metabolized within minutes to hours. Therefore, there remains a need for new and improved NIR imaging agents that are targeted to tumor or diseased tissues and / or have enhanced stability, can rapidly balance between intravascular and extravascular spaces, and are efficiently cleared, including through renal filtration.

[0009] Furthermore, while existing fluorophores have achieved some success in targeting specific antigens and other biomarkers expressed by various cancer cell types, there remains a need to improve the targeting of specific antigens to enhance the specific uptake of the fluorophore and thus increase signal intensity. For example, targeting vectors for prostate-specific membrane antigen (PSMA), bombesin receptor, somatostatin receptor, and fibroblast activation protein (FAP) would provide fluorophores with increased signal intensity for more accurate imaging of various tumors and benign but diseased tissues, as well as tumor stroma and other diseases characterized by tissue remodeling. The imaging agent of this invention addresses these and other needs.

[0010] Improved solubility, particularly in saline solutions, and enhanced targeting potential provide better identification of malignant tissue for resection and margin assessment, as well as improved visualization during minimally invasive laparoscopic surgery. Therefore, further improvements in solubility and target-to-background ratio remain in the art to help practitioners achieve complete and accurate resection of cancerous tissue during surgery. Summary of the Invention

[0011] This invention is based, at least in part, on the discovery that containing zwitterionic components based on N-oxides can improve the solubility of zwitterionic fluorophores without reducing the stability or clearance provided by other elements designed by the fluorophore.

[0012] The imaging agents disclosed herein are particularly advantageous because their in vivo behavior is believed to contribute to excellent solubility, superior optical imaging properties, and, in some cases, excellent stability. More specifically, charge balance is thought to confer good biodistribution and clearance properties and reduce unwanted nonspecific binding, while the inclusion of a targeting ligand increases circulation time and prevents further degradation after binding to target cells. These in vivo properties contribute to improved target-to-background ratio in applied and imaged tissues, resulting in higher sensitivity and higher resolution imaging.

[0013] In one aspect, this disclosure provides a developer dye comprising a charge-balanced developer conjugated to a targeting carrier, the developer dye having one or more zwitterionic groups based on N-oxides.

[0014] In one embodiment of the developer dye according to this disclosure, the charge-balancing developer is a reagent of formula (I):

[0015] L-RC-(-Sp-N + (CH2)2O - ) p

[0016] For equation (I):

[0017] L represents a linker group that can conjugate with a target carrier;

[0018] RC represents the resonant core;

[0019] Each Sp independently represents a spacer group; and

[0020] p represents an integer from 1 to 4.

[0021] In another embodiment of the developer dye according to this disclosure, the charge-balancing developer is a reagent of formula (II), or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof:

[0022]

[0023] For equation (II):

[0024] Each R1 is independently a -C1-C4 alkyl-N + (CH2)2O - ;

[0025] Each R2 is independently H, OR', halogen, sulfonate, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0026] Each R3 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0027] Alternatively, each group of R1 and R2 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atoms to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0028] Alternatively, each group of R1 and R3 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atom to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0029] Each Q is N + (CH2)2O - ;

[0030] X and Y are each independently represented as O, S, Se, C(R”)2, NR”';

[0031] Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, wherein R6 is an optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, alkyl-N3, aryl-N3, or aryl-halogen.

[0032] Each R' is independently H, alkyl, or aryl;

[0033] Each R” is independently H or alkyl;

[0034] Each R”' is independently H, alkyl, alkyl-SO3H or alkyl-COOH;

[0035] m is an integer from 0 to 3, and

[0036] Each n is an independent integer from 1 to 4; and

[0037] L is an anion;

[0038] Furthermore, the Z group can be conjugated with the target carrier.

[0039] In yet another embodiment of the developer dye according to this disclosure, the charge-balancing developer is a reagent of formula (III), or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof:

[0040]

[0041] For equation (III):

[0042] Each R1 is independently a -C1-C4 alkyl-N + (CH2)2O - ;

[0043] Each R2 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0044] Each R3 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0045] Alternatively, each group of R1 and R2 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atoms to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0046] Alternatively, each group of R1 and R3 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atom to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0047] Each Q is N + (CH2)2O - ;

[0048] X and Y are independently represented as O, S, Se, C(R”)2, NR;

[0049] Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, wherein R6 is an optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, alkyl-N3, aryl-N3, or aryl-halogen.

[0050] R is independently H, OR”'' (where R = H, alkyl or aryl, NH2, NHR, alkylNH2, alkylCOOH),

[0051] L is an anion;

[0052] Each R' is independently H, alkyl, or aryl;

[0053] Each R” is independently H or alkyl;

[0054] Each R”' is independently H, alkyl, alkyl-SO3H or alkyl-COOH;

[0055] Each R'”' is independently H, alkyl or aryl, NH2, NHR, alkyl-NH2 or alkyl-COOH;

[0056] Each n is an independent integer from 1 to 4; and

[0057] L is an anion;

[0058] Furthermore, the Z group can be conjugated with the target carrier.

[0059] In yet another embodiment of the developer dye according to this disclosure, the charge-balanced developer is:

[0060] ;

[0061] ;

[0062] ;or

[0063] .

[0064] In some embodiments of the developer dyes according to this disclosure, the targeting carrier is cRGD, dPSMA-617, KUE, FAP-binding carrier, octreotide, or bufotoxin.

[0065] In other embodiments of the developer dyes according to this disclosure, the charge-balanced developer is conjugated to the target carrier via a direct bond or via a linking group.

[0066] In another aspect, this disclosure provides a method for imaging tissues, cells, or cavities in an object, the method comprising:

[0067] (a) For an object, contacting a tissue, cell, or cavity with a developer comprising a dye containing a developer according to this disclosure.

[0068] (b) Irradiate the tissue, cell or cavity at a wavelength absorbed by the dye;

[0069] (c) and detecting signals from the imaging agent to image the tissue, cell or cavity.

[0070] In some embodiments of a method for imaging tissue, cells, or cavities in an object, the cells are tumor cells. In some embodiments, the object is a human being.

[0071] In some embodiments of methods for imaging tissues, cells, or cavities in an object, the imaging agent has a peak absorbance at approximately 600 nm to 850 nm.

[0072] In other embodiments of the method for imaging tissues, cells, or cavities in an object, in vivo imaging of tissues or cells is performed.

[0073] In some embodiments of methods for imaging tissues, cells, or cavities in an object, the imaging agent also comprises a PEG portion.

[0074] In other embodiments of the method for imaging tissues, cells, or cavities in an object, the imaging agent also contains a radioactive isotope for single-photon emission computed tomography (SPECT) or positron emission tomography (PET).

[0075] In some further embodiments of the method for imaging tissues, cells, or cavities in an object, the imaging agent contains a reactive linker group, such as an NHS ester, a sulfonated NHS ester, or a TFP ester.

[0076] In yet another aspect, this disclosure provides a method for treating cancer in a subject, the method comprising:

[0077] (a) Applying an effective amount of the imaging agent according to this disclosure to the object.

[0078] (b) Irradiating cells, tissues or organs of a suspected cancerous object with wavelengths absorbed by the imaging agent;

[0079] (c) Diagnosing cancer in the cells, tissues, or organs of the subject by detecting signals from the imaging agent; and

[0080] (d) Administering chemotherapy, radiation therapy or surgery to a subject to treat cancer.

[0081] Other features and advantages of the invention will become apparent from the following detailed description and from the claims.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting. Attached Figure Description

[0083] To enable those skilled in the art to readily understand how to manufacture and use the apparatus and methods disclosed herein without excessive experimentation, some other embodiments will be described in detail below with reference to certain accompanying drawings, wherein:

[0084] Figure 1 A schematic overview of the structure of the N-oxide zwitterionic NIR fluorophore of the present invention.

[0085] Figure 2 This is a schematic diagram of four specific N-oxide zwitterionic NIR fluorophores of the present invention.

[0086] Figure 3 This is a schematic diagram outlining the synthesis scheme of the first three stages for synthesizing the N-oxide zwitterionic NIR fluorophore of the present invention.

[0087] Figure 4 This is a schematic diagram outlining the synthetic scheme for the condensation stage of the N-oxide zwitterionic NIR fluorophore of the present invention. The scheme illustrates two different condensation reactions to provide different resonance nuclei to the fluorophore.

[0088] Figure 5 This is a schematic diagram outlining the synthesis schemes of two different N-oxide zwitterionic NIR fluorophores of the present invention via SN1 reaction—the N-oxide zwitterionic NIR fluorophores having CO or CS bonds.

[0089] Figure 6 This is a schematic diagram outlining the synthetic schemes of two different Suzuki coupling reaction schemes for N-oxide zwitterionic NIR fluorophores of the present invention—the N-oxide zwitterionic NIR fluorophores having C-C bonds. Detailed Implementation

[0090] The following is a detailed description provided to assist those skilled in the art in practicing the present disclosure. Modifications and variations to the embodiments described herein may be made by those skilled in the art without departing from the spirit or scope of this disclosure. All publications, patent applications, patents, drawings, and other references mentioned herein are expressly incorporated herein by reference in their entirety.

[0091] This disclosure relates in particular to imaging agents composed of dye molecules optionally conjugated to a targeting ligand via a linking group. For example, the imaging agents described herein can be used to detect abnormal or diseased biological tissues and cells. The conjugates are particularly useful for imaging the entire organism because they exhibit improved in vivo behavior, such as low nonspecific binding to non-target tissues and ultra-high stability, resulting in an improved target-to-background ratio associated with the detected optical signal. These improved in vivo properties are thought to arise from the balance of formal charges on the conjugate, thereby producing “charge-balanced” molecules with a neutral or near-neutral net charge.

[0092] Definitions and alternative implementation schemes

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terminology used in this specification is for describing particular embodiments only and is not intended to limit the scope of this disclosure.

[0094] For example, the following discussion includes a list of non-exhaustive definitions for several specific terms used in this disclosure (other terms may be defined or clarified elsewhere in this document). These definitions are intended to clarify the meaning of the terms used herein. Terms are assumed to be used in a manner consistent with their ordinary meaning, but their definitions are still provided herein for clarity.

[0095] The following definitions will help in understanding this invention.

[0096] In some embodiments, the imaging agents of the present invention may further comprise a targeting carrier for agricultural processes, chemical processes, disease or tissue-specific epitopes, such as the cyclic peptide cRGDyK (also known as cRGD) bound to the imaging agent. cRGD is a cyclic derivative of the tripeptide Arg-Gly-Asp, which may be conjugated to one or more imaging agents of the present invention. In still other embodiments, the targeting carrier is octreotide or bufotoxin. In yet other embodiments, the targeting carrier is KUE or dPSMA-617, a small molecule capable of targeting fibroblast activation protein (FAP), also known as an FAP inhibitor or FAPI, an amino acid or combination of amino acids, or a derivative thereof. In such embodiments, a targeting carrier-conjugate may be formed in place of one or more zwitterionic groups. In some embodiments, the targeting ligand comprises one or more of the following: for the diagnosis / treatment of melanoma, having LyP-1 peptide with a sequence that binds to P32; used for the diagnosis / treatment of breast tumors. A K237 peptide with a sequence that binds to VEGFR-2; used for the diagnosis / treatment of lung tumors, breast tumors, and colon tumors. IL4RPep-1 peptide with sequence and binding to IL4R; used for the diagnosis / treatment of breast tumors. mUNO peptide with sequence and binding to CD206; folic acid receptor for the diagnosis / treatment of ovarian and lung cancer; GE11 (a dodecapeptide) that binds to epidermal growth factor receptor (EGFR or ErbB1) for the diagnosis / treatment of epithelial-derived tumors.

[0097] An ideal zwitterionic imaging dye conjugated to a targeting carrier will utilize the total net charge of the targeting carrier. This is intentional because, in most cases, the charge on the targeting carrier is crucial for its ability to bind to its target. Therefore, the targeted zwitterionic imaging dye retains the key advantage of maximizing specific binding while minimizing non-specific binding. It will be apparent to those skilled in the art that additional charge can be added to the zwitterionic imaging dye if desired to balance the total surface charge to zero.

[0098] In some embodiments, the developing dye may contain a reactive linker group. Such a reactive linker group is typically an activated carboxylic acid derivative, such as n-hydroxysuccinimide (NHS) ester, sulfonated NHS ester, pentafluorophenyl (PFP) ester, hydroxybenzotriazole (HOBt) ester, hydroxyazabenzotriazole (HOAt) ester, tetrafluorophenyl (TFP) ester, anhydride, acidic azide, or acyl halide. As will be understood by those skilled in the art in synthesizing such compounds, such a reactive linker group can be attached to or substituted onto a chelating agent at any suitable structural position. Reactive linker groups also include, but are not limited to, alkynes, azides, maleimides, thiols, amines, alcohols, phenols, carbonyl groups, phosphines, alkenes, and tetraazines.

[0099] The term “comprising / including” as used herein is intended to mean that a composition and method includes the elements described, but does not exclude other elements. “consisting substantially of” when used to define a composition and method should mean excluding other elements that have any substantial significance to the composition. Therefore, a composition consisting substantially of the elements defined herein does not exclude trace contaminants from separation and purification methods, as well as pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, etc. “consisting of” should mean excluding other components besides trace elements and substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transitional terms are within the scope of the invention.

[0100] Unless the context clearly indicates otherwise, as used in this specification and claims, nouns without quantifiers include plural pronouns.

[0101] The ranges provided in this document are understood as abbreviations of all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange that comes from the following groups: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0102] Unless otherwise specified or obvious from the context, the term “or” as used herein shall be understood to be inclusive.

[0103] The listing of chemical groups in any definition of a variable herein includes any single group or combination thereof that defines the variable as a listed group. Descriptions of embodiments of a variable or aspect herein include embodiments as a single embodiment or in combination with any other embodiment or part thereof. The terms “object” or “patient” as used herein encompass both mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, parasites, microorganisms, etc.

[0104] As used herein, the term "administering" a compound to an object refers to providing the compound and / or its precursor drug of the present invention to an object requiring diagnosis or treatment.

[0105] As used in this article, the term "carrier" refers to a chemical compound or reagent that facilitates the incorporation of the compounds described herein into cells or tissues.

[0106] The term “acceptable” as used in this article with respect to the formulations, compositions or ingredients used herein means that it has no lasting harmful effect on the overall health of the person receiving treatment.

[0107] As used herein, the term "diluent" refers to a chemical compound used to dilute the compounds described herein prior to delivery. Diluents may also be used to stabilize the compounds described herein.

[0108] As used herein, the term "contact" refers to bringing substances together in a manner that allows them to interact with each other. For example, when an imaging agent is "contacted" with a tissue or cell, the tissue or cell can interact with the imaging agent, such as allowing the feasibility of binding interactions between the reagent and molecular components of the tissue or cell. "Contact" also includes the application of a substance, such as the imaging agent of this invention, to an organism. Application can be, for example, oral or extragastric.

[0109] As used herein, the term "ionic group" refers to a portion containing one or more charged substituents. A "charged substituent" is a functional group that is typically an anionic or cationic when subjected to substantially neutral aqueous conditions (e.g., pH approximately 6.5 to 8.0 or approximately physiological pH (7.4)). As mentioned above, some examples of charged anionic substituents include anions of inorganic and organic acids, such as sulfonates (-SO3-). 1-The substituents include sulfite, carboxylate, phosphonite, phosphonate, phosphate, and their esters (e.g., alkyl esters). In some embodiments, the charged substituent is a sulfonate. Some examples of charged cationic substituents include quaternary ammonium (-NR3+), where R is independently selected from C1-6 alkyl, aryl, and arylalkyl groups. Other charged cationic substituents include protonated primary, secondary, and tertiary amines, as well as guanidine. In some embodiments, the charged substituent is N(CH3)3+.

[0110] As used herein, the phrase "nonionic oligomeric or polymeric solubilizing group" refers to soluble polymers such as polyethylene glycol, polypropylene glycol, copolymers of polyethylene oxide and propylene oxide, carbohydrates, dextran, polyacrylamide, etc. The solubilizing group can be linked in any desired manner. Linkage points can be, for example, carbon-carbon bonds, carbon-oxygen bonds, or nitrogen-carbon bonds. Linking groups can be, for example, ester groups, carbonate groups, ether groups, thioether groups, amino groups, alkylene groups, amide groups, carbonyl groups, or phosphate groups.

[0111] Some examples of solubilizing groups include polyethylene glycol, such as (CH2CH2O)aH, -OC(=O)O(CH2CH2O)aH, -OC(=O)O(CH2CH2O)aCH3, -O(CH2CH2O)aCH3, and -S(CH2CH2O)2CH3, where "a" means about 2 to about 1. The integer between 4 and 12 or 5 and 10. In some embodiments, "a" is 6, 7, or 8. Other examples of solubilizing groups include dextran, such as -OC(=O)O (dextran).

[0112] The absolute molecular weight of the solubilized portion can be from about 500 amu to about 100,000 amu, for example from about 1,000 amu to about 50,000 amu or from about 1,500 to about 25,000 amu.

[0113] Other examples of solubilizing groups include: -(CH2)c-(OCH2CH2)d-ORa, where "c" is 0 to 6, "d" is 1 to 200, and Ra is H or a C1-6 alkyl group. In some embodiments, "c" is 1 to 4, "d" is 1 to 10, and Ra is H. In some embodiments, "d" is 6 or 7.

[0114] See WO 2008 / 017074, U.S. Serial No. 12 / 376,243 (filed February 3, 2009) and U.S. Serial No. 12 / 376,225 (filed February 3, 2009), each of which is incorporated herein by reference in its entirety for further description of suitable nonionic oligomeric or polymeric solubilizing groups and methods for incorporating them into dyes.

[0115] It should also be understood that, for clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, multiple features of the invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination.

[0116] The compounds of this invention may also contain all isotopes of the atoms present in the intermediates or the final compound. Isotopes include those atoms with the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0117] Unless otherwise stated, chemical substances referred to in this document by name, chemical formula or structure are intended to include all their stereoisomers, geometric isomers, tautomers, resonance structures and isotopes.

[0118] The chemical substances described herein may be charged or contain substituents with a formal charge. When such a chemical substance is represented as charged, it should be understood that, unless otherwise stated, the charge is generally counteracted by a suitable counterion. For example, a chemical substance or functional group with a -1 charge is understood to be counteracted by an ion with a +1 charge. Suitable counterions with a +1 charge include Na+, K+, tetraalkylammonium ions, etc. Conversely, a chemical substance or functional group with a +1 charge is understood to be counteracted by an ion with a -1 charge. Suitable counterions with a -1 charge include F-, Cl-, Br-, I-, sulfate, phosphate, perchlorate, acetate, trifluoroacetate, maleate, fumarate, methanesulfonate, lactate, pyruvate, levulinate, gluconate, etc.

[0119] Imaging dyes

[0120] In at least one aspect, the present invention provides a developer dye comprising a charge-balanced developer conjugated to a targeting carrier. In some embodiments, the charge-balanced developer may have one or more N-oxide-based zwitterionic groups having the formula shown herein and in the accompanying drawings.

[0121] The imaging agent of the present invention is particularly advantageous because its behavior in vivo is believed to contribute to excellent optical imaging properties and excellent stability. More specifically, charge balance is thought to confer good biodistribution and clearance properties to the agent and reduce unwanted nonspecific binding, while the inclusion of a targeting ligand increases circulation time and contact time with the target. These in vivo properties contribute to improving the target-to-background ratio of the imaged tissue, resulting in higher resolution imaging.

[0122] In at least one aspect, the present invention provides a developer that may comprise a dye having one or more zwitterionic groups based on N-oxides. According to at least one aspect of the invention, the developing dye may be conjugated with a targeting ligand or a targeting carrier, as discussed in the following sections.

[0123] In at least one aspect, the developer used in this invention is a charge-balanced developer, which is a reagent of formula (I):

[0124] L-RC-(-Sp-N + (CH2)2O - ) p

[0125] For equation (I):

[0126] L represents a linker group that can conjugate with a target carrier;

[0127] RC represents the resonant core;

[0128] Each Sp independently represents a spacer group; and

[0129] p represents an integer from 1 to 4.

[0130] In at least one aspect, the developer used in this invention is a charge-balancing developer, which is a reagent of formula (II) below, or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof:

[0131] (II):

[0132]

[0133] For equation (II):

[0134] Each R1 is independently a -C1-C4 alkyl-N + (CH2)2O - ;

[0135] Each R2 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0136] Each R3 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0137] Alternatively, each group of R1 and R2 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atoms to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N+ (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0138] Alternatively, each group of R1 and R3 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atom to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0139] Each Q is N + (CH2)2O - ;

[0140] X and Y are each independently represented as O, S, Se, C(R”)2, NR”';

[0141] Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, wherein R6 is an optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, alkyl-N3, aryl-N3, or aryl-halogen.

[0142] Each R' is independently H, alkyl, or aryl;

[0143] Each R” is independently H or alkyl;

[0144] Each R”' is independently H, alkyl, alkyl-SO3H or alkyl-COOH;

[0145] m is an integer from 0 to 3, and

[0146] Each n is an independent integer from 1 to 4; and

[0147] L is an anion;

[0148] Furthermore, the Z group can be conjugated with the target carrier.

[0149] In at least one aspect, the developer used in this invention is a charge-balancing developer, which is a reagent of formula (III) below, or a salt, solvate, hydrate, polymorph, prodrug, or stereoisomer thereof:

[0150]

[0151] For equation (III):

[0152] Each R1 is independently a -C1-C4 alkyl-N +(CH2)2O - ;

[0153] Each R2 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0154] Each R3 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl;

[0155] Alternatively, each group of R1 and R2 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atoms to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0156] Alternatively, each group of R1 and R3 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atom to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H;

[0157] Each Q is N + (CH2)2O - ;

[0158] X and Y are independently represented as O, S, Se, C(R”)2, NR;

[0159] Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, wherein R6 is an optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, alkyl-N3, aryl-N3, or aryl-halogen.

[0160] R is independently H, OR”'' (where R = H, alkyl or aryl, NH2, NHR, alkylNH2, alkylCOOH),

[0161] L is an anion;

[0162] Each R' is independently H, alkyl, or aryl;

[0163] Each R” is independently H or alkyl;

[0164] Each R”' is independently H, alkyl, alkyl-SO3H or alkyl-COOH;

[0165] Each R'”' is independently H, alkyl or aryl, NH2, NHR, alkyl-NH2 or alkyl-COOH;

[0166] Each n is an independent integer from 1 to 4; and

[0167] L is an anion;

[0168] Furthermore, the Z group can be conjugated with the target carrier.

[0169] In some embodiments of the invention, the developing dye according to the invention has the following structure (either as a salt, solvate, or hydrate, or as a dimer):

[0170]

[0171] ZN-800-1

[0172] In some embodiments of the invention, the developing dye according to the invention has the following structure (either as a salt, solvate, or hydrate, or as a dimer):

[0173]

[0174] ZN-830-1

[0175] In some embodiments of the invention, the developing dye according to the invention has the following structure (either as a salt, solvate, or hydrate, or as a dimer):

[0176]

[0177] ZN-800-1-Forte

[0178] In some embodiments of the invention, the developing dye according to the invention has the following structure (either as a salt, solvate, or hydrate, or as a dimer):

[0179]

[0180] ZN-700-1-Forte

[0181] In some embodiments of the invention, the imaging dye according to the invention may comprise two or more imaging agents described herein. In some specific embodiments, the imaging dye according to the invention may comprise two or more imaging agents, wherein the imaging agent cores are the same but have different targeting carriers. In still other embodiments, the imaging dye according to the invention may comprise two or more imaging agents described herein, wherein the imaging agent cores are different but have the same targeting carrier—specifically, but not limited to, the imaging agent cores may be selected to absorb different wavelengths of radiation. In still other embodiments, the imaging dye according to the invention may comprise two or more imaging agents described herein, wherein the imaging agent cores are different and the targeting carriers are different—specifically, but not limited to, the imaging agent cores may be selected to absorb different wavelengths of radiation, while the targeting carriers may be selected to identify different cell / tumor types.

[0182] The imaging dye of the present invention can be used Figures 3 to 6 The synthesis is performed according to the scheme described in the illustration. Alternatively, the N-oxide functional group can be synthesized by replacing group R in the scheme with a suitable leaving group (e.g., a halide ion or a sulfonate ion) through nucleophilic substitution. The nucleophile in these transformations is an N,N-dialkylated hydroxylamine, which can be protected at the hydroxyl group.

[0183] Targeting ligands or vectors

[0184] At least one aspect of the invention provides an imaging agent comprising one of the aforementioned imaging dyes conjugated to a targeting ligand or targeting carrier. In some embodiments, the targeting carrier (or targeting ligand) may be cRGD, a PSMA-binding carrier (e.g., dPSMA-617 or KUE), an FAP-binding carrier (e.g., NH2-FAPI-74), a bufotin receptor-binding carrier, or a somatostatin receptor-binding carrier, or a corresponding homodimer or heterodimer thereof. In some embodiments, the targeting carrier is an agonist or antagonist of the target, including but not limited to agonists or antagonists of integrins, agonists or antagonists of FAP, agonists or antagonists of PSMA, agonists or antagonists of somatostatin receptors, and agonists or antagonists of GRPR. In still other embodiments, the targeting carrier may be a derivative of a targeting carrier, including but not limited to derivatives of cRGD, derivatives of FAP, derivatives of PSMA-binding carriers, derivatives of octreotide, and derivatives of bufotin.

[0185] In some embodiments, the charge-balancing imaging agent is conjugated to the target carrier via a direct bond or via a linker group, as shown in the formulas and figures described herein.

[0186] In some implementations, one or more imaging agent conjugates in combination form contain cRGD as a targeting ligand.

[0187] In some embodiments of the present invention, the targeting ligand according to the present invention may be a cyclic-RGD having the following structure:

[0188]

[0189] cRGD peptide targeting ligand

[0190] Below are some examples of cRGD conjugates for use in developing agents, including cRGD-ZW800-1, cRGD-ZW830-1, or cRGD-ZW700-1-Forte. The targeting ligand can be conjugated to the developing dye via an optional linking group or via a direct bond. As shown herein, the developing targeting ligand is conjugated to the developing dye via an amine bond. However, other conjugation bonds as determined by those skilled in the art can be used. Other cRGD conjugates can be prepared without departing from the spirit of the invention.

[0191] Integrins are a family of cell adhesion receptors that enable cell motility and invasion. The human integrin family comprises 18 α and β subunits that assemble into distinct functional heterodimers. Some integrins act as mediators of angiogenesis in solid tumors. Integrin expression is important for tumor progression and metastasis by promoting tumor cell migration, invasion, proliferation, and survival. Besides cancer, integrins are also highly expressed during many normal and abnormal processes such as fibrosis, wound healing, and inflammation. Integrin αvβ3 expression has been identified as a surrogate marker of angiogenic activity. Similarly, integrin αvβ6 is a subtype of the integrin family that is expressed only on epithelial cells. αvβ6 is typically expressed at low or undetectable levels in normal adult tissues, but can be highly upregulated during pathological and physiological processes such as wound healing, fibrosis, inflammation, and cancer. RGD mimics have been used as targeting components in some studies to deliver integrin antagonists to multiple cell types. However, continuous infusion of RGD peptides has been found to stimulate tumor growth (see Reynolds, LE et al., Enhanced pathological angiogenesis in mice lacking β3 integrin or β3 and β5 integrins. Nature Med. 8, 27–34 (2002)). Furthermore, many RGD binders have expensive, complex, time-consuming, and low-yield synthetic processes. Similarly, in imaging, the degradation of reagents and the accumulation of RGD binders in the bladder have been found to impair some imaging methods. Considering their stability and clearance, the conjugates of this invention have the potential to provide long-term visual identification and imaging of tumors overexpressing integrins while avoiding the obstacles of long-term systemic administration of RGD peptide ligands.

[0192] Therefore, in one specific aspect, charge-balanced imaging agents can be used to image or identify integrin overproduction in target cells (e.g., during surgery), for example when the imaging dye compound is conjugated with cRGD, as shown in the structure above.

[0193] In some embodiments of the invention, the targeting ligand according to the invention may be a prostate-specific membrane antigen (PSMA) binding carrier. For example, dPSMA-617 or Vipivotide tetraxetan. In some embodiments, the PSMA binding carrier may be F, a targeting carrier based on (((S)-5-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carbamate)-3-(naphth-2-yl)propamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid, which is a derivative of PSMA-617 or Vipivotide tetraxetan. This PSMA targeting carrier is referred to as dPSMA-617 in this disclosure (representing "a derivative of PSMA-617").

[0194] In some embodiments of the present invention, the targeting ligand according to the present invention may be dPSMA-617 having the following structure:

[0195]

[0196] dPSMA-617

[0197] The targeting ligand can be conjugated to the developing dye via an optional linking group or via a direct bond. As shown herein, the developing targeting ligand is conjugated to the developing dye via an amine bond. However, other conjugation bonds as determined by those skilled in the art can be used. Other dPSMA-617 conjugates can be prepared without departing from the spirit of the invention.

[0198] In some embodiments, the targeting ligand comprises one or more derivatives of bufotin, including but not limited to BIM26226, JMV594, JMV641, JMV736, JMV717, JMV845, and JMV542. Other bufotin derivatives, including bufotin agonists, bufotin antagonists, and bufotin pseudopeptides, can be found in Azay et al., Peptides, Vol. 19, No. 1, pp. 57-63, 1988—which is incorporated herein by reference.

[0199] In some embodiments of the present invention, the PSMA targeting carrier is (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid (KUE). The targeting ligand KUE may have the following structure:

[0200]

[0201] The targeting ligand can be conjugated to the developing dye via an optional linking group or via a direct bond. As shown herein, the developing targeting ligand is conjugated to the developing dye via a linking group (L). However, other conjugation bonds as determined by those skilled in the art can be used. Other KUE conjugates can be prepared without departing from the spirit of the invention.

[0202] In some implementations, the FAPI targeting ligand includes NH2-FAPI-74. Specific details about NH2-FAPI-74 are described in: Linder et al., “Radioligands Targeting Fibroblast Activation Protein (FAP)”, which is incorporated herein by reference in its entirety.

[0203] Generally speaking, NH2-FAPI-74 has the following structure:

[0204]

[0205] The targeting ligand can be conjugated to the developing dye via an optional linking group or via a direct bond. As shown herein, the developing targeting ligand is conjugated to the developing dye via a linking group (L). However, other conjugation bonds as determined by those skilled in the art can be used. Other FAP conjugates can be prepared without departing from the spirit of the invention.

[0206] In some embodiments of the present invention, the targeting ligand according to the present invention may be a toad peptide receptor binding vector.

[0207] Generally, the structure of bufotoxin (SEQ ID No.: 6) is as follows:

[0208]

[0209] or its derivatives.

[0210] The targeting ligand can be conjugated to the imaging dye via an optional linker group or via a direct bond. As shown herein, the imaging targeting ligand is conjugated to the imaging dye via a linker group (L). However, other conjugation bonds as determined by those skilled in the art can be used. Other bufotin conjugates can be prepared without departing from the spirit of the invention.

[0211] In some embodiments of the present invention, the targeting ligand according to the invention may be a somatostatin receptor binding vector. In some embodiments, the somatostatin receptor binding vector may be octreotide.

[0212] Generally, the structure of octreotide (SEQ ID No.: 5) is as follows:

[0213]

[0214] or its derivatives.

[0215] The targeting ligand can be conjugated to the developing dye via an optional linker group or via a direct bond. As shown herein, the developing targeting ligand is conjugated to the developing dye via a linker group (L). However, other conjugation bonds as determined by those skilled in the art can be used. Other octreotide conjugates can be prepared without departing from the spirit of the invention.

[0216] In some implementations, the targeting ligand comprises one or more of the following: [elements related to the diagnosis / treatment of melanoma]. LyP-1 peptide with a sequence that binds to P32; used for the diagnosis / treatment of breast tumors. A K237 peptide with a sequence that binds to VEGFR-2; used for the diagnosis / treatment of lung tumors, breast tumors, and colon tumors. IL4RPep-1 peptide with sequence and binding to IL4R; used for the diagnosis / treatment of breast tumors. mUNO peptide with sequence and binding to CD206; folic acid receptor for the diagnosis / treatment of ovarian and lung cancer; GE11 (a dodecapeptide) that binds to epidermal growth factor receptor (EGFR or ErbB1) for the diagnosis / treatment of epithelial-derived tumors.

[0217] Therefore, the present invention provides a developer dye comprising a charge-balancing developer conjugated to a targeting carrier. In certain embodiments of the developer dye according to the invention, the charge-balancing developer may be ZN-800-1, ZN-830-1, ZN-800-1-Forte, or ZN-700-1-Forte, and the targeting carrier may be a PSMA ligand (e.g., KUE or dPSMA-617), FAP ligand, bufotenide receptor ligand, or somatostatin receptor ligand, as shown and described. The charge-balancing developer may be conjugated to the targeting carrier via a direct bond or via a linker group.

[0218] Any suitable combination of charge-balanced imaging agents and targeting ligands can be used, for example, charge-balanced imaging agents discussed herein, such as ZN-800-1, ZN-830-1, ZN-800-1-Forte, or ZN-700-1-Forte, which can be used with any of the targeting vectors discussed herein, such as cRGD, PSMA-binding vectors (e.g., dPSMA-617 or KUE), FAP-binding vectors, bufotenide receptor-binding vectors, or somatostatin receptor-binding vectors.

[0219] In each combination, the corresponding targeting ligand or carrier can be covalently linked to the corresponding reactive linker group of the corresponding dye compound of the present invention via a standard coupling procedure. For example, the carboxyl group or activated carboxyl group of the reactive linker can react with a nucleophilic functional group (e.g., an amine or alkoxy derivative) on the targeting ligand to form an amide or ester bond. Further details regarding dye conjugation can be found in WO 2008 / 017074 and in Frangioni et al., Molecular Imaging, Vol. 1 (4), 354-364 (2002), both of which are incorporated herein by reference in their entirety.

[0220] In some embodiments, the targeting carrier may be a targeting ligand and may be covalently linked to a reactive group of the dye compound via a standard coupling procedure or via an optional linker group (L). For example, the carboxyl group or activated carboxyl group of the reactive linker group may react with a nucleophilic functional group (e.g., an amine or alkoxy derivative) on the targeting ligand to form an amide or ester bond. Further details regarding dye conjugation can be found in WO 2008 / 017074 and in Frangioni et al., Molecular Imaging, Vol. 1 (4), 354-364 (2002), both of which are incorporated herein by reference in their entirety.

[0221] As used herein, "linking group" refers to any molecular entity with a molecular weight of about 50 to about 500 Da capable of conjugating a targeting ligand (TL). Specifically, the linking group comprises at least one reactive group selected from carboxylic acid groups or their anhydrides or esters and isothiocyanate groups. In some embodiments, the linking group comprises a carboxylic acid group. In some embodiments, the linking group is a PEG moiety or a straight-chain or branched hydrocarbon moiety having 2 to 12 carbon atoms. In some embodiments, the linking group includes a branch point, a reactive linking group, or other reactive groups that can be used to further functionalize the developer, for example, for containing a radioactive isotope.

[0222] In some embodiments, the targeting ligand may also include a molecular scaffold portion that can be linked to the binding portion and other groups. For example, the molecular scaffold may have one or more of the following: (1) a portion designed to react with the reactive linking group of the dye to form a covalent bond, (2) a charge-balancing portion, such as any ionic group described herein, and (3) a portion that binds to the biological target. An example of a molecular scaffold is an adamantane derivative, such as that described in U.S. Patent Application Publication No. 2006 / 0063834, which is incorporated herein by reference in its entirety, and illustrate by way of example the preparation of a targeting ligand incorporated into an adamantane scaffold. In particular, the adamantane core contains (1) an amino group capable of reacting with the dye compound, (2) a charge-balancing portion that neutralizes the negative charge on the dye molecule, and (3) two portions that bind to the biological target PSMA. For a description of the portion relating to PSMA, see Humblet, V. et al. Mol. Imaging, 2005, 4: 448-62; Misra P. et al. J. Nucl. Med. 2007, 48: 1379-89; Chen, Y. et al. J. Med. Chem, 2008, 51: 7933-43; Chandran, SS, et al. Cancer Biol. Ther., 2008, 7:974-82; Banerjee, SR, J. Med. Chem. 2008, 51: 4504-17; Mease, RC, et al. Clin. Cancer Res., 2008, 14:3036-43; Foss, CA, et al. Clin. Cancer. Res., 2005, 11:4022-8, each of which is incorporated herein by reference in its entirety.

[0223] In some embodiments, ZN-800-1, ZN-830-1, ZN-800-1-Forte, or ZN-700-1-Forte, the targeting ligand, and the developer can be separated into salts, acids, bases, or combinations thereof. For example, dyes, conjugates, and developers having multiple charged substituents can be separated by introducing counterions and / or protons sufficient to counteract the charges of the multiple substituents typically present at neutral pH, such that the dye, conjugate, or developer can be separated, for example, as a solid substance.

[0224] In some embodiments, the imaging agent further comprises a PEG moiety. As will be understood by those skilled in the art in synthesizing such compounds, such a moiety can bind to the conjugate at any suitable structural position. In some embodiments, the agent further comprises a PEG moiety to alter circulation time in the blood. As will be understood by those skilled in the art in synthesizing such compounds, such a moiety can bind to the conjugate at any suitable structural position. Additionally, in some embodiments, using methods known to those skilled in the art, the PEG moiety can be included as an optional linking group between ZN-800-1, ZN-830-1, ZN-800-1-Forte, or ZN-700-1-Forte and the targeting ligand.

[0225] In some embodiments, the imaging agent may contain a radioactive isotope for single-photon emission computed tomography (SPECT) or positron emission tomography (PET). As will be understood by those skilled in the art in synthesizing such compounds, such a radioactive isotope can be bound to or further conjugated with the conjugate at any suitable structural location.

[0226] In some embodiments, the developer may contain a reactive linker group. Such a reactive linker group is typically an activated carboxylic acid derivative, such as n-hydroxysuccinimide (NHS) ester, sulfonyl-NHS ester, pentafluorophenyl (PFP) ester, hydroxybenzotriazole (HOBt) ester, hydroxyazabenzotriazole (HOAt) ester, tetrafluorophenyl (TFP) ester, anhydride, acidic azide, or acyl halide. As will be understood by those skilled in the art in synthesizing such compounds, such a reactive linker group can be attached to or substituted onto a chelating agent at any suitable structural position. Reactive linker groups also include, but are not limited to, alkynes, azides, maleimides, thiols, amines, alcohols, phenols, carbonyl groups, phosphines, alkenes, and tetrazines.

[0227] Imaging methods, methods for identifying and imaging tumors, and methods for treating cancer.

[0228] Embodiments comprising imaging agents conjugated with dye compounds that target antigens specifically can be particularly advantageous because the behavior of such imaging agents in vivo can contribute to superior optical imaging properties, such as producing higher resolution imaging by significantly increasing the target-to-background ratio of the imaged tissue, and ultimately providing better identification of malignant tissue for resection and margin assessment, as well as improved visualization, for example, during minimally invasive laparoscopic surgery. Complete and accurate resection of cancerous tissue during surgery is crucial; therefore, the higher target-to-background ratio observed in embodiments using imaging agents in conjunction with one or more embodiments of the methods of the invention described herein provides clinicians with better visualization to ensure complete resection.

[0229] Furthermore, embodiments of the imaging agent according to the invention include unstable linkages that, when broken, cause the fluorophore to cease fluorescence in the NIR spectrum and dissociate the targeting ligand from the fluorophore. The imaging dye is stable if the targeting ligand binds to the cell and is internalized in lysosomes or other acidic intracellular compartments. The NIR fluorophore of the present invention is highly stable in acidic environments and therefore exhibits maximum NIR fluorescence. Conversely, unbound targeting fluorophores degrade over time in the blood, significantly reducing background. This combination of increased NIR fluorophore stability after targeting and internalization and decreased stability in the blood leads to counterintuitive results, forming the basis of the present invention.

[0230] In at least one aspect, the present invention provides a method for imaging cells (e.g., tumor cells) in an object, wherein the cells may be tumor cells, inflammatory cells or cells undergoing angiogenesis, and the object may be a human object, and further, the cells may be imaged in vivo.

[0231] Methods for imaging tissues, cells, or cavities include the following basic steps:

[0232] (a) Contacting a tissue, cell or cavity with a developer containing a dye conjugate, the conjugate containing a PSMA, FAP, bufotenide receptor or somatostatin receptor targeting ligand that binds to the dye.

[0233] (b) Irradiating tissues or cells at wavelengths absorbed by the conjugate; and

[0234] (c) Detecting optical signals from irradiated tissues or cells.

[0235] In some embodiments, the method according to the present invention may include the following steps:

[0236] a) Applying an effective amount of the imaging agent according to any one or more embodiments of the invention described herein to the object;

[0237] b) Two to four hours after application of the imaging agent, irradiate the area of ​​the subject where tumor cells are expected to be found with a wavelength absorbed by the imaging agent; and

[0238] c) Detect the signal from the developer.

[0239] In at least one aspect, the present invention provides a method for imaging cells, and also provides a method for treating cancer in a subject.

[0240] In some embodiments of the method according to the invention, the method for treating cancer may further include imaging cells, which may include the following steps:

[0241] a) Applying an imaging-effective amount of an imaging agent according to any one or more embodiments of the invention described herein (e.g., an imaging agent comprising a dye conjugate containing a PSMA, FAP, bufotoxin receptor, or somatostatin receptor-targeting ligand bound to the dye); and

[0242] b) Irradiate cells, tissues, or organs of a suspected cancerous object with wavelengths absorbed by the developing agent. In some embodiments, the developing agent has a peak absorbance of about 600 nm to 850 nm.

[0243] Methods for treating cancer may also include the following steps:

[0244] c) Diagnosing cancer in the cells, tissues, or organs of a subject by detecting signals from the imaging agent; and

[0245] d) Treating cancer by administering chemotherapy, radiation therapy, or surgery to the subject.

[0246] In some implementations, this method can be applied to imaging abnormal but non-malignant tissues, such as using FAP as a targeting ligand for defects in the musculoskeletal system, using cRGD as a targeting ligand for the vascular system, using LyP-1 peptide as a targeting ligand for melanoma, using K237 peptide and / or mUNO as a targeting ligand for breast cancer, and using IL4RPep-1 as a targeting ligand for imaging lung tumors, breast tumors, and colon tumors.

[0247] In some embodiments, the method can be applied to imaging cancer, tumors, or growths. In other embodiments, the cancer is selected from ocular cancer or eye cancer, rectal cancer, colon cancer, cervical cancer, prostate cancer, breast cancer and bladder cancer, oral cancer, benign and malignant tumors, gastric cancer, liver cancer, pancreatic cancer, lung cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, brain / cns cancer (e.g., glioma), throat cancer, melanoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms' tumor, neuroblastoma, oropharyngeal cancer, esophageal cancer, laryngeal cancer, lymphoma, neurofibromatosis, tuberous sclerosis, hemangioma, and lymphangiogenesis.

[0248] In some implementations, the cancer cells are adult solid tumor cells or pediatric solid tumor cells. Some non-limiting examples of such cells include melanoma cells, neuroblastoma cells, lung cancer cells, adrenal cancer cells, colon cancer cells, colorectal cancer cells, ovarian cancer cells, prostate cancer cells, liver cancer cells, subcutaneous cancer cells, squamous cell carcinoma cells, colon cancer cells, retinoblastoma cells, cervical cancer cells, glioma cells, breast cancer cells, pancreatic cancer cells, Ewing sarcoma cells, rhabdomyosarcoma cells, osteosarcoma cells, retinoblastoma cells, Wilms tumor cells, and pediatric brain tumor cells.

[0249] In some specific implementation schemes, the cancer cells are prostate cancer cells.

[0250] In some implementations, the biological sample is part or all of the object. In some implementations, the biological sample is obtained from the object.

[0251] In some specific embodiments, the method includes step (a): contacting a biological sample with the combination of the above-described imaging agent conjugates, wherein each of the combined imaging agent conjugates contains a targeting ligand linked to a charge-balanced imaging agent. The charge-balanced imaging agent can be detected by one or more conventional scanning methods.

[0252] In some embodiments, the compound is administered via parenteral, intranasal, sublingual, rectal, or percutaneous delivery. In some such embodiments, the compound is administered intravenously. In some embodiments, the compound is administered intratumorally.

[0253] In some specific embodiments, the cells are tumor cells. For example, and not limited to, the tumor cells are melanoma cells, sarcoma cells, musculoskeletal tumor cells, breast cancer cells, renal cell carcinoma cells, rectal cancer cells, bone metastases, neuroendocrine tumor cells, brain metastases, glioblastoma multiforme (GBM) cells, squamous cell carcinoma cells of the head and neck (SCCHN) cells, or non-small cell lung cancer (NSCLC) cells. Therefore, the present invention provides a method for imaging such tumors in a subject.

[0254] In some implementations, the cells are associated with inflammation or angiogenesis. For example, and not limited to, the cells may be associated with inflammatory lesions, endometriosis, ischemic injury, cardiovascular disease, neurovascular disease, myocardial infarction, moyamoya disease, stroke, atherosclerosis, or rheumatoid arthritis.

[0255] In some embodiments, and in at least one aspect, the present invention provides a method for imaging cells, the method comprising: contacting cells with an embodiment of an imaging agent according to any one or more embodiments of the invention described herein (e.g., an embodiment having one of the formulas shown and described herein); irradiating the cells with a wavelength absorbed by the imaging agent; and detecting a signal from the imaging agent, thereby imaging the cells. The imaged cells may be tumor cells, inflammatory cells, or cells undergoing angiogenesis.

[0256] In some embodiments of the method for imaging cells according to the present invention, an imaging agent is applied to an organism containing or suspected of containing said cells. The imaging agent described herein is a substance that can be used to image tissues or cells (e.g., tissues or cells of a living organism) for purposes such as diagnosis, treatment, image-guided surgery, etc. In some embodiments, the organism is a mammal, such as a human.

[0257] Some embodiments of the imaging agent of the present invention comprise a dye capable of absorbing electromagnetic radiation, typically in the ultraviolet (UV), visible, or near-infrared (NIR) range. The imaging agent may also be capable of emitting fluorescence, for example, in the visible or NIR range. The optical signal detected from the dye or conjugate may be, for example, absorption or fluorescence emission. In some embodiments, fluorescence emission from the dye is the primary optical signal detected for imaging purposes. In some embodiments, the dye has peak absorbance at about 525 nm to about 850 nm, about 550 nm to about 825 nm, about 600 nm to about 825 nm, about 700 nm to about 825 nm, or about 750 nm to about 825 nm. In some embodiments, the dye has peak fluorescence emission at about 700 nm to 875 nm, about 725 nm to about 850 nm, about 750 nm to about 850 nm, or about 775 nm to about 850 nm. In some specific embodiments, the conjugated imaging agent of the present invention has peak fluorescence emission at about 700 nm.

[0258] Unless otherwise stated, certain embodiments of the developer are typically “charge-balanced,” meaning they have a net total charge of zero or near zero, such as +1 or -1. Charge balance occurs when negatively charged substituents on the developer are offset by the same or nearly the same number of positively charged substituents present on the same molecule, and vice versa. In some embodiments, the net charge is 0 or +1. In some embodiments, the net charge is 0. In some embodiments, the net charge is +1. In other embodiments, the net charge is -1. The value “n” in the formulas provided herein represents the net charge.

[0259] Compared to currently known fluorescence imaging agents, certain embodiments of the imaging agents described herein typically exhibit an improved target-to-background ratio (TBR). This improvement in TBR is considered a result of improved in vivo properties due to "charge balance." TBR is a measure of the intensity of the fluorescence signal (peak signal) obtained from the target (the tissue or cells targeted by the imaging agent) divided by a measure of the intensity of the fluorescence signal (background signal) obtained near the target. TBR measurements can be readily obtained using standard measurement procedures. For fluorescence imaging systems and other types of optical systems, recording digital images of the optical signal of the target facilitates TBR measurements. Higher TBR values ​​are desirable, leading to higher resolution imaging of the tissue. In some embodiments, the imaging agent achieves a TBR of at least about 1.1 (i.e., the peak signal is at least 10% higher than the background signal).

[0260] Some embodiments of the developer of this invention typically include one or more ionic groups. In some embodiments, the developer includes two or more, three or more, four or more, or five or more ionic groups. The ionic groups are used to improve the solubility of the conventional hydrophobic dye moiety of the developer, thereby improving biodistribution. The ionic groups can be located on any part of the developer, such as the dye moiety, the targeting ligand, or both.

[0261] The term "ionic group" refers to a moiety containing one or more charged substituents. A "charged substituent" is a functional group that is typically an anionic or cationic under substantially neutral aqueous conditions (e.g., pH about 6.5 to 8.0 or preferably about physiological pH (7.4)). Some examples of charged anionic substituents include anions of inorganic and organic acids, such as sulfonates (-SO3-). 1- ), sulfite, carboxylate, phosphonite, phosphonate, phosphate, and their esters (e.g., alkyl esters). In some embodiments, the charged substituent is a sulfonate. Some examples of charged cationic substituents include quaternary ammonium (-NR3). + ), where R is independently selected from C 1-6 Alkyl, aryl, and arylalkyl groups. Other charged cationic substituents include protonated primary, secondary, and tertiary amines, as well as guanidine. In some embodiments, the charged substituent is -N(CH3)3. + Other examples of ionic groups are described below.

[0262] Certain embodiments of the developing agent of the present invention generally exhibit good solubility in substantially neutral aqueous media, and particularly in blood and serum. In some embodiments, the developing agent has a solubility of at least about 10 μM in a 10 mM HEPES solution at pH 7.4. In other embodiments, the developing agent has a solubility of at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 40 μM, or at least about 50 μM in a 10 mM HEPES solution at pH 7.4.

[0263] Some embodiments of the developer of this invention are typically neutral molecules or salts. For example, if the dye or dye conjugate is charged, the developer may be or contain a salt or acid (or combination thereof) of the dye or dye conjugate. For positively charged dyes or conjugates, suitable counterions include anions such as fluoride, chloride, bromide, iodide, acetate, perchlorate, and PF6. - For negatively charged dyes or conjugates, suitable counterions include cations, such as Na+. + K + And quaternary ammonium.

[0264] Certain embodiments of the imaging agent of the present invention exhibit significantly improved stability over time, allowing for significantly improved operability and use in imaging and mapping. Similarly, the stability of the imaging agent allows for increased accuracy during surgery because the signal does not weaken over time.

[0265] The charge-balanced imaging agents of the present invention are particularly advantageous because their behavior in vivo is believed to contribute to excellent optical imaging properties. More specifically, charge balance is thought to endow the agents with good biodistribution and clearance properties, and reduce unwanted nonspecific binding. These in vivo properties contribute to improved target-to-background ratios in imaging cells and tissues, resulting in higher resolution imaging. Furthermore, the charge-balanced imaging agents of the present invention are primarily or solely cleared by the kidneys.

[0266] In some embodiments, a tumor or cells are present in the object. In many of its embodiments, the object to be treated by the currently disclosed methods is intended to be a human object, although it should be understood that the methods described herein are effective for all vertebrate species, which are intended to be included in the term "object". Thus, "object" can include human objects for medical purposes (e.g., for treating an existing condition or disease or for preventative treatment to prevent the onset of a condition or disease) or animal (non-human) objects for medical, veterinary, or developmental purposes. Suitable animal subjects include mammals, including but not limited to primates such as humans, monkeys, and apes; bovines such as cattle and oxen; ovines such as sheep; caprines such as goats; porcines such as pigs and hogs; equines such as horses, donkeys, and zebras; felines, including wildcats and domestic cats; canines, including dogs; rabbits, including rabbits and hares; and rodents, including mice and rats. Animals may be transgenic. In some embodiments, the subject is a human being, including but not limited to fetuses, newborns, infants, adolescents, and adults. Furthermore, "subject" may include patients who have or are suspected of having a condition or disease. Therefore, the terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject is a human being. In other embodiments, the subject is a non-human being.

[0267] Dosage form and administration method

[0268] In the method of the present invention, the imaging agent can be administered at a predetermined dose. There is no particular limitation on the amount of the predetermined dose, provided that it is administered in at least the minimum amount that can be cleared by the kidneys within twelve hours.

[0269] In some embodiments, a detectable effective amount of the imaging agent of the currently disclosed method is administered to the subject. According to the currently disclosed subject matter, the “detectable effective amount” of the imaging agent is defined as an amount sufficient to produce acceptable images using clinically usable equipment. The detectable effective amount of the agent can be administered in more than one injection. The detectable effective amount of the imaging agent can vary depending on factors such as individual susceptibility, individual age, sex, and weight, individual idiosyncratic response, dosimetry, and instrument and film-related factors. Optimization of such factors is entirely within the scope of the art.

[0270] The reagents of this invention can be administered in any of the manner described herein and are generally acceptable to patients and those skilled in the art. In particular, the charge balance imaging agent is administered intravenously.

[0271] The predetermined target amount depends on a variety of factors, including but not limited to the type of tumor or lesion to be observed and the location of any cells to be imaged. Therefore, the predetermined amount is set by a person skilled in the art before administering the reagent. Such factors include, but are not limited to, the amount of the determined dose, the patient's height, weight, age, body mass index, and sex, or any combination thereof.

[0272] In some specific embodiments, when the predetermined dose is from about 2.5 mg to about 5.0 mg or more, the predetermined target dose is 50% of the predetermined dose. In other embodiments, when the predetermined dose is from about 0.5 mg to about 2.5 mg, the predetermined target dose is 60% of the predetermined dose. In still other embodiments, when the predetermined dose is about 0.5 mg or less, the predetermined target dose is 80% of the predetermined dose.

[0273] The imaging agent of this invention can be administered using any suitable technique, including both enteral and parenteral methods. In some embodiments, the imaging agent can be formulated into a pharmaceutically acceptable preparation and administered intravenously to the organism for imaging. The administered organism can be imaged using, for example, the FLARE™ Image-Guided Surgery System, a continuous-wave (CW) intraoperative imaging system capable of simultaneously acquiring and displaying color video (i.e., surgical anatomy) and two channels of invisible NIR fluorescence (700 nm and 800 nm) light in real time. The imaging system can irradiate the administered organism with radiation absorbed by the imaging agent and detect optical signals, such as NIR fluorescence, emitted from the target site of the imaging agent-containing organism. The detected signals can be recorded and analyzed by obtaining digital images or videos of the target organism, thereby facilitating diagnostic procedures and image-guided medical technologies.

[0274] Any route of administration may be suitable for administering the disclosed reagent to a subject. In one embodiment, the disclosed imaging agent may be administered to the subject via intravenous injection. In another embodiment, the disclosed imaging agent may be administered to the subject via any other suitable systemic delivery method (e.g., parenteral, intranasal, sublingual, rectal, or percutaneous).

[0275] Applications, features, and components

[0276] The conjugates described herein can be used, for example, in planar optics, optical tomography, endoscopy, photoacoustics, and acoustic fluorescence applications for the detection, imaging, and treatment of tumors and other abnormalities. The conjugates can also be used for local treatment. This can be accomplished, for example, by directing the conjugate to a desired target site or allowing the conjugate to selectively accumulate at the target site; or by irradiating the agent with light of an appropriate wavelength to activate the agent. Therefore, the novel conjugates can be used in tissue sections, for example, for the detection, imaging, and treatment of tumors.

[0277] In addition, by monitoring the blood clearance profile of the conjugate, the conjugate can be used to detect the presence of tumors and other abnormalities, for laser-assisted guided surgery to detect small micrometastases, such as those in somatostatin subtype 2 (SST-2) positive tumors, and for the diagnosis of atherosclerotic plaques and blood clots.

[0278] Conjugates can be formulated into diagnostic and therapeutic compositions for enteral or parenteral administration. Typically, these compositions contain an effective amount of the conjugate, along with conventional drug carriers and excipients suitable for the intended type of administration. For example, parenteral formulations contain the conjugate in a sterile aqueous solution or suspension. Parenteral compositions can be injected directly into the desired site on the subject, or mixed with larger volumes of parenteral compositions for systemic administration. Such solutions may also contain pharmaceutically acceptable buffers and optional electrolytes, such as sodium chloride.

[0279] Formulations for enteral administration typically comprise a liquid containing an effective amount of the desired dye or dye conjugate in an aqueous solution or suspension. Such enteral compositions may optionally contain buffers, surfactants, and thixotropic agents. Compositions for oral administration may also contain flavoring agents and other ingredients to enhance their sensory qualities.

[0280] Generally, diagnostic compositions are administered at a dose that effectively achieves the desired signal intensity for detection. Such a dose can vary depending on the organ or tissue to be imaged and the imaging device used. For example, Zeheer et al., Nature Biotechnology, 19, 1148-1154 (2001), used 0.1 μmol / kg as the dose of the IRDye78 conjugate in vivo. Diagnostic compositions can be administered systemically or locally to the patient's organ or tissue to be imaged, followed by an imaging procedure.

[0281] Generally speaking, conjugates or dye compounds absorb and emit light in the visible and infrared regions of the electromagnetic spectrum; for example, they can emit green, yellow, orange, red, or near-infrared light (“NIR”).

[0282] In some embodiments, the ZW-800-1, ZW-830-1, and ZW-700-1-Forte dyes emit and / or absorb radiation at wavelengths from about 300 nm to about 1000 nm, for example, from about 400 nm to about 900 nm, or from about 450 μm to about 850 nm. In some specific embodiments, the ZW700-1 Forte dye emits and / or absorbs radiation at wavelengths from about 700 nm.

[0283] In some embodiments, the maximum excitation and / or maximum emission of the conjugate and dye compound, measured in a 10 mM HEPES solution at pH 7.4, is about 525 nm to about 875 nm, for example, about 550 nm to about 825 nm, or about 550 nm to about 800 nm.

[0284] The invention will be described in more detail by way of specific embodiments. The following embodiments are provided for illustrative purposes and are not intended to limit the invention or the claims in any way. Several non-critical parameters in these embodiments may be changed or modified to produce substantially the same results.

[0285] Example

[0286] Example 1: Imaging of organisms

[0287] The FLARE™ image-guided surgery system is a continuous-wave (CW) intraoperative imaging system capable of simultaneously acquiring and displaying color video (i.e., surgical anatomy) and two channels of invisible NIR fluorescence (700 nm and 800 nm) light in real time. The theory, design, and operation of this imaging system have been described in detail previously. See Tanaka, E., HS Choi, H. Fujii, MG Bawendi, and JV Frangioni, Image-guided oncologic surgery using invisible light completed: pre-clinical development for sentinel lymph nodemapping. Ann Surg Oncol, 2006. 13: 1671-81; De Grand, AM, and JV Frangioni, An operational near-infrared fluorescence imaging system prototype for largeanimal surgery. Technol Cancer Res Treat, 2003. 2: 553-562; and Nakayama, A., F. del Monte, RJ Hajjar, and JV Frangioni, Functional near-infrared fluorescence imaging for cardiac surgery and targeted gene therapy. Molecular Imaging, 2002. 1: 365-377, each of which is incorporated herein by reference.

[0288] The specifications of the FLARE™ image-guided surgical system are provided in Table 1 below.

[0289]

[0290]

[0291]

[0292] Example 2: In vivo characterization of dyes and conjugates

[0293] For in vivo characterization, 40 pmol / g (average 10 nmol) of the targeted dye IV of this invention can be injected into 25 g athymic nude mice bearing xenograft human tumors. The FLARE™ imaging system can be set to an excitation dose rate of 1 mW / cm² at 665 nm. Synchronous color video and NIR fluorescence (700 nm) images can be acquired before injection, every second for the first 20 seconds, and every minute thereafter for 2 hours. A constant camera acquisition time (typically 100 ms) can be maintained, and selections can be made to ensure that all intensity measurements are within the linear range of the 12-bit Orca-AG (Hamamatsu) NIR camera. Blood can be collected via tail vein at 0, 1, 2, 5, 10, 15, 30, 60, and 120 minutes. Intensity-time curves for all major organs and tissues can be quantified. The peak fluorescence intensity and time for each tumor / tissue / organ, as well as the intensity of each tumor / tissue / organ 1 hour after injection, can be determined.

[0294] Other implementation plans

[0295] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0296] Those skilled in the art will recognize or be able to determine many equivalent schemes of the specific embodiments of the invention described herein using only conventional experiments.

[0297] Such equivalents are intended to be covered in the appended claims.

Claims

1. A developer dye comprising a charge-balanced developer conjugated to a targeting carrier, said developer dye having one or more zwitterionic groups based on N-oxides.

2. The developer dye according to claim 1, wherein the charge-balancing developer is a reagent of formula (I): L-RC-(-Sp-N + (CH2)2O - ) p For equation (I): L represents a linker group that can conjugate with a target carrier; RC represents the resonant core; Each Sp independently represents a spacer group; and p represents an integer from 1 to 4.

3. The developer dye according to claim 1, wherein the charge-balancing developer is a reagent of formula (II), or its salt, solvate, hydrate, polymorph, prodrug, or stereoisomer: For equation (II): Each R1 is independently a -C1-C4 alkyl-N + (CH2)2O - ; Each R2 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl; Each R3 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl; Alternatively, each group of R1 and R2 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atoms to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H; Alternatively, each group of R1 and R3 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atom to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H; Each Q is N + (CH2)2O - ; X and Y are each independently represented as O, S, Se, C(R”)2, NR”'; Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, wherein R6 is an optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, alkyl-N3, aryl-N3, or aryl-halogen. Each R' is independently H, alkyl, or aryl; Each R” is independently H or alkyl; Each R”' is independently H, alkyl, alkyl-SO3H or alkyl-COOH; m is an integer from 0 to 3, and Each n is an independent integer from 1 to 4; and L is an anion; Furthermore, the Z group can be conjugated with the target carrier.

4. The developer dye according to claim 1, wherein the charge-balancing developer is a reagent of formula (III), or its salt, solvate, hydrate, polymorph, prodrug, or stereoisomer: For equation (III): Each R1 is independently a -C1-C4 alkyl-N + (CH2)2O - ; Each R2 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl; Each R3 is independently H, OR', halogen, sulfonate / ester, substituted or unsubstituted amino, C(O)NH-C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy or phenyl; Alternatively, each group of R1 and R2 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atoms to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H; Alternatively, each group of R1 and R3 bonded to the same ring can form a 5- to 6-membered aryl or heteroaryl ring together with the carbon atom to which they are attached, the 5- to 6-membered aryl or heteroaryl ring being bound by at least one -C1-C4 alkyl-N + (CH2)2O - The group is substituted, and optionally further substituted by halogen, alkyl, alkoxy, hydroxyl, -SO2OH or -CO2H; Each Q is N + (CH2)2O - ; X and Y are independently represented as O, S, Se, C(R”)2, NR; Z is H, halogen, CN, R6, OR6, SR6, NHR6 or CH2R6, wherein R6 is an optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, alkyl-N3, aryl-N3, or aryl-halogen. R is independently H, OR”'' (where R = H, alkyl or aryl, NH2, NHR, alkylNH2, alkylCOOH), L is an anion; Each R' is independently H, alkyl, or aryl; Each R” is independently H or alkyl; Each R”' is independently H, alkyl, alkyl-SO3H or alkyl-COOH; Each R'”' is independently H, alkyl or aryl, NH2, NHR, alkyl-NH2 or alkyl-COOH; Each n is an independent integer from 1 to 4; and L is an anion; Furthermore, the Z group can be conjugated with the target carrier.

5. The developer dye according to claim 1, wherein the charge-balancing developer is: ； ； ;or 。 6. The developer dye according to claim 1, wherein the targeting carrier is cRGD, dPSMA-617, KUE, FAP binding carrier, octreotide, or bufotoxin.

7. The developer dye of claim 1, wherein the charge-balancing developer is conjugated to the target carrier via a direct bond or via a linking group.

8. A method for imaging tissue, cells, or cavities in an object, the method comprising: (a) For the object, causing the tissue, cell, or cavity to... The developer contact comprising a dye containing a developer, as described in claim 1. (b) Irradiate the tissue, cell or cavity at a wavelength absorbed by the dye; (c) and detecting signals from the imaging agent to image the tissue, cell or cavity.

9. The method for imaging tissue, cells, or cavities in an object according to claim 8, wherein the cells are tumor cells.

10. The method of claim 8, wherein the object is a person.

11. The method of claim 8, wherein the developer has a peak absorbance at about 600 nm to 850 nm.

12. The method of claim 8, wherein the tissue or cells are imaged in vivo.

13. The method of claim 8, wherein the developer further comprises a PEG portion.

14. The method of claim 8, wherein the imaging agent further comprises a radioisotope for single-photon emission computed tomography (SPECT) or positron emission tomography (PET).

15. The method of claim 8, wherein the developer comprises a reactive linker group, such as an NHS ester, a sulfonated NHS ester, or a TFP ester.

16. A method of treating cancer in a subject, the method comprising: (a) Applying an effective amount of the imaging agent according to claim 1 to the object. (b) Irradiating cells, tissues or organs of a suspected cancerous object with wavelengths absorbed by the imaging agent; (c) Diagnosing cancer in the cells, tissues, or organs of the subject by detecting signals from the imaging agent; and (d) Treating the cancer by administering chemotherapy, radiation therapy or surgery to the subject.

Images