Hemicyanine compounds having optoacoustic properties and methods of use

Abstract

Hemicyanine-based compounds having optoacoustic properties and methods of their use as imaging and diagnostic agents and as conjugates with targeting and / or drug compounds.

Description

HEMI CYANINE COMPOUNDS HAVING OPTOACOUSTIC PROPERTIES AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Patent Application Ser. No.63 / 741,219, filed January 2, 2025, the content of which is hereby expressly incorporated herein by reference in its entirety.STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Contract Numbers CA205941 and EB034731 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Commonly used imaging modalities (e.g., MRI, CT, Ultrasound, PET) have various limitations for simultaneously detecting multiple molecular features within various diseases, i.e., cancer, inflammation, and infectious diseases, among others. While optoacousticphotoacoustic imaging is a newer modality with potential to overcome some limitations observed with more commonly used imaging modalities, there are few, if any, small molecule dyes / compounds that have been engineered to optimize photoacoustic signals and allow for multiple.'non-overlapping exogenous reporters. It is to addressing this need that the present disclosure is directed.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0005] FIG. 1A shows UV-VIS absorbance spectra of compound 11A in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.14936-0208-1406, v. 1

[0006] FIG. IB shows UV-VIS absorbance spectra of compound 11B in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.

[0007] FIG. 1C shows UV-VIS absorbance spectra of compound 12A in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.

[0008] FIG. ID shows UV-VIS absorbance spectra of compound 12B in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.

[0009] FIG. IE shows UV-VIS absorbance spectra of compound 13A in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.

[0010] FIG. IF shows UV-VIS absorbance spectra of compound 13B in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.

[0011] FIG. 1G shows UV-VIS absorbance spectra of compound 14A in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.

[0012] FIG. 1H shows UV-VIS absorbance spectra of compound 14B in 50% EtOH in water (orange) and acetic acid (blue). Solvent polarity did not alter absorbance spectra of the compound.

[0013] FIG. 2 A shows optoacoustic spectra for compound 11A at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.

[0014] FIG. 2B shows optoacoustic spectra for compound 11B at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.

[0015] FIG. 2C shows optoacoustic spectra for compound 12A at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.

[0016] FIG. 2D shows optoacoustic spectra for compound 12B at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.

[0017] FIG. 2E shows optoacoustic spectra for compound 13A at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.

[0018] FIG. 2F shows optoacoustic spectra for compound 13B at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.24936-0208-1406, v. 1

[0019] FIG. 2G shows optoacoustic spectra for compound 14A at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.

[0020] FIG. 2H shows optoacoustic spectra for compound 14B at concentrations 1 mM, 100 µM, 10 µM, and 1 µM to evaluate aggregation-based spectral shifts.

[0021] FIG. 3 A shows optoacoustic signals for compound 11 A as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0022] FIG. 3B shows optoacoustic signals for compound 1 IB as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0023] FIG. 3C shows optoacoustic signals for compound 12A as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0024] FIG. 3D shows optoacoustic signals for compound 12B as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0025] FIG. 3E shows optoacoustic signals for compound 13A as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0026] FIG. 3F shows optoacoustic signals for compound 13B as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0027] FIG. 3G shows optoacoustic signals for compound 14A as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0028] FIG. 3H shows optoacoustic signals for compound 14B as a function of wavelength in solvents of varying dielectric constant (i.e., polarity), including 50% EtOH, acetone, dichloromethane, acetic acid, and diethyl ether.

[0029] FIG. 4A shows results of ATPLite cellular viability assays of compounds 11A-14B (200 pg / mL) on human kidney cells (cell line 293) versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).34936-0208-1406, v. 1

[0030] FIG. 4B shows results of ATPLite cellular viability assays of compounds 11A-14B (20 pg / mL) on human kidney cells (cell line 293) versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).

[0031] FIG. 4C shows results of ATPLite cellular viability assays of compounds 11A-14B (200 pg / mL) on human liver cells (cell line HEP3B) versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).

[0032] FIG. 4D shows results of ATPLite cellular viability assays of compounds 11A-14B (20 pg / mL) on human liver cells (cell line HEP3B)versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).

[0033] FIG. 4E shows results of ATPLite cellular viability assays of compounds 11A-14B (200 pg / mL) on human lung cells (cell line A427) versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).

[0034] FIG. 4F shows results of ATPLite cellular viability assays of compounds 11A-14B (20 pg / mL) on human lung cells (cell line A427) versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).

[0035] FIG. 4G shows results of ATPLite cellular viability assays of compounds 11A-14B (200 pg / mL) on human umbilical vein endothelial cells (HUVEC) versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).

[0036] FIG. 4H shows results of ATPLite cellular viability assays of compounds 11A-14B (20 pg / mL) on human umbilical vein endothelial cells (HUVEC) versus standard infrared dyes indocyanine green and methylene blue compared to positive (untreated) controls. No significance was observed (p>0.05).

[0037] FIG. 5A shows results of optoacoustic tomographic slices within mice treated with compounds 11A-14B. Higher slice numbers (top) indicate cross sections closer to the tail of mice.

[0038] FIG. 5B shows quantification of signals from the tomographic slices of FIG. 5 A. * = p<0.05, ** = p<0.01, *** = pcO. OOL44936-0208-1406, v. 1

[0039] FIG. 5C shows whole-body images of near-infrared fluorescence of the treated mice of FIG. 5 A.

[0040] FIG. 5D shows quantification of signals from the animals of FIG. 5C. * = p<0.05, ** = pcO. Ol, *** = pcO. OOl.

[0041] FIG. 6A shows ex vivo visualization of NIR fluorescence signal in the pancreas, spleen, liver, kidney, heart, and lung of mice treated with compounds 11 A-14B.

[0042] FIG. 6B shows quantification of NIR fluorescence from FIG. 6 A. * = p<0.05, ** = pcO. Ol, *** =p<0.001.DETAILED DESCRIPTION

[0043] The present disclosure is directed to novel hemicyanine (HC) compounds which show optoacoustic / photoacoustic spectra that are red-shifted from their absorbance spectra. This allows for the compounds to be multiplexed with existing NIR ZW-800 and ZW-700 contrast agents (dyes), for example, without overlap. The HC compounds (also referred to herein as HC-based compounds) can be used as reporter dye, •contrast agents for any disease targeted peptide, antibody, or nanoparticle. The novel HC compounds can also be used with fluorescent imaging applications. The HC compounds can be conjugated to targeting moieties such as peptides, monoclonal antibodies, or monoclonal antibody fragments and derivatives to enable their use in imaging and detecting particular diseased cells and tissues, image-guided surgery, and monitoring of treatment response.

[0044] Before further describing various embodiments of the present disclosure in more detail by way of exemplary description, examples, and results, it is to be understood that the compounds, compositions, and methods of present disclosure are not limited in application to the details of specific embodiments and examples as set forth in the following description. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. As such, the language used herein is intended to be given the broadest possible scope and meaning, and the embodiments and examples are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description only and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the present disclosure. However, it will be apparent to a person having ordinary skill in the art that the present disclosure may be practiced without these specific details. In other instances, features54936-0208-1406, v. 1which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. It is intended that all alternatives, substitutions, modifications, and equivalents apparent to those having ordinary skill in the art are included within the scope of the present disclosure. All of the compounds, compositions, and methods and application and uses thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure. Thus, while the compounds, compositions, and methods of the present disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compounds, compositions, and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the inventive concepts.

[0045] All patents, published patent applications, and non-patent publications including published articles mentioned in the specification or referenced in any portion of this application, including U. S. Provisional Patent Application Ser. No. 63 / 741,219, filed January 2, 2025, are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0046] The following abbreviations may be used herein:ANOVA: Analysis of Variance,a.u.: Atomic units,Br: Bromine,CDCl3: Deuterated chloroform,Cl: Chlorine,CO2: Carbon dioxide,CT: Computed tomography,DCM: Dichloromethane,DEE: Diethyl ether,DMEM: Dulbecco’s modified eagle medium,DMSO: Dimethyl sulfoxide,EtOAc: Ethyl acetate,EtOH: Ethanol,F: Fluorine,FBS: Fetal bovine serum,64936-0208-1406, v. 1H: Hydrogen,HC: Hemicyanine,H& E: Hematoxylin and eosin,HC1: Hydrochloric acid,HUVEC: Human umbilical vein cells,ICG: Indocyanine green,I: Iodine,K2CO3: Potassium carbonate,MeOH: Methanol,MRI: Magnetic resonance imaging,MSOT: Multispectral optoacoustic tomography,MTS assay: MTS tetrazolium reduction assay,MTT assay: MTT tetrazolium reduction assay,NaCOv Sodium carbonate,NaOAc: Sodium acetate,NIR: Near Infrared,NMR: Nuclear magnetic resonance,PET: Positron emission tomography,SnCl2: Stannous chloride,TEA: Triethylamine,TICT: Twisted intramolecular charge transfer,TMS: Tetramethylsilane,UV-VIS: Ultraviolet- visible light.

[0047] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those having ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Where used herein, the specific term “single” is limited to “only one.”

[0048] As utilized in accordance with the methods, compounds, and compositions of the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.74936-0208-1406, v. 1

[0049] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer inclusive therein. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0050] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0051] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. A range is intended to include any sub-range therein, although that sub-range may not be explicitly designated herein. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 2-125 therefore includes 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and 125, as well as sub-ranges within the greater range, e.g., for 2-125, sub-ranges include but are not limited to 2-50, 5-50, 10-60, 5-45, 15-60, 10-40, 15-30, 2-85, 5-85, 20-75, 5-70, 10-70, 28-70, 14-56, 2- 100, 5-100, 10-100, 5-90, 15-100, 10-75, 5-40, 2-105, 5-105, 100-95, 4-78, 15-65, 18-88, and 12-56. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,84936-0208-1406, v. 114, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, a range of 1-1,000 includes, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, and includes ranges of 1-20, 10-50, 50-100, 100-500, and 500- 1,000. The range 100 units to 2000 units therefore refers to and includes all values or ranges of values of the units, and fractions of the values of the units and integers within said range, including for example, but not limited to 100 units to 1000 units, 100 units to 500 units, 200 units to 1000 units, 300 units to 1500 units, 400 units to 2000 units, 500 units to 2000 units, 500 units to 1000 units, 250 units to 1750 units, 250 units to 1200 units, 750 units to 2000 units, 150 units to 1500 units, 100 units to 1250 units, and 800 units to 1200 units. Any two values within the range of about 100 units to about 2000 units therefore can be used to set the lower and upper boundaries of a range in accordance with the embodiments of the present disclosure. More particularly, a range of 10-12 units includes, for example, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, and 12.0, and all values or ranges of values of the units, and fractions of the values of the units and integers within said range, and ranges which combine the values of the boundaries of different ranges within the series, e.g., 10.1 to 11.5. Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10 includes 9, 8, 7, etc. all the way down to the number one (1).

[0052] The terms “increase,” “increasing,” "enhancing," or "enhancement" are defined as indicating a result that is greater in magnitude than a control number derived from analysis of a cohort, for example, the result can be a positive change of at least 5%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more in comparison with the control number. Similarly, the terms “decrease,” “decreasing,” ‘lessening," or "reduction" are defined as indicating a result that is lesser in magnitude than a control number, for example, the result can be a negative change of at least 5%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more in comparison with the control number.

[0053] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is94936-0208-1406, v. 1important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0054] Throughout this application, the terms “about” or “approximately” are used to indicate that a value includes the inherent variation of error for the composition, the method used to administer the composition, or the variation that exists among the study subjects. As used herein the qualifiers “about” or “approximately” are intended to include not only the exact value, amount, degree, orientation, or other qualified characteristic or value, but are intended to include some slight variations due to measuring error, manufacturing tolerances, stress exerted on various parts or components, observer error, wear and tear, and combinations thereof, for example. The terms “about” or “approximately,” where used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass, for example, variations of ± 25%, or ± 24%, or ± 23%, or ± 22%, or ± 21%, or ± 20%, or ± 19%, or ± 18%, or ± 17%, or ± 16%, or ± 15%, or ± 14%, or ± 13%, or ± 12%, or ± 11%, or ± 10%, or ± 9%, or ± 8%, or ± 7%, or ± 6%, or ± 5%, or ± 4%, or ± 3%, or ± 2%, or ± 1.5%, or ± 1%, or ± 0.5%, or ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0055] As used herein, the term “substantially” means that the thing referred to, such as, for example, a value, amount, degree, temperature, dimension, measurement, orientation, event, circumstance, rate, or parameter, or other qualified characteristic completely occurs, or occurs to a great extent or degree. For example, the term “substantially” means that the subsequently described value, amount, degree, temperature, dimension, measurement, orientation, event, circumstance, rate, or parameter or other qualified characteristic occurs at least 75% of the time, at least 80% of the time, at least 90% of the time, at least 91% of the time, at least 92% of the time, at least 93% of the time, at least 94% of the time, at least 95% of the time, at least 96% of the time, at least 97% of the time, at least 98% of the time, or at least 99% of the time.

[0056] The terms “significant” and “significantly” when used non-statistically in reference to a quantitative reference measure, are defined as meaning at least 5% of a reference measure, or at least 10% of a reference measure, or at least 20% of a reference measure,104936-0208-1406, v. 1or at least 30% of a reference measure, or at least 40% of a reference measure, or at least 50% of a reference measure, or at least 60% of a reference measure, or at least 70% of a reference measure, or at least 80% of a reference measure, or at least 90% of a reference measure, or at least 95% of a reference measure, including 100% of a reference measure.

[0057] As used herein any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment and may be included in other embodiments. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment and are not necessarily limited to a single or particular embodiment. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0058] Where used herein, the pronouns “we” or “us” or the possessive determiner “our” are intended to refer to all persons involved in a particular aspect of the investigation disclosed herein and as such may include non-inventor laboratory assistants and non-inventor collaborators working under the supervision of the inventor(s).

[0059] The term “pharmaceutically acceptable” refers to compounds and compositions which are suitable for administration to humans and / or animals without undue adverse side effects such as toxicity, irritation and / or allergic response commensurate with a reasonable benefit / risk ratio. The compounds or conjugates of the present disclosure may be combined with one or more pharmaceutically-acceptable excipients, including carriers, vehicles, and diluents which may improve solubility, deliverability, dispersion, stability, and / or conformational integrity of the compounds or conjugates thereof.

[0060] The term “active agent” as used herein is intended to refer to a substance which possesses a chemical or biological activity relevant to the present disclosure, and particularly refers to therapeutic, diagnostic, and / or imaging substances which may be used in methods described in the present disclosure. By “biologically active” is meant the ability to modify the physiological system of a cell, (issue, or organism without reference to how the active agent has its physiological effects.

[0061] As used herein, “pure” or “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other object species in the composition thereof), and particularly a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis)114936-0208-1406, v. 1of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, more particularly more than about 85%, more than about 90%, more than about 95%, or more than about 99%. The term “pure” or “substantially pure” also refers to preparations where the object species is at least 60% (w / w) pure, or at least 70% (w / w) pure, or at least 75% (w / w) pure, or at least 80% (w / w) pure, or at least 85% (w / w) pure, or at least 90% (w / w) pure, or at least 92% (w / w) pure, or at least 95% (w / w) pure, or at least 96% (w / w) pure, or at least 97% (w / w) pure, or at least 98% (w / w) pure, or at least 99% (w / w) pure, or 100% (w / w) pure.

[0062] As used herein the term “subject” refers to an animal and more particularly may refer to a mammal. Non-limiting examples of mammals include dogs, cats, rats, mice, guinea pigs, chinchillas, rabbits, horses, donkeys, mules, goats, cattle, sheep, camelids, zoo animals, Old and New World monkeys, non-human primates, and humans.

[0063] “Treatment” refers to therapeutic treatments. “Prevention” refers to prophylactic or preventative treatment measures or reducing the onset of a condition or disease. The term “treating” refers to administering the composition to a subject for therapeutic purposes and / or for prevention.

[0064] The terms “therapeutic coinposi lion” and “pharmaceutical composition” refer to an active agent-containing composition that may be administered to a subject by any method known in the art or otherwise contemplated herein, wherein administration of the composition brings about a therapeutic effect as described elsewhere herein. In addition, the compositions of the present disclosure may be designed to provide delayed, controlled, extended, and / or sustained release using formulation techniques which are well known in the art.

[0065] The term “effective amount” refers to an amount of an active agent which is sufficient to exhibit a detectable therapeutic or treatment effect in a subject without excessive adverse side effects (such as substantial toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner of the present disclosure. The effective amount for a subject will depend upon the subject’s type, size, and health, the nature and severity of the condition to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. The effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.124936-0208-1406, v. 1

[0066] The term “ameliorate” means a detectable or measurable improvement in a subject’ s condition, disease, or symptom thereof. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit, or control in the occurrence, frequency, severity, progression, or duration of the condition or disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease. A successful treatment outcome can lead to a “therapeutic effect” or “benefit” of ameliorating, decreasing, reducing, inhibiting, suppressing, limiting, controlling, or preventing the occurrence, frequency, severity, progression, or duration of a disease or condition, or consequences of the disease or condition in a subject.

[0067] A decrease or reduction in worsening, such as stabilizing the condition or disease, is also a successful treatment outcome. A therapeutic benefit therefore need not be complete ablation or reversal of the disease or condition, or any one, most, or all adverse symptoms, complications, consequences, or underlying causes associated with the disease or condition. Thus, a satisfactory endpoint may be achieved when there is an incremental improvement such as a partial decrease, reduction, inhibition, suppression, limit, control, or prevention in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal of the condition or disease (e.g., stabilizing), over a short or long duration of time (hours, days, weeks, months, etc.). Effectiveness of a method or use, such as a treatment that provides a potential therapeutic benefit or improvement of a condition or disease, can be ascertained by various methods and testing assays.

[0068] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where context excludes that possibility), and the method can also include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all of the defined steps (except where context excludes that possibility).

[0069] Where used herein, the pronouns “we” or “us” or the possessive determiner “our” are intended to refer to all persons involved in a particular aspect of the investigation disclosed herein and as such may include non-inventor laboratory personnel, assistants, technicians, collaborators and / or contributors who worked under the supervision of the inventor(s), and thus are not intended to represent an inventorship role by said laboratory personnel, assistants, technicians, collaborators, and / or contributors in any subject matter disclosed herein.

[0070] In certain embodiments, the active agents of the present disclosure may comprise formulations or treatments that are synergistic. As used herein the terms “synergism,”134936-0208-1406, v. 1“synergistic,” or "synergistic effect" refers to a therapeutic, diagnostic, or imaging effect or result that is greater than the addi li vc effects of each active agent used individually. Presence or absence of a synergistic effect for a particular combination of treatment substances can be quantified by using the Combination Index (CI) (e.g., Chou, Pharmacol Rev, 2006. 58(3): 621-81), wherein CI values lower than 1 indicate synergy and values greater than 1 imply antagonism. Combinations of conjugates comprising the presently disclosed HC compounds conjugated to various cell inhibitors, antagonists, or agonists can be tested in vitro for synergistic cell growth or growth inhibition using standard cell lines, e.g., for particular cancers, or in vivo using standard animal models. A synergistic effect of a combination described herein can permit, in some embodiments, the use of lower levels of dosages of one or more of the components of the combination. A synergistic effect can also permit, in some embodiments, less frequent administration of at least one of the administered active agents. Such lower dosages and reduced frequency of administration can reduce the toxicity associated with the administration of at least one of the therapies to a subject without reducing the efficacy of the treatment.

[0071] The term "coadministration" refers to administration of two or more active agents, e.g., HC compounds as described herein. The timing of coadministration depends in part of the combination and compositions administered and can include administration at the same time, just prior to, or just after the administration of one or more additional therapies Coadministration is meant to include simultaneous or sequential administration of the compound and / or composition individually or in combination. Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). For example, the compositions described herein can be used in combination with one another, or with other active agents.

[0072] In at least certain compounds of the present disclosure, polyethylene glycol (PEG) molecules (also known as polyethylene oxide) and poly(oxyethylene)) are used, for example as linkers to link other compounds together to form drug conjugates. PEG comprises repeating units of ethylene glycol, and is available in different average molecular weights (MW) based on the average number of ethylene glycol units in the PEG molecules of the particular PEG composition. For example, PEGss, a PEG molecule with 2 ethylene glycol units, has a MW of 88 Daltons (Da). PEG400, a PEG molecule with about 8 ethylene glycol units, has a MW of 400 Daltons (Da). PEGeo.ooo, a PEG molecule with about 1364 ethylene glycol units, has a MW of about 60,000. The PEG molecule may comprise up to 30,000 ethylene glycol units, Other144936-0208-1406, v. 1examples include, but are not limited to, PEG200 having an average MW of about 200 Daltons (Da), PEG300 having an average MW of about 300 Da, PEG400 having an average MW of about 400 Da, PEG500 having an average MW of about 500 Da, PEG750 having an average MW of about 750 Da, PEG1000 having an average MW of about 1000 Da, PEG1500 having an average MW of about 1500 Da, PEG2000 having an average MW of about 2000 Da, PEG3000 having an average MW of about 3000 Da, PEG3350 having an average MW of about 3350 Da, PEG3500 having an average MW of about 3500 Da, PEG4ooo having an average MW of about 4000 Da, PEG5000 having an average MW of about 5000 Da, PEGeooo having an average MW of about 6000 Da, PEG7500 having an average MW of about 7500 Da, PEG 10.000 having an average MW of about 10,000 Da, PEG 15.000 having an average MW of about 15,000 Da, PEG2o,ooo having an average MW of about 20,000 Da, PEG25,ooo having an average MW of about 25,000 Da, PEGso.ooo having an average MW of about 30,000 Da, PEG4o,ooo having an average MW of about 40,000 Da, PEGso.ooo having an average MW of about 50,000 Da, and PEGeo.ooo having an average MW of about 60,000 Da. Where used herein the term PEG is intended to refer to any of the examples of PEG listed above, and to PEGs having MWs in the range of 88 and 60,000, unless a particular MW is specified. In other embodiments, the linker molecule may be an amino acid, a peptide, or a polypeptide,

[0073] In various non-limiting embodiments, the conjugates of the present disclosure include an HC compound as described herein, which are linked via a linker (e.g., a PEG, amino acid, peptide or polypeptide) to an anchor-solubilizing moiety such as a phosphatidylethanolamine (PE). Examples of such PEs include but are not limited to distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, diarachidylphosphatidylethanolamine, dilaurylphosphatidylethanolamine, dioleylphosphatidylethanolamine, palmitoylstearoylphosphatidylethanolamine, myristoylstearoylphosphatidylethanolamine, arachidylstearoylphosphatidylethanolamine, laurylstearoylphosphatidylethanolamine, oleylstearoylphosphatidylethanolamine, myristoylpalmitoylphosphatidylethanolamine, arachidylpalmitoylphosphatidylethanolamine, laurylpalmitoylphosphatidylethanolamine, arachidylmyristoylphosphatidylethanolamine, laurylmyristoylphosphatidylethanolamine, laurylarachidylphosphatidylethanolamine, oleylpalmitoylphosphatidylethanolamine, oleylmyristoylphosphatidylethanolamine, oleylarachidylphosphatidylethanolamine, and lauryloleylphosphatidylethanolamine,. In other embodiments, anchoring / solubilizing moiety may comprise any one of the above moieties wherein the ethanolamine is substituted with154936-0208-1406, v. 1serine (forming a phosphatidylserine (PS)) or choline (forming a phosphatidylcholine (PC)), such as distearoylphosphatidylserine or distearoylphosphatidylcholine. In other embodiments, the anchoring / solubilizing moiety may comprise a combination of two or more of the above moieties. In other embodiments, anchoring / solubilizing moiety may comprise a single saturated, unsaturated, or polyunsaturated lipid molecule comprising 2-28 carbon atoms, particularly 10-18 carbon atoms, such as a saturated, unsaturated, or polyunsaturated fatty acid. The anchor-solubilizing moiety may comprise a PE, PS or PC with a single fatty acid or two fatty acids, which may be selected from the group of saturated, unsaturated, and polyunsaturated fatty acids.

[0074] In particular, non-limiting examples, the HC compounds or conjugates of the present disclosure are carried in liposomes. In addition to other pharmaceutically acceptable carrier(s), the liposome may contain amphipathic agents such as lipids which exist in an aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, combinations thereof, and the like. Preparation of such liposomal formulations is well within the level of ordinary skill in the art, as disclosed, for example, in U. S. Patent No. 4,235,871; U. S. Patent No. 4,501,728; U. S. Patent No. 4,837,028; and U. S. Patent No. 4,737,323; the entire contents of each of which are incorporated herein by reference. As used herein, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Eiposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the acli vc agent to be delivered. Eiposomes can be made from phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC) or other similar lipids. Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example (but not by way of limitation), soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and / or phosphatidylcholine and / or cholesterol.

[0075] Compositions or methods "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition that comprises an HC164936-0208-1406, v. 1compound may contain the HC compound alone or in combination with other ingredients. The phrase "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of an HC compound or a conjugate thereof, or agent administered with the HC compound. Exemplary salts include sulfate, citrate, acetate, oxalate, fluoride, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l, T-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as (but not limited to) an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0076] The term "antibody" as used herein can refer to both intact “full length” antibodies as well as to antigen-binding fragments thereof (unless otherwise explicitly noted). The aforementioned antigen-binding fragments may also be referred to herein as antigen binding fragments, antigen binding compounds, antigen binding portions, binding fragments, binding portions, or antibody fragments. Also, as used herein, the term "antibody" includes, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker, i.e., single-chain Fv (scFv) fragments, bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fab fragments, Fab' fragments, F(ab') fragments, F(ab')2 fragments, F(ab)2 fragments, disulfide-linked Fvs (sdFv) (including bi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, dAb fragments, nanobodies, diabodies, triabodies, tetrabodies, linear antibodies, isolated CDRs, and epitopebinding fragments of any of the above. Regardless of structure, an antibody fragment refers to an isolated portion of the antibody that binds to the same antigen that is recognized by the intact antibody.

[0077] The antibodies of several embodiments provided herein may be monospecific, bispecific, trispecific, or of greater inullispccilicily, such as multispecific antibodies formed174936-0208-1406, v. 1from antibody fragments. The term "antibody" also includes a diabody (homodimeric Fv fragment) or a minibody (VL- H-CHS), a bispecific antibody, or the like. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Multispecific antibodies may be specific for different epitopes of a polypeptide or may be specific for both a polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present disclosure (e.g., see, for example, International Patent Apphcation Publication Nos. WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; and WO 92 / 05793; and U. S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819).

[0078] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by the hybridoma method first described by Kohler et al. (Nature, 256:495 (1975)), or may be made by recombinant DNA methods (see, for example, U. S. Patent No. 4,816,567).

[0079] An "isolated" antibody refers to an antibody that has been identified and separated and / or recovered from components of its natural environment and / or an antibody that is recombinantly produced. A "purified antibody" is an antibody that is typically at least 50% w / w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of pharmaceutical acceptable carrier(s) or other vehicle(s) intended to facilitate its use. Interfering proteins and other contaminants can include, for example, cellular components of the cells from which an antibody is isolated or recombinantly produced. Sometimes monoclonal antibodies are at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% w / w pure of interfering proteins and contaminants from production or purification. The antibodies and antigen binding compounds described herein can be provided in isolated and / or purified form.

[0080] In at least certain embodiments of the present disclosure, the term "therapeutic agent" refers to an active agent comprising an antibody and / or antibody-derived compound or other compound as described herein.184936-0208-1406, v. 1

[0081] A "diagnostic agent," which may also be referred to herein as an imaging agent, is a substance that is useful in diagnosing a disease or imaging a cell or tissue. Useful diagnostic agents of the present disclosure may include antibodies and antibody-derived compounds described herein, and may further comprise by linkage or other association radioisotopes, dyes, contrast agents, fluorescent compounds or molecules, and enhancing agents (e.g., paramagnetic ions).

[0082] An "immunoconjugate" or “antibody-drug conjugate” (ADC) is a conjugate of an antibody or antibody-derived compound with an atom, molecule, or a higher-ordered structure (e.g., with a liposome), a therapeutic agent, and / or a diagnostic agent, such as an HC compound of the present disclosure. In certain embodiments, the ADC comprises an antibody or antibody-derived compound conjugated directly or indirectly to a therapeutic agent, and an imaging agent such as a HC compound as described herein. In other embodiments, the conjugate comprises another targeting agent such as a receptor ligand (e.g., a peptide, oligopeptide, or polypeptide) conjugated directly or indirectly to a therapeutic agent and an imaging agent such as a HC compound as described herein.

[0083] As used herein, the term "antibody fusion protein" is a recombinantly produced antigen-binding molecule in which an antibody or antibody fragment is linked to another protein or peptide, such as the same or different antibody or antibody fragment. The fusion protein may comprise a single antibody component, a multivalent or multispecific combination of different antibody components, or multiple copies of the same antibody component, or other component described elsewhere herein. The fusion protein may additionally comprise an antibody or an antibody fragment and a therapeutic agent.

[0084] The basic structural unit of an antibody is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region (variable heavy chain and variable light chain) of about 100 to 120 or more amino acids, which include portions called complementarity determining regions (CDRs) as described below, which are primarily responsible for antigen recognition. The three CDRs of the variable heavy chain may be referred to herein as CDRH1, CDRH2, and CDRH3. The three CDRs of the variable light chain may be referred to herein as CDRL1, CDRL2, and CDRL3. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a “light chain mature variable region” means a light chain variable region without the194936-0208-1406, v. 1light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. A CDR is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, a CDR is sometimes referred to as hypervariable region. A variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest.

[0085] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a " J" region of about 12 or more amino acids, with the heavy chain also including a " D" region of about 10 or more amino acids.

[0086] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, which as noted above are known as CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0087] The assignment of amino acids to each domain (FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4) is done in accordance with the protocols of the IMGT, e.g., see Ehrenmann, F., Kaas, Q. and Lefranc, M.-P., “IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF,” Nucl. Acids Res., 38(Sl): D301-D307 (2010). DOI:10.1093 / nar / kgp946. PMID: 19900967, and Ehrenmann, F. and Lefranc, M.-P. Cold Spring Harb. Protocols, 201 l(6):737-749. DOI:10.1101 / pdb.prot5636. PMID:21632775.

[0088] In other embodiments, the assignment of amino acids to each domain may be done in accordance with the protocols of Kabat (Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk (J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989)). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number.

[0089] The term "epitope" refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed204936-0208-1406, v. 1by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., “Epitope Mapping Protocols,” in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0090] Also within the scope of the present disclosure are antibodies or antibody-derived compounds thereof in which specific amino acids have been substituted, deleted, or added. These alternations do not have a substantial effect on the peptide's biological properties, such as (but not limited to) binding activity. For example, antibodies may have amino acid substitutions in the framework region, such as to improve binding to the antigen. In another example, a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids. The donor framework can be a mature or germline human antibody framework sequence or a consensus sequence. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in the following: Bowie et al. (Science, 247: 1306-1310 (1990)); Cunningham et al. (Science, 244: 1081-1085 (1989)); Ausubel (ed.) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994)); Manialis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N. Y. (1989)); Pearson (Methods Mol. Biol. 243:307-31 (1994)); and Gonnet et al. (Science, 256:1443-45 (1992)).

[0091] For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped in one non-li ini ling embodiment as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same group. Non-conservative substitutions constitute exchanging a member of one of these groups for a member of another.

[0092] Tables of conservative amino acid substitutions have been constructed and are known in the art. In other embodiments, examples of interchangeable amino acids include, but are not limited to, the following: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. In other non-limiting214936-0208-1406, v. 1embodiments, the following substitutions can be made: Ala (A) by leu, ile, or val; Arg (R) by gin, asn, or lys; Asn (N) by his, asp, lys, arg, or gin; Asp (D) by asn or glu; Cys (C) by ala or ser; Gin (Q) by glu or asn; Glu (E) by gin or asp; Gly (G) by ala; His (H) by asn, gin, lys, or arg; lie (I) by val, met, ala, phe, or leu; Leu (L) by val, met, ala, phe, or ile; Lys (K) by gin, asn, or arg; Met (M) by phe, ile, or leu; Phe (E) by leu, val, ile, ala, or tyr; Pro (P) by ala; Ser (S) by thr; Thr (T) by ser; Trp (W) by phe or tyr; Tyr (Y) by trp, phe, thr, or ser; and Val (V) by ile, leu, met, phe, or ala.

[0093] Other considerations for amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent- (i.e., externally) exposed. Lor interior residues, conservative substitutions include for example: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; lie and Val; Val and Leu; Leu and lie; Leu and Met; Phe and Tyr; and Tyr and Trp. Lor solvent-exposed residues, c nservali vc substitutions include for example: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and lie; lie and Val; and Phe and Tyr.

[0094] Compositions or methods "comprising" one or more recited elements may include other elements not specifically recited. Lor example, a composition that comprises an HC compound may contain the HC compound alone or in combi nalion with other ingredients. The phrase "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of an HC compound or a conjugate thereof, or agent administered with the HC compound. Exemplary salts include sulfate, citrate, acetate, oxalate, fluoride, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as (but not limited to) an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.224936-0208-1406, v. 1

[0095] A chimeric antibody is a molecule in which different portions are derived from different animal species. For example, an antibody may contain a variable region derived from a murine mAh and a human immunoglobulin constant region. Chimeric antibodies can be produced by recombinant DNA techniques, e.g., see Morrison et al. (Proc Natl Acad Sci, 81:6851-6855 (1984)). For example, a gene encoding a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted. Chimeric antibodies can also be created by recombinant DNA techniques where DNA encoding murine variable regions can be ligated to DNA encoding the human constant regions, e.g., see International Patent Publication Nos. WO 87 / 002671 and WO 86 / 01533, and U. S. Patent No. 4,816,567.

[0096] A chimeric antibody is a recombinant protein that contains the variable domains including the CDRs of an antibody derived from one species, for example a rodent or rabbit antibody, while the constant domains of the antibody molecule are generally derived from those of a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of other species, such as but not limited to, a cat, dog, or horse.

[0097] A chimeric antibody can be humanized by replacing the sequences of, for example, a murine FR in the variable domains of the chimeric antibody with one or more different human FR sequences. Specifically, mouse CDRs are transferred from heavy and light variable chains of the mouse immunoglobulin into the corresponding variable domains of a human antibody. As simply transferring mouse CDRs into human FRs may result in a reduction of antibody affinity, addi lional modifications might be required in order to restore the original affinity of the murine antibody. This can be accomplished by the replacement of one or more human residues in the FR regions with their murine counterparts to obtain an antibody with enhanced binding affinity to the DIR epitope or D2R epitope (e.g., see Tempest et al. (Biotechnology, 9:266 (1991)) and Verhoeyen et al. (Science, 239: 1534 (1988))). Techniques for producing humanized antibodies are known to persons having ordinary skill in the art and are disclosed, for example, by Jones et al. (Nature, 321: 522 (1986)), Riechmann et al. (Nature, 332: 323 (1988)), Verhoeyen et al. (Science, 239: 1534 (1988)), Carter et al. (Proc. Nat'l Acad. Sci. USA, 9: 4285 (1992)), Sandhu (Crit. Rev. Biotech. 2 A 1 (1992)), and Singer et al. (J. Immun. 150: 2844 (1993)).

[0098] As noted, an antibody’s light chain variable region contains an FR interrupted by three different CDRs (CDRL1, CDRL2, and CDRL3), and a heavy chain region contains an FR interrupted by three different CDRs (CDRH1, CDRH2, and CDRH3). In one non-limiting234936-0208-1406, v. 1embodiment, humanized antibodies are antibody molecules from non-human species having one, two, three, four, live or all six CDRs from the non-human species and a framework region from a human immunoglobulin molecule.

[0099] A humanized antibody is a genetically engineered antibody in which the variable heavy and variable light CDRs from a non-human "donor" antibody are grafted into human "acceptor" antibody sequences (see for example, U. S. Patent Nos. 5,530,101; 5,585,089; 5,225,539; 6,407,213; 5,859,205; and 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from a non-human donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence, and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain.[000100] As noted, humanized antibodies can be generated by replacing framework sequences of the variable region that are not directly involved in antigen binding with equivalent sequences from human variable regions. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of variable regions from at least one of a heavy or light chain. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Each antibody light and heavy chain variable region consists of a framework region interrupted by the three corresponding CDRs. In one non-limiting embodiment, humanized antibodies are antibody molecules from non-human species having one, two, or all CDRs from the non-human species and a framework region from a human immunoglobulin molecule. Therefore, humanized antibodies can be generated by replacing framework sequences of the variable region that are not directly involved in antigen binding with equivalent sequences from human variable244936-0208-1406, v. 1regions. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of variable regions from at least one of a heavy or light chain. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.[000101] The light and heavy chain variable regions can optional ly be ligated to corresponding constant regions. CDR-grafted antibody molecules can be produced by CDR-grafting or CDR substitution. One, two, or all CDRs of an immunoglobulin chain can be replaced. For example, all of the CDRs of a particular antibody may be from at least a portion of a non-human animal (e.g., mouse, such as (but not limited to) CDRs shown herein), or only some of the CDRs may be replaced. It is only necessary to keep the CDRs which are required for specific and high binding affinity of the antibody to a target. Once expressed, antibodies can be purified according to standard procedures of the art, including but not limited to HPLC purification, column chromatography, and gel electrophoresis. Methods for producing human antibodies include, but are not limited to, those shown in U. S. Patent Nos.4,634,664; 4,634,666; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126; 5,569,825; 5,545,806; 5,877,218; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and 5,565,332; and International Patent Application Publication Nos. WO 91 / 17271; WO 92 / 01047; and WO93 / 12227.[000102] A fully human antibody can be obtained from a transgenic non-human animal (see, e.g., Mendez et al. (Nature Genetics, 15: 146-156, 1997); and U. S. Patent No. 5,633,425). Methods for producing fully human antibodies using either combinatorial approaches or transgenic animals transformed with human immunoglobulin loci are known in the art (e.g., Mancini et al. (New Microbiol., 27:315-28 (2004)); Conrad and Scheller (Comb. Chem. High Throughput Screen. 8:117-26 (2005)); and Brekke and Uoset (Curr. Opin. Pharmacol., 3:544-50 (2003)). Such fully human antibodies are expected to exhibit even fewer side effects than chimeric or humanized antibodies and to function in vivo as essentially endogenous human antibodies. In certain non-limiting embodiments, the claimed methods and procedures may utilize human antibodies produced by such techniques.[000103] A CDR in a humanized or human antibody may be defined as “substantial ly derived from” or “substantially identical to” a corresponding CDR in a non-human antibody when at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs. In some non-limiting embodiments, a CDR in a humanized antibody or human antibody is substantially derived from or substantially identical254936-0208-1406, v. 1to a corresponding CDR in a non-human antibody when there are no more than one, two, or three conservative amino acid substitutions in any given CDR. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are “substantially from” a human variable region framework sequence or human constant region, respectively, when at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of corresponding residues (as defined by Kabat numbering) are identical. As noted elsewhere herein, although humanized antibodies often incorporate all six CDRs (e.g., as defined elsewhere herein) from a non-human (e.g., mouse or rabbit) antibody, they can also be made with less than all of the non-human CDRs (e.g., at least 2, 3, 4, or 5).[000104] Heavy and light chain variable regions of humanized antibodies can be linked to at least a portion of a human constant region, for example, for human antibody isotypes IgGl, IgG2, IgG3, or IgG4. Light chain constant regions can be lambda or kappa. Antibodies can be expressed as, for example (but not by way of limitation): tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which heavy and light chain variable domains are linked through a spacer. All antibody isotypes are encompassed by the present disclosure, including (but not limited to) IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgA (IgAl, IgA2), IgD, or IgE. The antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, rabbit, human) antibodies or antigen-binding portions thereof.[000105] As mentioned above, mAbs and mAb-derived compounds can be dcrivalizcd or linked to, e.g., conjugated to, the HC compounds disclosed herein, for example, directly or indirectly, by covalent bonding or by noncovalent interactions.[000106] Particularly suitable (but non-limiting) moieties for conjugation to the HC compounds or the targeting moiety conjugated to the HC compound include cytotoxic agents (e.g., chemotherapeutic agents), prodrug converting enzymes, radionuclides such as (but not limited to) radioactive isotopes or compounds, an immunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, a cytokine, a chemokine, a drug, a hormone, an siRNA, an enzyme, a growth factor, a prodrug, an oligonucleotide, a pro-apoptotic agent, an interference RNA, a photoactive therapeutic agent, a tyrosine kinase inhibitor, a Bruton kinase inhibitor, a sphingosine inhibitor, a cytotoxic agent, or toxins (these moieties being collectively referred to as therapeutic agents or drugs). Examples of useful classes of cytotoxic agents include (but are not limited to) DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Exemplary cytotoxic264936-0208-1406, v. 1agents include (but are not limited to) auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines (e.g., pyrrolo[l,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids. Techniques for conjugating therapeutic agents to proteins, and in particular to antibodies, are well-known (e.g., see, Carter, PJ and Senter PD, “Antibody-Drug Conjugates for Cancer Therapy.” Cancer J., 14(3): 154- 169 (2008)).[000107] Examples of diagnostic agents that may be used with the HC compounds of the present disclosure include, but are not limited to: radionuclides, contrast agents, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photoactive diagnostic agents.[000108] Examples of radionuclides include, but are not limited to:inIn,niAt,177Lu,211Bi,212Bi,213Bi,211At,62Cu,67Cu,90Y,125I,131I,1331,32P,33P,47SC,111Ag,67Ga,153Sm,161Tb,152Dy,166Dy,161HO,166HO,186Re,188Re,189Re,211Pb,212Pb,223Ra,225Ac,227Th,77As,89Sr, " Mo,105Rh, 149pni 194jj, 58QQ 80mgj. 99mrp^ 103IHRJ^ ^^mOs ^FmnC,13N,15O,75Br,198Au,199Au,224Ac,77Br,113mIn,95Ru,97Ru,103Ru,105Ru,107Hg,203Hg,i2im'j’g i22m'j’g --"'Hi i25m'j’gl97l inl98l in197Pll(l9Pd142Pr143Pr161Tb32Co38Co51Cr,59Fe,75Se,2O1T1,76Br,169Yb,110In,18F,52Fe,64Cu,68Ga,86Y,89Zr,94mTc,94Tc,1201,123I,124I,154-158Gd,32F,nC,13N,51Mn,52mMn,55Co,72As,75Br,76Br,82mRb,83Sr, or other gamma-, beta-, or positron-emitters.[000109] Examples of paramagnetic ions include, but are not limited to: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).[000110] Examples of fluorescent labeling diagnostic agents include, but are not limited to: fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine; or a chemiluminescent labeling compound selected from the group comprising luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt, and an oxalate ester; or a bioluminescent compound selected from the group comprising luciferin, luciferase, and aequorin.[000111] Therapeutic and / or diagnostic agents may include, without limitation, immunomodulators, cytokines (and their inhibitors), chemokines (and their inhibitors), tyrosine kinase inhibitors, growth factors, hormones and certain enzymes (i.e., those that do not induce local necrosis), or their inhibitors.14936-0208-1406, v. 1[000112] Examples of therapeutic agents that can be used as cargo molecules in the liposomes and compositions of the present disclosure include, but are not limited to, drugs to treat myocardial infarction, myocardial ischemia, reperfusion injury, congestive heart failure (CHF), cardiomyopathies, coronary artery disease (CAD), atrial fibrillation, inflammation, atherosclerosis, unstable angina, arrhythmias, valve diseases, congenital and inherited heart conditions, and heart infections.[000113] Examples of types of drugs for treating heart failure that may be used in the compositions of the present disclosure include but are not limited to ACE inhibitors, such as benazepril, captopril, cilazapril, delapril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, spirapril, trandolapril, and zofenopril; angiotensin-2 receptor blockers (ARBs) such as azilsartan, candesartan, eprosartan, fimasartan, forasartan, irbesartan, losartan, olmasartan, pratosartan, saparasartan, saralasin, tasosartan, telmisartan, and valsartan; beta blockers such as atenolol, acebutolol, bisoprolol, bucindolol, carvedilol (Coreg), celiprolol, esmolol, labetolol, metoprolol, nadolol, nebivolol, pindolol, propranolol, sotalol, and timolol; calcium channel blockers such as amlodipine, diltiazem (Cardizem), felodipine, isradipine, nicardipine, nifedipine (Procardia), nisoldipine (Sular), verapamil (Calan SR); mineralocorticoid receptor antagonists such as canrenone, eplerenone, finerenone, and spironolactone; ivabradine; sacubitril valsartan; hydralazine with nitrate; and digoxin.[000114] Examples of other therapeutic agents that may be used include, but are not limited to: 5 -fluorouracil, aplidin, azaribine, anastrozole, anthracy clines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carinusline, celecoxib, chlorambucil, cisplalinuin, Cox-2 inhibitors, CPT-11 SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), pro-2P-DOX, cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, paclitaxel, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil284936-0208-1406, v. 1mustard, vinorelbine, vinblastine, vincristine, a vinca alkaloid, a tyrophostin, canertinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, leflunomide, nilotinib, pazopanib, semaxinib, sorafenib, sunitinib, sutent, vatalanib, PCI-32765 (ibrutinib), PCI-45292, GDC-0834, LFM-A13, and RN486.[000115] Examples of toxins include, but are not limited to, ricin, abrin, alpha toxin, saporin, ribonuclease (RNase; e.g., onconase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.[000116] Immunomodulators include, but are not limited to, cytokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSF), interferons (IFN), erythropoietins, thrombopoietins, and combinations thereof. Specifically useful are lymphotoxins such as (but not limited to) tumor necrosis factor (TNF); hematopoietic factors such as (but not limited to) interleukin (IE); colony stimulating factors such as (but not limited to) granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF); interferons such as (but not limited to) interferons-alpha, -beta, -lambda, or -gamma; and stem cell growth factors such as (but not limited to) that designated " SI factor.” Included among the cytokines are growth hormones such as (but not limited to): human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as (but not limited to) follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (EH); hepatic growth factor; prostaglandin; fibroblast growth factor; prolactin; placental lactogen; OB protein; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; aclivin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as (but not limited to) NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as (but not liinilcd to) TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as (but not limited to) interferonalpha, -beta, -lambda, and -gamma; colony stimulating factors (CSFs) such as (but not limited to) macrophage-CSF (M-CSF); interleukins (ILs) such as (but not limited to) IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25; leukemia inhibitory factor (LIP); kit-ligand or FLT-3 ligand; angiostatin; thrombospondin; endostatin; tumor necrosis factor; and lymphotoxin. Chemokines of use include (but are not liinilcd to): RANTES, MGAL, MIPl-alpha, MIPl-Beta, and IP-10.294936-0208-1406, v. 1[000117] In certain non-limiting embodiments, therapeutic radionuclides have a decay-energy in the range of 20 to 6,000 keV, such as (but not limited to) in the ranges of: 60 to 200 keV for an Auger emitter; 100-2,500 keV for a beta emitter; and 4,000-6,000 keV for an alpha emitter. Maximum decay energies of useful beta-particle-emitting nuclides may be, but are not limited to, 20-5,000 keV; 100-4,000 keV; or 500-2,500 keV. Also included are radionuclides that substantially decay with Auger-emitting particles, such as (but not limited to): Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m, and Ir-192. Decay energies of useful beta-particle-emitting nuclides may be (for example but not by way of limitation): <1,000 keV, <100 keV, or <70 keV Also included are radionuclides that substantially decay with generation of alpha-particles. Such radionuclides include, but are not limited to: Dy- 152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227, and Fm-255. Decay energies of useful alpha-particle-emitting radionuclides include (but are not limited to): 2,000-10,000 keV; 3,000-8,000 keV; or 4,000- 7,000 keV[000118] Furthermore, the compositions can be formulated into compositions in either neutral or salt forms. Pharmaceutically acceptable salts include (but are not limited to) the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.[000119] The HC compounds or compositions comprising the HC compounds can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight, and condition of the subject, the particular composition used, and the route of administration. In one non-limiting embodiment, a single dose of the composition according to the disclosure is administered. In other non-limiting embodiments, multiple doses are administered. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, or whether the composition is used for prophylactic or curative purposes or only diagnostic and imaging purposes. For example, in certain non-limiting embodiments, the composition is administered once per month, twice per month, three times per month, every other week, once per week, twice per week, three times per week, four times per week, five times per week, six limes per week, every other day, daily, twice a day, or three times a day. The duration of treatment (i.e.,304936-0208-1406, v. 1the period of time over which the compound or composition is administered) can vary, depending on any of a variety of factors, e.g., subject response. For example, the composition can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.[000120] The HC compounds or compositions can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, for example but not by way of limitation) stabilize or increase or decrease the absorption or clearance rates of the pharmaceutical compositions. Physiologically acceptable compounds can include, for example but not by way of limitation: carbohydrates, such as glucose, sucrose, or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; detergents; liposomal carriers; excipients; or other stabilizers and / or buffers. Other physiologically acceptable compounds include (but are not limited to) weting agents, emulsifying agents, dispersing agents, or preservatives.[000121] In one aspect, the pharmaceutical formulations comprising the HC compounds are incorporated in lipid monolayers or bilayers, such as (but not limited to) liposomes, such as shown in U. S. Patent Nos. 6,110,490; 6,096,716; 5,283,185; and 5,279,833. In other aspects, non-liiniling embodiments of the disclosure include formulations in which the HC compounds have been atached to the surface of the monolayer or bilayer of the liposomes. Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art, such as (but not limited to) those disclosed in U. S. Patent Nos.4,235,871; 4,501,728; and 4,837,028, and as described elsewhere herein.[000122] The compositions may be administered in solution. The formulation thereof may be in a solution having a suitable pharmaceutically acceptable buffer, such as (but not limited to) phosphate, Tris (hydroxymethyl) aminomethane-HCl, or citrate, and the like. Buffer concentrations should be in the range of 1 to 100 mM. The formulated solution may also contain a salt, such as (but not limited to) sodium chloride or potassium chloride in a concentration of 50 to 150 mM. An effective amount of a stabilizing agent such as (but not limited to) mannitol, trehalose, sorbitol, glycerol, albumin, a globulin, a detergent, a gelatin, a protamine, or a salt of protamine may also be included.314936-0208-1406, v. 1[000123] The composition is formulated to contain an effective amount of the presently disclosed active agent, wherein the amount depends on the animal to be treated and the condition to be treated. In certain non-limiting embodiments, the active agent is administered at a dose ranging from about 0.001 mg to about 10 g, from about 0.01 mg to about 10 g, from about 0.1 mg to about 10 g, from about 1 mg to about 10 g, from about 1 mg to about 9 g, from about 1 mg to about 8 g, from about 1 mg to about 7 g, from about 1 mg to about 6 g, from about 1 mg to about 5 g, from about 10 mg to about 10 g, from about 50 mg to about 5 g, from about 50 mg to about 5 g, from about 50 mg to about 2 g, from about 0.05 pg to about 1.5 mg, from about 10 pg to about 1 mg protein, from about 30 pg to about 500 pg, from about 40 pg to about 300 pg, from about 0.1 pg to about 200 mg, from about 0.1 pg to about 5 pg, from about 5 pg to about 10 pg, from about 10 pg to about 25 pg, from about 25 pg to about 50 pg, from about 50 pg to about 100 pg, from about 100 pg to about 500 pg, from about 500 pg to about 1 mg, or from about 1 mg to about 2 mg.[000124] The dosage of an administered active agent for humans will vary depending upon factors such as (but not limited to) the patient's age, weight, height, sex, general medical condition, and previous medical history. In certain non-limiting embodiments, the recipient is provided with a dosage of the active agent that is in the range of from about 1 mg to about 1000 mg as a single infusion or single or multiple injections, although a lower or higher dosage also may be administered. In certain non-limiting embodiments, the dosage may be in the range of from about 25 mg to about 100 mg of the active agent per square meter (m2) of body surface area for a typical adult, although a lower or higher dosage also may be administered. Non-limiting examples of dosages of the active agent that may be administered to a human subject further include 1 to 500 mg, 1 to 70 mg, or 1 to 20 mg, although higher or lower doses may be used. Dosages may be repeated as needed, for example (but not by way of limitation), once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. It may also be given less frequently, such as (but not limited to) every other week for several months, or more frequently, such as twice weekly or by continuous infusion.[000125] In some non-limiting embodiments, the effective amount of a cardiac-targeting moiety of the present disclosure is in a concentration of about 1 nM, about 5 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 500 nM, about 550 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 2 pM, about 3 pM, about 4324936-0208-1406, v. 1pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 60 pM, about 70 pM, about 75 pM, about 80 pM, about 90 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 750 pM, about 800 pM, about 900 pM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM, about 300 mM, about 400 mM, about 500mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, about 10 M, about 15 M, about 20 M, about 25 M, about 30 M, about 35 M, about 40 M, about 45 M, about 50 M, about 75 M, about 100 M, or any range in between any two of the aforementioned concentrations, including said two concentrations as endpoints of the range, or any number in between any two of the aforementioned concentrations.[000126] In some non-limiting methods, the patient is administered the active agent each one, two, three, or four weeks, for example. The dosage depends on the frequency of administration, condition of the patient, response to prior treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, among other factors.[000127] Administration can be (for example but not by way of limitation) parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Administration can also be localized directly into a tumor, for example. Administration into the systemic circulation by intravenous or subcutaneous administration is typical. Intravenous administration can be, for example (but not by way of limitation), by infusion over a period such as (but not limited to) 30-90 min or by a single bolus injection.[000128] The number of dosages of the HC compound administered depends on the severity and temporal nature of the disorder (e.g., whether presenting acute or chronic symptoms) and the334936-0208-1406, v. 1response of the disorder to the treatment with which the HC compound is being used. For acute disorders or acute exacerbations of a chronic disorder, between 1 and 10 doses may be used. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder.[000129] In certain non-limiting embodiments, pharmaceutical compositions are sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries. The formulation depends on the route of administration chosen. For injection, the active agent can be formulated in aqueous solutions, such as (but not limited to) in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active agent can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The concentration of the active agent in a liquid formulation can be for example (but not by way of limitation) 0.01-10 mg / ml, such as 1.0 mg / ml.[000130] Returning now to the inventive concepts of the present disclosure, in at least certain non-limiting embodiments, the HC-based compounds of the present disclosure comprise Chemical Structure I, as shown below.whereinX is selected from the group consisting of H, F, Cl, Br, and I;344936-0208-1406, v. 1Y is selected from the group consisting of methyl (CH3-), ethyl (CH3CH2-), propyl (CH3CH2CH2-), amino (NH2-), methylamino (CH3NH-), ethylamino (CH3CH2NH-), propylamino (CH3CH2CH2NH-), dimethylamino ((CHs)2N-), diethylamino ((CHsCIrb^N-), dipropylamino ((CHsQHC^ N-), sulfur trioxide (-SO3 )- quaternary carbon-trimethyl ((CHsYC-)- quaternary carbon-triethyl ((CHsCTb^C-)), benzene (CeHs-), indolium ring, oxygen (double bonded, O=), and hydroxyl (OH); andR is selected from the group consisting of methyl, ethyl, propyl, trimethylpropylammonium bromide (TMAB), benzene, propyl benzene, a trimethyl benzene, and propyl sulfur trioxide.[000131] Chemical structural representations of several of the potential R-group substitutions are shown below:HNMethyl aminoHNEthyl aminoHNPropyl aminoDimethyl amino354936-0208-1406, v. 1Diethyl aminoDipropyl aminoQuaternary carbon-trimethyl (tert-butyl)Quaternary carbon-triethyl 0" Sulfur trioxidePropyl sulfur trioxide364936-0208-1406, v. 1Propyl benzeneIndolium ringTrimethyl benzene374936-0208-1406, v. 1[000132] Particular embodiments of the HC compounds of the present disclosure are shown in Tables 1A-5G.Table 1A, Compounds with Chemical Structure I where X=H, R=MethylX Y R H Methyl MethylH Ethyl MethylH Propyl MethylH Amino MethylH Dimethylamino MethylH Diethylamino MethylH Dipropylamino MethylH Methylamino MethylH Ethylamino MethylH Propylamino MethylH Sulfur trioxide MethylH Quaternary carbon (trimethyl) MethylH Quaternary carbon (triethyl) MethylH Benzene MethylH Indolium ring MethylH Double bonded oxygen MethylH Hydroxyl MethylTable IB, Compounds with Chemical Structure I where X=H, R=EthylX Y R H Methyl EthylH Ethyl EthylH Propyl EthylH Amino EthylH Dimethylamino EthylH Diethylamino EthylH Dipropylamino EthylH Methylamino EthylH Ethylamino EthylH Propylamino EthylH Sulfur trioxide EthylH Quaternary carbon (trimethyl) EthylH Quaternary carbon (triethyl) EthylH Benzene EthylH Indolium ring EthylH Double bonded oxygen EthylH Hydroxyl Ethyl384936-0208-1406, v. 1Table 1C. Compounds with Chemical Structure I where X=H, R=Propyl X Y R H Methyl PropylH Ethyl PropylH Propyl PropylH Amino PropylH Dimethylamino PropylH Diethylamino PropylH Dipropylamino PropylH Methylamino PropylH Ethylamino PropylH Propylamino PropylH Sulfur trioxide PropylH Quaternary carbon (trimethyl) PropylH Quaternary carbon (triethyl) PropylH Benzene PropylH Indolium ring PropylH Double bonded oxygen PropylH Hydroxyl PropylTable ID. Compounds with Chemical Structure I where X=H, R=TMAB X Y R H Methyl TMABH Ethyl TMABH Propyl TMABH Amino TMABH Dimethylamino TMABH Diethylamino TMABH Dipropylamino TMABH Methylamino TMABH Ethylamino TMABH Propylamino TMABH Sulfur trioxide TMABH Quaternary carbon (trimethyl) TMABH Quaternary carbon (triethyl) TMABH Benzene TMABH Indolium ring TMABH Double bonded oxygen TMABH Hydroxyl TMAB394936-0208-1406, v. 1Table IE, Compounds with Chemical Structure I where X=H, R= Propyl benzene X Y R H Methyl Propyl benzeneH Ethyl Propyl benzeneH Propyl Propyl benzeneH Amino Propyl benzeneH Dimethylamino Propyl benzeneH Diethylamino Propyl benzeneH Dipropylamino Propyl benzeneH Methylamino Propyl benzeneH Ethylamino Propyl benzeneH Propylamino Propyl benzeneH Sulfur trioxide Propyl benzeneH Quaternary carbon (trimethyl) Propyl benzeneH Quaternary carbon (triethyl) Propyl benzeneH Benzene Propyl benzeneH Indolium ring Propyl benzeneH Double bonded oxygen Propyl benzeneH Hydroxyl Propyl benzeneTable IF, Compounds with Chemical Structure I where X=H, R= Propyl sulfur trioxide X Y R H Methyl Propyl sulfur trioxideH Ethyl Propyl sulfur trioxideH Propyl Propyl sulfur trioxideH Amino Propyl sulfur trioxideH Dimethylamino Propyl sulfur trioxideH Diethylamino Propyl sulfur trioxideH Dipropylamino Propyl sulfur trioxideH Methylamino Propyl sulfur trioxideH Ethylamino Propyl sulfur trioxideH Propylamino Propyl sulfur trioxideH Sulfur trioxide Propyl sulfur trioxideH Quaternary carbon (trimethyl) Propyl sulfur trioxideH Quaternary carbon (triethyl) Propyl sulfur trioxideH Benzene Propyl sulfur trioxideH Indolium ring Propyl sulfur trioxideH Double bonded oxygen Propyl sulfur trioxideH Hydroxyl Propyl sulfur trioxide404936-0208-1406, v. 1Table 1G. Compounds with Chemical Structure I where X=H, R= Benzene X Y R H Methyl BenzeneH Ethyl BenzeneH Propyl BenzeneH Amino BenzeneH Dimethylamino BenzeneH Diethylamino BenzeneH Dipropylamino BenzeneH Methylamino BenzeneH Ethylamino BenzeneH Propylamino BenzeneH Sulfur trioxide BenzeneH Quaternary carbon (trimethyl) BenzeneH Quaternary carbon (triethyl) BenzeneH Benzene BenzeneH Indolium ring BenzeneH Double bonded oxygen BenzeneH Hydroxyl BenzeneTable 2A, Compounds with Chemical Structure I where X=F, R=Methyl X Y R F Methyl MethylF Ethyl MethylF Propyl MethylF Amino MethylF Dimethylamino MethylF Diethylamino MethylF Dipropylamino MethylF Methylamino MethylF Ethylamino MethylF Propylamino MethylF Sulfur trioxide MethylF Quaternary carbon (trimethyl) MethylF Quaternary carbon (triethyl) MethylF Benzene MethylF Indolium ring MethylF Double bonded oxygen MethylF Hydroxyl Methyl414936-0208-1406, v. 1Table 2B, Compounds with Chemical Structure I where X=F, R=Ethyl X Y R F Methyl EthylF Ethyl EthylF Propyl EthylF Amino EthylF Dimethylamino EthylF Diethylamino EthylF Dipropylamino EthylF Methylamino EthylF Ethylamino EthylF Propylamino EthylF Sulfur trioxide EthylF Quaternary carbon (trimethyl) EthylF Quaternary carbon (triethyl) EthylF Benzene EthylF Indolium ring EthylF Double bonded oxygen EthylF Hydroxyl EthylTable 2C. Compounds with Chemical Structure I where X=F, R=Propyl X Y R F Methyl PropylF Ethyl PropylF Propyl PropylF Amino PropylF Dimethylamino PropylF Diethylamino PropylF Dipropylamino PropylF Methylamino PropylF Ethylamino PropylF Propylamino PropylF Sulfur trioxide PropylF Quaternary carbon (trimethyl) PropylF Quaternary carbon (triethyl) PropylF Benzene PropylF Indolium ring PropylF Double bonded oxygen PropylF Hydroxyl Propyl424936-0208-1406, v. 1Table 2D. Compounds with Chemical Structure I where X=F, R=TMABX Y R F Methyl TMABF Ethyl TMABF Propyl TMABF Amino TMABF Dimethylamino TMABF Diethylamino TMABF Dipropylamino TMABF Methylamino TMABF Ethylamino TMABF Propylamino TMABF Sulfur trioxide TMABF Quaternary carbon (trimethyl) TMABF Quaternary carbon (triethyl) TMABF Benzene TMABF Indolium ring TMABF Double bonded oxygen TMABF Hydroxyl TMABTable 2E, Compounds with Chemical Structure I where X=F, R= Propyl benzene X Y R F Methyl Propyl benzeneF Ethyl Propyl benzeneF Propyl Propyl benzeneF Amino Propyl benzeneF Dimethylamino Propyl benzeneF Diethylamino Propyl benzeneF Dipropylamino Propyl benzeneF Methylamino Propyl benzeneF Ethylamino Propyl benzeneF Propylamino Propyl benzeneF Sulfur trioxide Propyl benzeneF Quaternary carbon (trimethyl) Propyl benzeneF Quaternary carbon (triethyl) Propyl benzeneF Benzene Propyl benzeneF Indolium ring Propyl benzeneF Double bonded oxygen Propyl benzeneF Hydroxyl Propyl benzene434936-0208-1406, v. 1Table 2F, Compounds with Chemical Structure I where X=F, R= Propyl sulfur trioxide X Y R F Methyl Propyl sulfur trioxideF Ethyl Propyl sulfur trioxideF Propyl Propyl sulfur trioxideF Amino Propyl sulfur trioxideF Dimethylamino Propyl sulfur trioxideF Diethylamino Propyl sulfur trioxideF Dipropylamino Propyl sulfur trioxideF Methylamino Propyl sulfur trioxideF Ethylamino Propyl sulfur trioxideF Propylamino Propyl sulfur trioxideF Sulfur trioxide Propyl sulfur trioxideF Quaternary carbon (trimethyl) Propyl sulfur trioxideF Quaternary carbon (triethyl) Propyl sulfur trioxideF Benzene Propyl sulfur trioxideF Indolium ring Propyl sulfur trioxideF Double bonded oxygen Propyl sulfur trioxideF Hydroxyl Propyl sulfur trioxideTable 2G. Compounds with Chemical Structure I where X=F, R= BenzeneX Y R F Methyl BenzeneF Ethyl BenzeneF Propyl BenzeneF Amino BenzeneF Dimethylamino BenzeneF Diethylamino BenzeneF Dipropylamino BenzeneF Methylamino BenzeneF Ethylamino BenzeneF Propylamino BenzeneF Sulfur trioxide BenzeneF Quaternary carbon (trimethyl) BenzeneF Quaternary carbon (triethyl) BenzeneF Benzene BenzeneF Indolium ring BenzeneF Double bonded oxygen BenzeneF Hydroxyl Benzene444936-0208-1406, v. 1Table 3A. Compounds with Chemical Structure I where X=C1, R=Methyl X Y RCl Methyl MethylCl Ethyl MethylCl Propyl MethylCl Amino MethylCl Dimethylamino MethylCl Diethylamino MethylCl Dipropylamino MethylCl Methylamino MethylCl Ethylamino MethylCl Propylamino MethylCl Sulfur trioxide MethylCl Quaternary carbon (trimethyl) MethylCl Quaternary carbon (triethyl) MethylCl Benzene MethylCl Indolium ring MethylCl Double bonded oxygen MethylCl Hydroxyl MethylTable 3B, Compounds with Chemical Structure I where X=C1, R=Ethyl X Y RCl Methyl EthylCl Ethyl EthylCl Propyl EthylCl Amino EthylCl Dimethylamino EthylCl Diethylamino EthylCl Dipropylamino EthylCl Methylamino EthylCl Ethylamino EthylCl Propylamino EthylCl Sulfur trioxide EthylCl Quaternary carbon (trimethyl) EthylCl Quaternary carbon (triethyl) EthylCl Benzene EthylCl Indolium ring EthylCl Double bonded oxygen EthylCl Hydroxyl Ethyl454936-0208-1406, v. 1Table 3C. Compounds with Chemical Structure I where X=C1, R=Propyl X Y RCl Methyl PropylCl Ethyl PropylCl Propyl PropylCl Amino PropylCl Dimethylamino PropylCl Diethylamino PropylCl Dipropylamino PropylCl Methylamino PropylCl Ethylamino PropylCl Propylamino PropylCl Sulfur trioxide PropylCl Quaternary carbon (trimethyl) PropylCl Quaternary carbon (triethyl) PropylCl Benzene PropylCl Indolium ring PropylCl Double bonded oxygen PropylCl Hydroxyl PropylTable 3D. Compounds with Chemical Structure I where X=C1, R=TMAB X Y RCl Methyl TMABCl Ethyl TMABCl Propyl TMABCl Amino TMABCl Dimethylamino TMABCl Diethylamino TMABCl Dipropylamino TMABCl Methylamino TMABCl Ethylamino TMABCl Propylamino TMABCl Sulfur trioxide TMABCl Quaternary carbon (trimethyl) TMABCl Quaternary carbon (triethyl) TMABCl Benzene TMABCl Indolium ring TMABCl Double bonded oxygen TMABCl Hydroxyl TMAB464936-0208-1406, v. 1Table 3E, Compounds with Chemical Structure I where X=C1, R= Propyl benzene X Y RCl Methyl Propyl benzeneCl Ethyl Propyl benzeneCl Propyl Propyl benzeneCl Amino Propyl benzeneCl Dimethylamino Propyl benzeneCl Diethylamino Propyl benzeneCl Dipropylamino Propyl benzeneCl Methylamino Propyl benzeneCl Ethylamino Propyl benzeneCl Propylamino Propyl benzeneCl Sulfur trioxide Propyl benzeneCl Quaternary carbon (trimethyl) Propyl benzeneCl Quaternary carbon (triethyl) Propyl benzeneCl Benzene Propyl benzeneCl Indolium ring Propyl benzeneCl Double bonded oxygen Propyl benzeneCl Hydroxyl Propyl benzeneTable 3F. Compounds with Chemical Structure I where X=C1, R= Propyl sulfur trioxide X Y RCl Methyl Propyl sulfur trioxideCl Ethyl Propyl sulfur trioxideCl Propyl Propyl sulfur trioxideCl Amino Propyl sulfur trioxideCl Dimethylamino Propyl sulfur trioxideCl Diethylamino Propyl sulfur trioxideCl Dipropylamino Propyl sulfur trioxideCl Methylamino Propyl sulfur trioxideCl Ethylamino Propyl sulfur trioxideCl Propylamino Propyl sulfur trioxideCl Sulfur trioxide Propyl sulfur trioxideCl Quaternary carbon (trimethyl) Propyl sulfur trioxideCl Quaternary carbon (triethyl) Propyl sulfur trioxideCl Benzene Propyl sulfur trioxideCl Indolium ring Propyl sulfur trioxideCl Double bonded oxygen Propyl sulfur trioxideCl Hydroxyl Propyl sulfur trioxide474936-0208-1406, v. 1Table 3G. Compounds with Chemical Structure I where X=C1, R= Benzene X Y RCl Methyl BenzeneCl Ethyl BenzeneCl Propyl BenzeneCl Amino BenzeneCl Dimethylamino BenzeneCl Diethylamino BenzeneCl Dipropylamino BenzeneCl Methylamino BenzeneCl Ethylamino BenzeneCl Propylamino BenzeneCl Sulfur trioxide BenzeneCl Quaternary carbon (trimethyl) BenzeneCl Quaternary carbon (triethyl) BenzeneCl Benzene BenzeneCl Indolium ring BenzeneCl Double bonded oxygen BenzeneCl Hydroxyl BenzeneTable 4A. Compounds with Chemical Structure I where X=Br, R=Methyl X Y RBr Methyl MethylBr Ethyl MethylBr Propyl MethylBr Amino MethylBr Dimethylamino MethylBr Diethylamino MethylBr Dipropylamino MethylBr Methylamino MethylBr Ethylamino MethylBr Propylamino MethylBr Sulfur trioxide MethylBr Quaternary carbon (trimethyl) MethylBr Quaternary carbon (triethyl) MethylBr Benzene MethylBr Indolium ring MethylBr Double bonded oxygen MethylBr Hydroxyl Methyl484936-0208-1406, v. 1Table 4B, Compounds with Chemical Structure I where X=Br, R=Ethyl X Y RBr Methyl EthylBr Ethyl EthylBr Propyl EthylBr Amino EthylBr Dimethylamino EthylBr Diethylamino EthylBr Dipropylamino EthylBr Methylamino EthylBr Ethylamino EthylBr Propylamino EthylBr Sulfur trioxide EthylBr Quaternary carbon (trimethyl) EthylBr Quaternary carbon (triethyl) EthylBr Benzene EthylBr Indolium ring EthylBr Double bonded oxygen EthylBr Hydroxyl EthylTable 4C. Compounds with Chemical Structure I where X=Br, R=Propyl X Y RBr Methyl PropylBr Ethyl PropylBr Propyl PropylBr Amino PropylBr Dimethylamino PropylBr Diethylamino PropylBr Dipropylamino PropylBr Methylamino PropylBr Ethylamino PropylBr Propylamino PropylBr Sulfur trioxide PropylBr Quaternary carbon (trimethyl) PropylBr Quaternary carbon (triethyl) PropylBr Benzene PropylBr Indolium ring PropylBr Double bonded oxygen PropylBr Hydroxyl Propyl494936-0208-1406, v. 1Table 4D. Compounds with Chemical Structure I where X=Br, R=TMABX Y RBr Methyl TMABBr Ethyl TMABBr Propyl TMABBr Amino TMABBr Dimethylamino TMABBr Diethylamino TMABBr Dipropylamino TMABBr Methylamino TMABBr Ethylamino TMABBr Propylamino TMABBr Sulfur trioxide TMABBr Quaternary carbon (trimethyl) TMABBr Quaternary carbon (triethyl) TMABBr Benzene TMABBr Indolium ring TMABBr Double bonded oxygen TMABBr Hydroxyl TMABTable 4E. Compounds with Chemical Structure I where X=Br, R= Propyl benzene X Y RBr Methyl Propyl benzeneBr Ethyl Propyl benzeneBr Propyl Propyl benzeneBr Amino Propyl benzeneBr Dimethylamino Propyl benzeneBr Diethylamino Propyl benzeneBr Dipropylamino Propyl benzeneBr Methylamino Propyl benzeneBr Ethylamino Propyl benzeneBr Propylamino Propyl benzeneBr Sulfur trioxide Propyl benzeneBr Quaternary carbon (trimethyl) Propyl benzeneBr Quaternary carbon (triethyl) Propyl benzeneBr Benzene Propyl benzeneBr Indolium ring Propyl benzeneBr Double bonded oxygen Propyl benzeneBr Hydroxyl Propyl benzene504936-0208-1406, v. 1Table 4F, Compounds with Chemical Structure I where X=Br, R= Propyl sulfur trioxide X Y RBr Methyl Propyl sulfur trioxideBr Ethyl Propyl sulfur trioxideBr Propyl Propyl sulfur trioxideBr Amino Propyl sulfur trioxideBr Dimethylamino Propyl sulfur trioxideBr Diethylamino Propyl sulfur trioxideBr Dipropylamino Propyl sulfur trioxideBr Methylamino Propyl sulfur trioxideBr Ethylamino Propyl sulfur trioxideBr Propylamino Propyl sulfur trioxideBr Sulfur trioxide Propyl sulfur trioxideBr Quaternary carbon (trimethyl) Propyl sulfur trioxideBr Quaternary carbon (triethyl) Propyl sulfur trioxideBr Benzene Propyl sulfur trioxideBr Indolium ring Propyl sulfur trioxideBr Double bonded oxygen Propyl sulfur trioxideBr Hydroxyl Propyl sulfur trioxideTable 4G. Compounds with Chemical Structure I where X=Br, R= BenzeneX Y RBr Methyl BenzeneBr Ethyl BenzeneBr Propyl BenzeneBr Amino BenzeneBr Dimethylamino BenzeneBr Diethylamino BenzeneBr Dipropylamino BenzeneBr Methylamino BenzeneBr Ethylamino BenzeneBr Propylamino BenzeneBr Sulfur trioxide BenzeneBr Quaternary carbon (trimethyl) BenzeneBr Quaternary carbon (triethyl) BenzeneBr Benzene BenzeneBr Indolium ring BenzeneBr Double bonded oxygen BenzeneBr Hydroxyl Benzene514936-0208-1406, v. 1Table 5 A, Compounds with Chemical Structure I where X=I, R=Methyl X Y R I Methyl MethylI Ethyl MethylI Propyl MethylI Amino MethylI Dimethylamino MethylI Diethylamino MethylI Dipropylamino MethylI Methylamino MethylI Ethylamino MethylI Propylamino MethylI Sulfur trioxide MethylI Quaternary carbon (trimethyl) MethylI Quaternary carbon (triethyl) MethylI Benzene MethylI Indolium ring MethylI Double bonded oxygen MethylI Hydroxyl MethylTable 5B, Compounds with Chemical Structure I where X=I, R=Ethyl X Y R I Methyl EthylI Ethyl EthylI Propyl EthylI Dimethylamino EthylI Amino EthylI Diethylamino EthylI Dipropylamino EthylI Methylamino EthylI Ethylamino EthylI Propylamino EthylI Sulfur trioxide EthylI Quaternary carbon (trimethyl) EthylI Quaternary carbon (triethyl) EthylI Benzene EthylI Indolium ring EthylI Double bonded oxygen EthylI Hydroxyl Ethyl524936-0208-1406, v. 1Table 5C. Compounds with Chemical Structure I where X=I, R=Propyl X Y R I Methyl PropylI Ethyl PropylI Propyl PropylI Amino PropylI Dimethylamino PropylI Diethylamino PropylI Dipropylamino PropylI Methylamino PropylI Ethylamino PropylI Propylamino PropylI Sulfur trioxide PropylI Quaternary carbon (trimethyl) PropylI Quaternary carbon (triethyl) PropylI Benzene PropylI Indolium ring PropylI Double bonded oxygen PropylI Hydroxyl PropylTable 5D. Compounds with Chemical Structure I where X=I, R=TMAB X Y R I Methyl TMABI Ethyl TMABI Propyl TMABI Amino TMABI Dimethylamino TMABI Diethylamino TMABI Dipropylamino TMABI Methylamino TMABI Ethylamino TMABI Propylamino TMABI Sulfur trioxide TMABI Quaternary carbon (trimethyl) TMABI Quaternary carbon (triethyl) TMABI Benzene TMABI Indolium ring TMABI Double bonded oxygen TMABI Hydroxyl TMAB534936-0208-1406, v. 1Table 5E, Compounds with Chemical Structure I where X=I, R= Propyl benzene X Y R I Methyl Propyl benzeneI Ethyl Propyl benzeneI Propyl Propyl benzeneI Amino Propyl benzeneI Dimethylamino Propyl benzeneI Diethylamino Propyl benzeneI Dipropylamino Propyl benzeneI Methylamino Propyl benzeneI Ethylamino Propyl benzeneI Propylamino Propyl benzeneI Sulfur trioxide Propyl benzeneI Quaternary carbon (trimethyl) Propyl benzeneI Quaternary carbon (triethyl) Propyl benzeneI Benzene Propyl benzeneI Indolium ring Propyl benzeneI Double bonded oxygen Propyl benzeneI Hydroxyl Propyl benzeneTable 5F, Compounds with Chemical Structure I where X=I, R= Propyl sulfur trioxide X Y R I Methyl Propyl sulfur trioxideI Ethyl Propyl sulfur trioxideI Propyl Propyl sulfur trioxideI Amino Propyl sulfur trioxideI Dimethylamino Propyl sulfur trioxideI Diethylamino Propyl sulfur trioxideI Dipropylamino Propyl sulfur trioxideI Methylamino Propyl sulfur trioxideI Ethylamino Propyl sulfur trioxideI Propylamino Propyl sulfur trioxideI Sulfur trioxide Propyl sulfur trioxideI Quaternary carbon (trimethyl) Propyl sulfur trioxideI Quaternary carbon (triethyl) Propyl sulfur trioxideI Benzene Propyl sulfur trioxideI Indolium ring Propyl sulfur trioxideI Double bonded oxygen Propyl sulfur trioxideI Hydroxyl Propyl sulfur trioxide544936-0208-1406, v. 1Table 5G. Compounds with Chemical Structure I where X=I, R= BenzeneX Y R I Methyl BenzeneI Ethyl BenzeneI Propyl BenzeneI Amino BenzeneI Dimethylamino BenzeneI Diethylamino BenzeneI Dipropylamino BenzeneI Methylamino BenzeneI Ethylamino BenzeneI Propylamino BenzeneI Sulfur trioxide BenzeneI Quaternary carbon (trimethyl) BenzeneI Quaternary carbon (triethyl) BenzeneI Benzene BenzeneI Indolium ring BenzeneI Double bonded oxygen BenzeneI Hydroxyl BenzeneEXPERIMENTAL[000146] Having generally described particular embodiments of HC compounds, a fuller understanding of the presently disclosed technology can be obtained by reference to certain specific examples which are provided below for purposes of illustration only and are not intended to be limiting of the inventive concepts described herein. Several embodiments of the above-listed HC compounds of the present disclosure were synthesized, analyzed, and tested as described below.Materials and MethodsChemicals[000147] Chemicals used are ACS or HPLC grade, purchased from Sigma Aldrich (Saint Louis, MO), Thermo Fisher Scientific and TCI America (Waltham, MA).1H-NMR (400 MHz) and13C-NMR (100 MHz) spectra were recorded using a Bruker Avance spectrometer with DMSO-d6 (Cambridge Isotope Laboratories, Andover, MA) and CDCl3 (Sigma-Aldrich, Burlington, MA) containing TMS as a calibration standard. The melting points were measured with open capillary tubes and Thomas Hoover apparatus. The UV-vis absorptions and fluorescence emission were measured using a Varian Cary 50 spectrophotometer (Santa Clara, CA) and Shimadzu RF-5301 PC spectrofluorometer, respectively. VWR disposable554936-0208-1406, v. 1polystyrene cuvettes with path length 1 cm were utilized to dissolve the dye in solvents for measurement. The quantum yields of dyes were measured according to the reported method with reference to ICG.Compound synthesisSynthetic procedure of heptamethine cyanine dyes (9 and 10)[000148] Heptamethine cyanine dyes 9 and 10 were synthesized The heptamethine cyanine dyes were formed by the condensation of substituted indolium salt and the Vilsmeier Haack linker. The indolium salt (2 mol), substituted linker 3 (2 mol), and NaOAc (3 mol) were added into a round bottomed flask and dissolved with 5 mL of acetic anhydride. The reaction was refluxed for 2-4 h at 70 °C. The reaction was monitored by UV spectrophotometer. After completion, the mixture was cooled down and precipitated with ether to crystallize. The solid was filtered and recrystallized with methanol and ethyl acetate (10:100). The products 9 and 10 yielded green solids.Synthetic procedure for HC compounds 11A and 11B[000149] The addition of resorcinol to heptamethine dye under basic conditions allowed the retro-Knoevenagel to occur. The resorcinol (2.5 mmol) was dissolved in acetonitrile (3 mL) under nitrogen atmosphere. TEA (2.5 mmol) was added to the mixture and stirred for 15 min. Heptamethine cyanine dye (9 or 10, 1 mol) was added to the mixture. The reaction was refluxed at 80 °C for 2-4 h. The reaction was monitored with UV. After completion, the mixture was reduced and purify with silica gel column chromatography using DCM-MeOH (90:10). The fractions were collected and dried with the vacuum. The solid was recrystallized and filtered. The product yielded dark blue solids.Synthetic procedure for HC compounds 12A and 12B[000150] 3-nitro phenol (3 mmol) and K2CO3 (3 mmol) were dissolved in acetonitrile (3 mL) by stirring for 15 mins at room temperature under nitrogen atmosphere. Then, the heptamethine cyanine dyes (9 or 10, 1 mmol) dissolved in acetonitrile were added to the reaction flask and refluxed at 80 °C for 12 hours. The reaction monitored by UV-vis spectroscopy and upon completion, the solvent was evaporated. To reduce the dye, it was dissolved in 20 mL MeOH and SnCl2 (10 mmol) in diluted HC1 solution (2 mL) was added. The mixture refluxed at 80 °C overnight. The solution was neutralized by NaHCO3 and filtered. The filtrate was collected by564936-0208-1406, v. 1washing several times with DCM and solvent evaporated. The solid was purified by silica gel column chromatography using DCM: MeOH (95:5) and recrystallized with MeOH: EtOAc (5:95) to yield green solids.Synthetic procedure for HC compounds 13A, 13B, 14A, and 14B[000151] The 3-(dimethylamino) phenol for 13a and 13b, and 3-(dimethylamino) phenol for 14a and 14b, (2.5 mmol) was dissolved in acetonitrile (3 mL). TEA (2.5 mol) was added to the mixture and stirred for 15 min. Heptamethine cyanine dye (1 mmol) was also added to the mixture. The reaction was refluxed at 80°C for 4 h. The reaction was monitored with UV. After completion, the mixture was reduced and purified with silica gel column chromatography using DCM-MeOH (90:10). The fractions were collected and dried under vacuum. The solid was recrystallized and filtered. The product yielded dark blue solids.[000152] Examples of the X, Y, and R side groups of compounds 11A-14B of the present disclosure are shown in Table 6.Table 6. X, Y, and R of Compounds 11A-14B according to Chemical Structure I.Compound No. X Y R11A H OH ethyl11B Br OH ethyl12A H NH2ethyl12B Br NH2ethyl13A H dimethylamino ethyl13B Br dimethylamino ethyl14A H diethylamino ethyl14B Br diethylamino ethylCompound characterizationMolecular properties[000153] Molecular properties of HC compounds were calculated with ChemDoodle Web Components.574936-0208-1406, v. 1Absorbance[000154] Following compound synthesis, each were diluted in EtOH (50%) to a dilution of 1 mM. Compounds were further diluted to lesser concentrations of 100 pM, 10 pM, and 1 pM. The absorbance of each of the dilutions were measured and recorded using a UV-vis Varian Cary 50 spectrophotometer.Optoacoustic spectra acquisition[000155] Dilutions that were utilized in absorbance measurements were similarly used for optoacoustic signal quantification. 200 pL of solutions noted above were loaded into 0.5 mL microcentrifuge tubes and encapsulation within agar-based tissue mimicking phantoms. Samples were quantified using an iThera Medical InVision 512 TF MSOT. Samples were irradiated with a pulsed, tunable laser source at wavelengths at 10 nm intervals between 680 nm and 900 nm to measure optoacoustic signal as a function of wavelength of irradiated light. Single-wavelength measurements at 710, 730, 740, 810, and 830 nm were also acquired.Evaluation of solvent effects[000156] To evaluate the effects of solvent polarity on the stabilization of the TICT state, multiple solvents with varying polarities were used to solvate compounds prior to absorbance and optoacoustic signal quantification. Solvents used and respective dielectric constants include: DEE - 4.3; Acetic Acid - 6; DCM - 9.1; Acetone - 18; EtOH (50%) - 45. As compounds varied in solubility depending on solvents, spectra were normalized to allow for comparison of spectral shape rather than magnitude. In addition, DEE, DCM, and acetone were unable to produce stable absorbance measurements, and therefore were included only in optoacoustic measurements.Evaluation of metal sensitivity[000157] To evaluate if amino lone pairs results in varied optoacoustic / absorbance spectra, multiple concentrations (1:1, 10:1) of copper, zinc, iron, and sodium were added to 1 mM solutions of compounds. Absorbance and optoacoustic spectra were recorded with each sample.Cell lines[000158] Human kidney cells (293), human liver cells (HEP3B), human lung cells (A427), and HUVEC were acquired from American Type Culture Collection. All cells were grown in584936-0208-1406, v. 1standard DMEM supplemented with 10% FBS and 1% L-glutamine and incubated at 37 °C with 5% CO2.Cellular viability[000159] Cell lines were plated at 5000 cells per well in a 96 well plate and incubated for 24 h to allow for adherence. Cells were treated with 2.5 pF compounds, as well as methylene blue and indocyanine green controls at 200 pg / mE and 20 pg / mL. 24 h post treatment ATP levels were measured via ATPlite™ to evaluate cellular viability. To avoid potential interactions with other dye-based viability assays (MTT and MTS assays) ATPLite™ was utilized.Murine model[000160] Female athymic nude mice aged 4 weeks were used for this study. Without hair, athymic mice are preferred for optoacoustic imaging due to a smooth connection between skin and ultrasonic transducers. Immunocompetent mice with hair would require shaving / hair removal to eliminate the possibility of obscured signal. All experiments outlined herein are in strict adherence to the Oklahoma University Health Science Center Animal Care and Use Committee approved protocol (IACUC 302166). Mice were fed a special diet (2920 X alfalfa free feed and ultrapure water) to reduce background in vivo signal.In vivo compound characterization[000161] Mice were anesthetized with isoflurane and the abdomen was cleaned with betadine. An incision of 1 cm was made in the upper left quadrant of the abdomen. Using forceps, the spleen was retracted to expose the pancreas. 1 mM of the HC compounds were diluted to 100 pM in sterilized ultrasound gel, and 100 pF of diluted compounds were injected into a secluded region of the pancreas with a 25-gauge needle. Injected compounds were deemed successful if the injection site and compounds within could be visually identified. The organs were returned to the abdomen and the incisions were closed using 5-0 prolene sutures.[000162] Mice were placed into the InVision 512 TF MSOT (iThera Medical, Munich, Germany) ventral side up and delivered constant anesthesia (1.5% isoflurane in 0.8 U / min medical air and 0.2 U / min oxygen). Serial slices of the abdomen were acquired at axial slices of 0.1 mm at intervals of 10 nm from 700 nm to 900 nm. 20 acquisitions (10 ps each) of each wavelength were recorded at each axial slice to minimize breathing artifacts. In addition, single wavelength signals were acquired at 710, 730, 740, 810, and 830 nm at a single slice with594936-0208-1406, v. 1maximum compound presence. Through direct video feed of the animal, and through observation of breathing according to optoacoustic imaging, signs of distress were constantly monitored throughout optoacoustic imaging.[000163] Following optoacoustic imaging, mice were imaged using near-infrared fluorescence (AMI, Spectral Imaging Instruments, Tucson, AZ, USA). Mice were anesthetized as previously mentioned and imaged using multiple excitation and emission filters between 640 nm and 850 nm at 5 s of exposure for each image to confirm the presence of compounds.Ex vivo organ analysis[000164] Following in vivo imaging, mice were euthanized by isoflurane overdose and cervical dislocation. Vital organs including the pancreas, liver, kidney, spleen, heart, and lung were excised and evaluated using NIR fluorescence to confirm the presence or absence of compounds in relationship to in vivo NIR fluorescence and optoacoustic imaging. Organs were also fixed in formalin and H& E stained to assess toxicity.Statistics[000165] Differences in optoacoustic signal of compounds were evaluated with ANOVA and post hoc comparisons. Tukey and Bonferroni corrections were utilized to adjust p-values due to multiple comparisons. Significance in this context: (* - p<0.08, ** - p<0.01, *** - p<0.001).Results and DiscussionDye synthesis[000166] The development of unique optoacoustic contrast agents is a necessity for advancement of optoacoustic imaging as a clinically relevant imaging modality. HC dyes have shown favorable features as both fluorescence and optoacoustic imaging agents due to strong absorbance in the near-infrared window, and ease of tunability. Therefore, an HC scaffold was derivatized with high polarity functional groups for the purposes of causing a TICT state.[000167] Heptamethine compounds 9 and 10 were synthesized by condensation of substituted indolium salt and the Vilsmeier Haack linker. Indolium salt, linker 3, and NaOAc were dissolved with acetic anhydride, and refluxed, cooled, and filtered. From compounds 9 and 10, compounds 11A-14B had slightly different synthesis procedures. For compounds 11A and 11B, resorcinol was added to TEA and compound 9 or 10. Compounds 11A and 11B were yielded following reflux and purification. 3-nitro phenol and K2CO3 were added to compound604936-0208-1406, v. 19 and 10 to yield compounds 12A and 12B. Dimethylamino phenol or diethylamino phenol were added with TEA and compounds 9 and 10 to yield compound 13A and compound 13B or compound 14A and compound 14B.Compound characterization[000168] Molecular properties of the various synthesized HC compounds are outlined in Table 7. Following compound synthesis and purification, each molecule was characterized in terms of molecular properties.Table 7. Molecular properties ofHC compounds 11A-14BMolecule Rotatable Polarizability | Refractivity Polar Surface VolumeBonds (A3) | (cm3 / mol) Area (A2) (A3) LogP11A 3 47.28 | 124.39 32.47 348.93 4.8911B 3 49.75 | 132.16 32.47 369.88 5.6912A 3 47.82 I 126.54 38.26 349.48 4.4812B 3 50.26 | 134.31 38.26 370.42 5.2713A 4 51.51 | 135.82 15.48 377.42 5.5013B 4 53.94 | 143.59 15.48 398.36 6.3014A 6 55.20 I 145.09 15.48 405.36 6.9014B 6 57.61 | 152.86 15.48 426.30 7.69[000169] Absorbance measurements of compounds 11A-14B reveal dual absorbance peaks for each molecule, which are relatively consistent between the A and B versions of each compound (FIGS. 1A-1H). Compounds 11A-B show absorbance peaks at approximately 610 nm and 660 nm, 12A-B at about 632 nm and 691 nm, 13A-B at about 665 nm and 725 nm, and 14A-B at approximately 666 nm and 718 nm. Overall, dimethyl amino and diethyl amino functionalized HCs (Compounds 13A-B and 14A-B) exhibit very similar dual absorbance peaks, while compounds 11 and 12 are less red-shifted.[000170] Subsequent to absorbance measurements, optoacoustic signals were measured for each of compounds 11A-14B (FIGS. 2A-2H). Optoacoustic spectra often correspond to absorbance measurements, due to immediate relaxation of excited molecules, and thus, generation of ultrasonic waves through non-radiative decay. However, several compounds exhibit red-shifted optoacoustic signal compared to absorbance. To ensure the phenomenon was not occurring due to aggregation, multiple wavelengths were used to confirm. Eargely, concentration of HCs resulted in only optoacoustic intensity differences, and not spectral shifts.614936-0208-1406, v. 1Compound 13A was the sole exception to this observation, and shows a maximum peak shift due to formations of J-aggregates.[000171] Stabilization by polar solvent is required to observe molecule effects while in a TICT state. In addition to ensuring metal sensing through nitrogen lone pair interactions were not responsible for red-shifted optoacoustic signal, compounds 11A-14B were measured in solvents of varying dielectric constants, thus, polarity (FIGS. 3A-3H). Compounds 11 and 12 had insignificant changes in terms of spectral shifts dependent on solvent polarity. However, compounds 13 and 14 exhibited large differences in spectral signature. Notably, high polarity solvents (50% EtOH, 45 dielectric constant) in compound 14 result in red-shifted optoacoustic signal in which a maximum peak of approximately 810 nm was observed. As polarity of solvents were lowered, optoacoustic spectral signatures became blue-shifted; acetone (18 dielectric constant) peaked at 785 nm and dichloromethane (9.1 dielectric constant), acetic acid (6 dielectric constant), and diethyl ether (4.3 dielectric constant) each peaked at 735 nm. FIGS.1A-1H indicated no changes in absorbance were observed between 50% EtOH and acetic acid, indicating the shift in optoacoustic signal was unable to be explained by solvent-driven absorbance shifts. Further, single-wavelength measurements were acquired for each compound and solvent to eliminate the possibility of delayed non-radiative responses from previous irradiation. Through stabilization of the TICT state with a high polarity solvent, non-radiative decay of compounds 13 and 14 result in large red-shifts in optoacoustic signal; potentially establishing each of the compounds as a relevant contrast agent for optoacoustic imaging in vivo.Cellular viability[000172] Prior to evaluation of the synthesized HC compounds in vivo, cellular viability was evaluated in vitro using human cell lines from kidney, liver, lung, and umbilical vein. Cells were treated with each HC compound (11A-14B) in addition to standard, commercial control dyes, methylene blue and indocyanine green. 24 h post-treatment, cells were evaluated for viability using ATPLite. FIGS. 4A-4H show that at both 200 pg / mL and 20 pg / mL concentrations, compounds 11A-14B had little impact of the viability of cell lines utilized. While umbilical vein cells are often utilized in viability assays due to results being transferable to other endothelial cell types, kidney, liver, and lung cells are major locations of drug accumulation following intravenous injection. The results of the viability assay depicted in624936-0208-1406, v. 1FIGS. 4A-4H support the use of HC derivative compounds as useful in vivo contrast agents for optoacoustic imaging.In vivo imaging[000173] In vivo evaluation of the synthesized HC compounds was determined with MSOT following injection of each HC compound into the pancreas of the mouse. Each animal received approximately 100 pF of 100 pM compounds that were mixed with sterilized ultrasound gel to increase structural stability and reduce chance of leakage. Results were consistent with prior findings; as compound 14B exhibited increased signal at aforementioned, red-shifted wavelengths as signals were spectrally unmixed utilizing the reference spectra corresponding to each compound (FIGS. 5A-5B). With an average signal of 0.737 a.u., compound 11A showed significantly more signal than each other the other compounds (11B - 0.307 a.u., p<0.01; 12A - 0.187 a.u„ pcO.001; 12B - 0.095 a.u„ pcO.001; 13A- 0.078 a.u„ pcO.001; 13B -0.097 a.u., p<0.001; 14A-0.061, p<0.001; and 14B -0.410, p<0.08). In addition, compound 14B showed significantly more signal than compounds 12B, 13A, 13B, and 14A (all p<0.05). Of note, in vivo data inherently compared signal amplitude, rather than emphasizing red-shifted signal observed with diethyl and dimethyl amino groups. While not a perfect comparison, all compounds were also quantified using the spectra of compound 14B to specifically evaluate red-shifted signal portrayed in compounds 13 and 14. These data confirm that compound 14B provides the most optoacoustic signal generation at wavelengths deeper into the NIR window. This may be due in part to HC compounds 13 and 14 being capable of reaching the TICT state, thus occluding typical optoacoustic signal generation at absorbance wavelengths.[000174] Following optoacoustic imaging, whole-body NIR fluorescence was used to confirm presence of compounds in the pancreas of the animals (FIGS. 5C-5D). Differences in fluorescence signal were revealed, however, such differences were inconsistent compared to optoacoustic signal. For example, compounds 12A and 12B, which were among the lowest optoacoustic signal, were significantly higher in terms of fluorescence signal than other compounds. Specifically, compound 12A (1.61xl09counts) was significantly higher than all other compounds excluding compound 12B (1.41xl09counts) (p<0.001). Further, compound 12B is also significantly higher than all other compounds excluding 12A and 14B (8.96xl08counts) (p<0.001). Direct comparison between optoacoustic and fluorescence values may be nuanced and difficult, however, there appears to be an inverse trend, which is consistent with TICT state theory and potential pathways of energy dissipalion following excitation. Data634936-0208-1406, v. 1exhibited herein support the use of TICT state to increase optoacoustic signal generation at higher wavelength for creation and optimization of unique optoacoustic contrast agents.Ex vivo confirmation[000175] Subsequent to in vivo evaluation of the synthesized HC compounds, organs were excised and imaged with NIR fluorescence to further confirm presence of compounds in the pancreas exclusively. As expected, fluorescence signal from the pancreas of animals treated with compounds 11A-14B were significantly higher than all other organs tested (kidney, liver, spleen, lung, heart) (p<0.001) (FIGS. 6A-6B).[000176] Following full in vivo and ex vivo experimentation, portions of harvested organs (pancreas, spleen, liver, kidney, heart, and lung) were fixed in formalin and standard H& E stained. Histological e valuation showed that the HC compounds were non-toxic to all animals. No morphological changes were observed, indicating no tissue damage.Conclusions[000177] The present work focused on developing functionalized HC derivatives for use as unique optoacoustic contrast agents. MSOT is a developing modality that has shown high potential in a clinical setting due to intrinsic advantages over many optical-based imaging modality such as increased imaging depth. However, a lack of unique contrast agents has limited MSOT. The HC compounds disclosed herein exploit TICT to shift optoacoustic signal generation to a unique wavelength range, given an appropriate solvent polarity. Data was validated in vivo in the pancreas of a murine model. Utilization of the TICT state thus can be incorporated into optoacoustic contrast agents for use in optoacoustic imaging applications in clinical settings.[000178] While the present disclosure has been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the present disclosure be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications, and equivalents are included within the scope of the present disclosure as defined herein. Thus the embodiments described above, which include particular embodiments, will serve to illustrate the practice of the inventive concepts of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments only and are presented in the cause of providing what is believed to be the most useful and readily644936-0208-1406, v. 1understood description of methods and procedures as well as of the principles and conceptual aspects of the present disclosure. Changes may be made in the formulations of the various compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the present disclosure. Further, while various embodiments of the present disclosure have been described in exemplary claims herein below, it is not intended that the present disclosure be limited to these particular exemplary claims.654936-0208-1406, v. 1

Claims

What is claimed is:

1. A hemicyanine-based compound, comprising chemical structure I:wherein:X is selected from the group consisting of H, F, Cl, Br, and I;Y is selected from the group consisting of methyl (CH3-), ethyl (CH3CH2-), propyl (CH3CH2CH2-), amino (NH2-), methylamino (CH3NH-), ethylamino (CH3CH2NH-), propylamino (CH3CH2CH2NH-), dimethylamino ((CH₃)₂N-), diethylamino ((CH₃CH₂)₂N-), dipropylamino ((CH₃CH₂CH₂)₂N-), sulfur trioxide (-SO, ), quaternary carbon-trimethyl ((CH₃)₃C-), quaternary carbon-triethyl ((CH₃CH₂)₃C-), benzene (CgHs-), indolium ring, oxygen (double bonded, O=), and hydroxyl (OH); andR is selected from the group consisting of methyl, ethyl, propyl, trimethylpropylammonium bromide (TMAB), benzene, propyl benzene, and propyl sulfur trioxide.. The hemicyanine-based compound of claim 1, wherein X is H.

3. The hemicyanine-based compound of claim 1, wherein X is F.

4. The hemicyanine-based compound of claim 1, wherein X is Cl.

5. The hemicyanine-based compound of claim 1, wherein X is Br.664936-0208-1406, v.

16. The hemicyanine -based compound of claim 1, wherein X is I.

7. The hemicyanine -based compound of any one of claims 1-6, wherein R is methyl.

8. The hemicyanine -based compound of any one of claims 1-6, wherein R is ethyl.

9. The hemicyanine -based compound of any one of claims 1-6, wherein R is propyl.

10. The hemicyanine -based compound of any one of claims 1-6, wherein R is TMAB.

11. The hemicyanine-based compound of any one of claims 1-6, wherein R is benzene.

12. The hemicyanine-based compound of any one of claims 1-6, wherein R is propyl benzene.

13. The hemicyanine -based compound of any one of claims 1-6, wherein R is propyl sulfur trioxide.

14. The hemicyanine -based compound of claim 1, wherein X, Y, and R, respectively, are selected from the group consisting of (i) H, OH, and ethyl, and (ii) Br, OH, and ethyl.

15. The hemicyanine -based compound of claim 1, wherein X, Y, and R, respectively, are selected from the group consisting of (i) H, NH2, and ethyl, and (ii) Br, NH2, and ethyl.

16. The hemicyanine -based compound of claim 1, wherein X, Y, and R, respectively, are selected from the group consisting of (i) H, dimethylamino, and ethyl, and (ii) Br, dimethylamino, and ethyl.

17. The hemicyanine -based compound of claim 1, wherein X, Y, and R, respectively, are selected from the group consisting of (i) H, diethylamino, and ethyl, and (ii) Br, diethylamino, and ethyl.674936-0208-1406, v.

118. The hemicyanine -based compound of any one of claims 1-17, further comprising a targeting agent conjugated thereto.

19. The hemicyanine-based compound of claim 18, wherein the targeting agent is linked directly or indirectly via a linker to the hemicyanine-based compound.

20. The hemicyanine-based compound of claim 19, further comprising a therapeutic agent conjugated to the targeting agent or to the hemicyanine-based compound.

21. A liposome, comprising the hemicyanine-based compound of any one of claims 1-20.

22. The hemicyanine-based compound of any one of claims 1-20 for use in optoacoustically imaging a cell or tissue in a subject to which the hemicyanine-based compound is administered.

23. A method of optoacoustically imaging a cell or tissue in vitro or ex vivo, comprising administering to the cell or tissue the hemicyanine-based compound of any one of claims 1-20, irradiating the hemicyanine-based compound, and measuring a signal emitted from the hemicyanine-based compound.684936-0208-1406, v. 1

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