Patent Application
20230382934
Imaging-related medications or contrast agents are commonly utilized to improve the visualization of radiographic, computed tomography (CT), and magnetic resonance (MRI) images. Such contrast agents contain molecules that have high X-ray attenuation. For instance, iodine and barium-based contrast agents are used in medical applications owing to the high atomic number and high X-ray attenuation properties of iodine. These agents are essential to accurate diagnoses and are nearly always safe and effective when administered correctly.
Reactive oxygen species (ROS), including singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radical, have long been subjects of research due to their central role in cell signaling, the aging process, and the pathogenesis of a variety of diseases such as cardiovascular conditions, diabetes, cancer, Alzheimer's and Parkinson's diseases. However, ROS's high reactivity and short lifetime make them very challenging to detect with current magnetic resonance imaging (MRI) contrasting agents (Tain, R. W. et al., 2018, J. Magn. Reson. Imaging, 47:222).
There are several Food and Drug Administration (FDA)-approved contrast agents for nuclear imaging and diagnosis of disease. Magnetic resonance contrast agents, particularly gadolinium-based agents, are incredibly safe and lack the nephrotoxicity of iodinated contrast media. Seven gadolinium agents are available in one or more countries, of which five are available for clinical use in the United States (Gadopiclenol, Magnevist ProHance, Omniscan, & Optimark). (Hall, J. 2022, Diagnostic Imaging, www.diagnosticimaging.com/view/fda-approves-new-mri-contrast-agent-gadopiclenol; Pierre, V. C. et al., 2014, J. Biol. Inorg. Chem., 19:127) Several methods have been developed for ROS measurement, including optical redox scanning, exogenous contrast magnetic resonance (MR), Overhauser-enhanced MRI, proton-electron double resonance, and obtaining contrast from irradiated radicals. Still, they require exogenous reagents to trap such radicals. Other emerging MRI methods rely on exogenous contrast agents with paramagnetic metallic ions. An ideal ROS imaging should be endogenous and able to determine changes in vivo with high spatial resolution and sensitivity.
The over-production of ROS, such as H2O2 and O2− has been implicated in a wide array of pathologies, including a host of neurological and cardiovascular health conditions (Tretter, L. et al., 2004, Neurochem. Res., 29:569; Roberts, C. K. et al., 2009, Life Sci., 84, 705; Mosley, R. L. et al., 2006, Clin. Neurosci. Res., 6:261; Fearon, I. M. et al., 2009, J. Mol. Cell. Cardiol., 47:372, Eskici, G. et al., 2012, Biochemistry, 51:6289). Given the possible roles of ROS in disease, molecular probes that can be used to non-invasively monitor ROS concentrations in physiological environments are of increased interest. Such sensors could potentially distinguish pathologies with similar clinical symptoms and better inform preventative and ameliorative therapies. To this end, a series of complexes that respond to H2O2 with changes in their T1-weighted relaxivity (r1) have already been reported (Yu, M. et al., 2014, J. Am. Chem. Soc., 136:12836; Yu, M. et al., 2012, Inorg. Chem., 51:9153; Yu, M. et al., 2017, Inorg. Chem., 56:2812; Hutchinson, T. E. et al., 2019, Inorg. Chim. Acta, 496:119045).
Furthermore, although ROS such as H2O2 and O2− have been found to serve beneficial roles in biology, the over-accumulation of these species is often observed in a wide array of pathologies, including several rightly feared cardiovascular and neurological disorders (Laforge, M. et al., 2020, Nat. Rev. Immunol., 20:515; Roberts, C. K. et al., 2009, Life Sci., 84:705; Ahmed, M. I. et al., 2010, J. Am. Coll. Cardiol., 55:671; Sies, H., 2014, J. Biol. Chem., 289:8735). The involvement of ROS in disease has motivated the development of methods to detect ROS in biological settings (Chang, M. C. Y. et al., 2004, J. Am. Chem. Soc., 126:15392; Ekanger, L. A. et al., 2014, Chem. Commun., 50:14835; Lippert, A. R. et al., 2011, Acc. Chem. Res. 44:793; Lou, Z. et al., 2015, Acc. Chem. Res., 48:1358; Miller, E. W. et al., 2005, J. Am. Chem. Soc., 127:16652; Song, B. et al., 2013, Dalton Trans., 42:8066; Srikun, D. et al., 2008, J. Am. Chem. Soc., 130:4596; Tsitovich, P. B. et al., 2014, J. Inorg. Biochem., 133:143; Loving, G. S. et al., 2013, J. Am. Chem. Soc., 135:4620; Wang, H. et al., 2019, J. Am. Chem. Soc., 141:5916).
Thus, there is a need in the art for improved imaging agents for ROS disease diagnosis with MRI. This invention satisfies this unmet need.
In one aspect, the present invention relates to a compound comprising a metal M and a macrocyclic ligand represented by General Formula I, and any ionic variant thereof:
In one embodiment, X1 and X3 are represented by NR1 wherein R1 is represented by Formula A. In one embodiment, R2 are each hydrogen. In one embodiment, n is 1. In one embodiment, x is 2.
In one embodiment, the metal M is selected from the group consisting of manganese, iron, cobalt, nickel, molybdenum, technetium, ruthenium, cobalt, copper, and rhodium. In one embodiment, the metal is manganese or iron.
In one embodiment, Formula A is represented by Formula B:
In one embodiment, y is at least 1 and at least one R3 represents hydroxyl. In one embodiment, the OH is coordinated to the metal M. In one embodiment, X1 and X3 are both represented by NR1 wherein R1 is represented by Formula B. In one embodiment, both OH groups are coordinated to the metal M.
In one embodiment, Formula A is represented by Formula C:
In one embodiment, X1 and X3 are both represented by NR1 wherein R1 is represented by Formula C. In one embodiment, M is further bonded to water.
In one embodiment, a contrast agent comprises the compound.
In one embodiment, the contrast agent is used in an imaging technique selected from the group consisting of magnetic resonance imaging, photoacoustic imaging, thermal imaging, photothermal imaging, and any combination thereof.
In another aspect, the present invention provides a method of enhancing magnetic resonance imaging (MRI) in a living subject, the method comprising the steps of: administering to a subject an effective amount of a composition comprising a contrast agent, and imaging the patient by MRI, wherein the MRI image is enhanced as compared to an MRI image obtained without said contrast agent, wherein the contrast agent comprises a metal M and a macrocyclic ligand represented by General Formula I, or a salt thereof:
In one embodiment, the method further comprises the step of oxidizing the contrast agent with a reactive oxygen species.
In one embodiment, the reactive oxygen species comprises H2O2.
In one embodiment, the contrast agent is administered in a concentration of 0.10 to 1.00 mM.
In one embodiment, the contrast agent is administered subcutaneously, intravenously, peritoneally, orally, intramuscular, topical, nasally, intradermally, ocularly, rectally, vaginally, or combinations thereof.
The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
The present invention is based, in part, on the unexpected discovery and results of an effective method for sensing reactive oxygen species (ROS) in clinical imaging technology comprising a composition comprising a redox-active metal complex comprising at least one metal and at least one macrocyclic ligand. In various embodiments, the composition can undergo a reaction with reactive oxygen species. In some embodiments, the chelating ligand comprises at least one aryl group substituted with a hydroxyl. In some embodiments, the chelating ligand comprises at least one para-quinone. In various embodiments, the composition is used to enhance magnetic resonance imaging (MRI) in living subjects.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
The terms “halo,” “halogen,” or “halide” as used interchangeably and refer to fluorine, chlorine, bromine, and iodine.
The term “acyl” refers to a substituted carbonyl radical (C(O)—Rs).
The term “ester” refers to a substituted oxycarbonyl (—O—C(O)—Rs or —C(O)—O—Rs) radical.
The term “ether” refers to an —ORs radical.
The terms “sulfanyl” or “thio-ether” are used interchangeably and refer to a —SRs radical.
The term “sulfinyl” refers to a —S(O)—Rs radical.
The term “sulfonyl” refers to a −SO2—Rs radical.
The term “phosphino” refers to a —P(Rs)3 radical, wherein each Rs can be same or different.
The term “silyl” refers to a —Si(Rs)3 radical, wherein each Rs can be same or different.
In each of the above, Rs can be hydrogen or a substituent selected from the group consisting of deuterium, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, arylalkyl, alkoxy, aryloxy, amino, silyl, alkenyl, cycloalkenyl, heteroalkenyl, alkynyl, aryl, heteroaryl, and combination thereof. Preferred Rs is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, and combination thereof.
The term “alkyl” refers to and includes both straight and branched chain alkyl radicals. Preferred alkyl groups are those containing from one to fifteen carbon atoms and includes methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and the like. Additionally, the alkyl group may be optionally substituted.
The term “cycloalkyl” refers to and includes monocyclic, polycyclic, and spiro alkyl radicals. Preferred cycloalkyl groups are those containing 3 to 12 ring carbon atoms and includes cyclopropyl, cyclopentyl, cyclohexyl, bicyclo[3.1.1]heptyl, spiro[4.5]decyl, spiro[5.5]undecyl, adamantyl, and the like. Additionally, the cycloalkyl group may be optionally substituted.
The terms “heteroalkyl” or “heterocycloalkyl” refer to an alkyl or a cycloalkyl radical, respectively, having at least one carbon atom replaced by a heteroatom. Optionally the at least one heteroatom is selected from O, S, N, P, B, Si and Se, preferably, O, S or N. Additionally, the heteroalkyl or heterocycloalkyl group is optionally substituted.
The term “alkenyl” refers to and includes both straight and branched chain alkene radicals. Alkenyl groups are essentially alkyl groups that include at least one carbon-carbon double bond in the alkyl chain. Cycloalkenyl groups are essentially cycloalkyl groups that include at least one carbon-carbon double bond in the cycloalkyl ring. The term “heteroalkenyl” as used herein refers to an alkenyl radical having at least one carbon atom replaced by a heteroatom. Optionally the at least one heteroatom is selected from O, S, N, P, B, Si and Se, preferably, O, S or N. Preferred alkenyl, cycloalkenyl, or heteroalkenyl groups are those containing two to fifteen carbon atoms. Additionally, the alkenyl, cycloalkenyl, or heteroalkenyl group is optionally substituted.
The term “alkynyl” refers to and includes both straight and branched chain alkyne radicals. Preferred alkynyl groups are those containing two to fifteen carbon atoms. Additionally, the alkynyl group is optionally substituted.
The terms “aralkyl” or “arylalkyl” are used interchangeably and refer to an alkyl group that is substituted with an aryl group. Additionally, the aralkyl group is optionally substituted.
The term “heterocyclic group” refers to and includes aromatic and non-aromatic cyclic radicals containing at least one heteroatom. Optionally the at least one heteroatom is selected from O, S, N, P, B, Si and Se, preferably, O, S or N. Hetero-aromatic cyclic radicals may be used interchangeably with heteroaryl. Preferred hetero-non-aromatic cyclic groups are those containing 3 to 7 ring atoms which includes at least one hetero atom, and includes cyclic amines such as morpholino, piperidino, pyrrolidino, and the like, and cyclic ethers/thio-ethers, such as tetrahydrofuran, tetrahydropyran, tetrahydrothiophene, and the like. Additionally, the heterocyclic group may be optionally substituted.
The term “aryl” refers to and includes both single-ring aromatic hydrocarbyl groups and polycyclic aromatic ring systems. The polycyclic rings may have two or more rings in which two carbons are common to two adjoining rings (the rings are “fused”) wherein at least one of the rings is an aromatic hydrocarbyl group, e.g., the other rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles, and/or heteroaryls. Preferred aryl groups are those containing six to thirty carbon atoms, preferably six to twenty carbon atoms, more preferably six to twelve carbon atoms. Especially preferred is an aryl group having six carbons, ten carbons or twelve carbons. Suitable aryl groups include phenyl, biphenyl, triphenyl, triphenylene, tetraphenylene, naphthalene, anthracene, phenalene, phenanthrene, fluorene, pyrene, chrysene, perylene, and azulene, preferably phenyl, biphenyl, triphenyl, triphenylene, fluorene, and naphthalene.
Additionally, the aryl group may be optionally substituted.
The term “heteroaryl” refers to and includes both single-ring hetero-aromatic groups and polycyclic aromatic ring systems that include at least one heteroatom. The heteroatoms include, but are not limited to O, S, N, P, B, Si and Se. In many instances, 0, S or N are the preferred heteroatoms. Hetero-single ring aromatic systems are preferably single rings with 5 or 6 ring atoms, and the ring can have from one to six heteroatoms. The hetero-polycyclic ring systems can have two or more rings in which two atoms are common to two adjoining rings (the rings are “fused”) wherein at least one of the rings is a heteroaryl, e.g., the other rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles, and/or heteroaryls. The hetero-polycyclic aromatic ring systems can have from one to six heteroatoms per ring of the polycyclic aromatic ring system. Preferred heteroaryl groups are those containing three to thirty carbon atoms, preferably three to twenty carbon atoms, more preferably three to twelve carbon atoms. Suitable heteroaryl groups include dibenzothiophene, dibenzofuran, dibenzoselenophene, furan, thiophene, benzofuran, benzothiophene, benzoselenophene, carbazole, indolocarbazole, pyridylindole, pyrrolodipyridine, pyrazole, imidazole, triazole, oxazole, thiazole, oxadiazole, oxatriazole, dioxazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, oxazine, oxathiazine, oxadiazine, indole, benzimidazole, indazole, indoxazine, benzoxazole, benzisoxazole, benzothiazole, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, naphthyridine, phthalazine, pteridine, xanthene, acridine, phenazine, phenothiazine, phenoxazine, benzofuropyridine, furodipyridine, benzothienopyridine, thienodipyridine, benzoselenophenopyridine, and selenophenodipyridine, preferably dibenzothiophene, dibenzofuran, dibenzoselenophene, carbazole, indolocarbazole, imidazole, pyridine, triazine, benzimidazole, 1,2-azaborine, 1,3-azaborine, 1,4-azaborine, borazine, and aza-analogs thereof. Additionally, the heteroaryl group may be optionally substituted.
Of the aryl and heteroaryl groups listed above, the groups of triphenylene, naphthalene, anthracene, dibenzothiophene, dibenzofuran, dibenzoselenophene, carbazole, indolocarbazole, imidazole, pyridine, pyrazine, pyrimidine, triazine, and benzimidazole, and the respective aza-analogs of each thereof are of particular interest.
The terms alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heteroalkenyl, alkynyl, aralkyl, heterocyclic group, aryl, and heteroaryl, as used herein, are independently unsubstituted or substituted with one or more general substituents.
In many instances, the general substituents are selected from the group consisting of deuterium, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, arylalkyl, alkoxy, aryloxy, amino, cyclic amino, silyl, alkenyl, cycloalkenyl, heteroalkenyl, alkynyl, aryl, heteroaryl, acyl, carbonyl, carboxylic acid, ether, ester, nitrile, isonitrile, sulfanyl, sulfinyl, sulfonyl, phosphino, and combinations thereof.
In some instances, the preferred general substituents are selected from the group consisting of deuterium, fluorine, alkyl, cycloalkyl, heteroalkyl, alkoxy, aryloxy, amino, silyl, alkenyl, cycloalkenyl, heteroalkenyl, aryl, heteroaryl, nitrile, isonitrile, sulfanyl, and combinations thereof.
In some instances, the preferred general substituents are selected from the group consisting of deuterium, fluorine, alkyl, cycloalkyl, alkoxy, aryloxy, amino, silyl, aryl, heteroaryl, sulfanyl, and combinations thereof.
In yet other instances, the more preferred general substituents are selected from the group consisting of deuterium, fluorine, alkyl, cycloalkyl, aryl, heteroaryl, and combinations thereof.
The term “substituted” refers to a substituent other than H that is bonded to the relevant position, e.g., a carbon. For example, where R1 represents mono-substituted, then one R1 must be other than H. Similarly, where R1 represents di-substituted, then two of R1 must be other than H. Similarly, where R1 is unsubstituted, R1 is hydrogen for all available positions. The maximum number of substitutions possible in a structure (for example, a particular ring or fused ring system) will depend on the number of atoms with available valencies.
As used herein, “combinations thereof” indicates that one or more members of the applicable list are combined to form a known or chemically stable arrangement that one of ordinary skill in the art can envision from the applicable list. For example, an alkyl and deuterium can be combined to form a partial or fully deuterated alkyl group; a halogen and alkyl can be combined to form a halogenated alkyl substituent; and a halogen, alkyl, and aryl can be combined to form a halogenated arylalkyl. In one instance, the term substitution includes a combination of two to four of the listed groups. In another instance, the term substitution includes a combination of two to three groups. In yet another instance, the term substitution includes a combination of two groups. Preferred combinations of substituent groups are those that contain up to fifty atoms that are not hydrogen or deuterium, or those which include up to forty atoms that are not hydrogen or deuterium, or those that include up to thirty atoms that are not hydrogen or deuterium. In many instances, a preferred combination of substituent groups will include up to twenty atoms that are not hydrogen or deuterium.
The “aza” designation in the fragments described herein, i.e. aza-dibenzofuran, aza-dibenzothiophene, etc. means that one or more of the C—H groups in the respective fragment can be replaced by a nitrogen atom, for example, and without any limitation, azatriphenylene encompasses both dibenzo[f,h]quinoxaline and dibenzo[f,h]quinoline. One of ordinary skill in the art can readily envision other nitrogen analogs of the aza-derivatives described above, and all such analogs are intended to be encompassed by the terms as set forth herein.
As used herein, “deuterium” refers to an isotope of hydrogen. Deuterated compounds can be readily prepared using methods known in the art. For example, U.S. Pat. No. 8,557,400, Patent Pub. No. WO 2006/095951, and U.S. Pat. Application Pub. No. US 2011/0037057, which are hereby incorporated by reference in their entireties, describe the making of deuterium-substituted organometallic complexes. Further reference is made to Ming Yan, et al., Tetrahedron 2015, 71, 1425-30 and Atzrodt et al., Angew. Chem. Int. Ed. (Reviews) 2007, 46, 7744-65, which are incorporated by reference in their entireties, describe the deuteration of the methylene hydrogens in benzyl amines and efficient pathways to replace aromatic ring hydrogens with deuterium, respectively.
It is to be understood that when a molecular fragment is described as being a substituent or otherwise attached to another moiety, its name may be written as if it were a fragment (e.g. phenyl, phenylene, naphthyl, dibenzofuryl) or as if it were the whole molecule (e.g. benzene, naphthalene, dibenzofuran). As used herein, these different ways of designating a substituent or attached fragment are considered to be equivalent.
The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
The term “biological tissue” as used herein refers to a collection of interconnected cells and extracellular matrix that perform a similar function or functions within an organism Examples of biological tissues include, but are not limited to, connective tissue, muscle tissue, nervous tissue (of the brain, spinal cord, and nerves), epithelial tissue, organ tissue, cancer tissue, and any combination thereof. Connective tissue includes fibrous tissue like fascia, tendon, ligaments, heart valves, bone, and cartilage. Muscle tissue includes skeletal muscle tissue, smooth muscle tissue, such as esophageal, stomach, intestinal, bronchial, uterine, urethral, bladder, and blood vessel tissue, and cardiac muscle tissue. Epithelial tissue includes simple epithelial tissue, such as alveolar epithelial tissue, blood vessel endothelial tissue, and heart mesothelial tissue, and stratified epithelial tissue.
The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
“Cancer,” as used herein, refers to the abnormal growth or division of cells. Generally, the growth and/or life span of a cancer cell exceeds, and is not coordinated with, that of the normal cells and tissues around it. Cancers may be benign, pre-malignant or malignant. Cancer occurs in a variety of cells and tissues, including, but not limited to, the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gall bladder, pancreas, etc.), respiratory system (e.g., larynx, lung, bronchus, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, genital system, (e.g., uterus, ovary, prostate, testis, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eye, nervous system (e.g., brain, etc.), head and neck, endocrine system (e.g., thyroid, etc.), soft tissues (e.g., muscle, fat, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).
As used herein, the term “diagnosis” refers to the determination of the presence of a disease or disorder. In some embodiments of the present invention, methods for making a diagnosis are provided which permit determination of the presence of a particular disease or disorder.
As used herein, “treating a disease or disorder” means reducing the severity and/or frequency with which a sign or symptom of the disease or disorder is experienced by a subject.
A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.
The term “derivative” refers to a small molecule that differs in structure from the reference molecule, but retains the essential properties of the reference molecule. A derivative may change its interaction with certain other molecules relative to the reference molecule. A derivative molecule may also include a salt, an adduct, tautomer, isomer, or other variant of the reference molecule.
The term “tautomers” are constitutional isomers of organic compounds that readily interconvert by a chemical process (tautomerization).
The term “isomers” or “stereoisomers” refer to compounds, which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
The term “effective amount” refers to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
The term “solvate” in accordance with this invention should be understood as meaning any form of the active compound in accordance with the invention in which the said compound is bonded by a non-covalent bond to another molecule (normally a polar solvent), including especially hydrates and alcoholates.
The terms “effective amount” and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
Disclosed are the components to be used to prepare the compositions of the disclosure as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions disclosed herein. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods disclosed herein.
A compound comprising a metal M and a macrocyclic ligand represented by General Formula I, and any salt thereof:
Exemplary metals M of the composition include but are not limited to titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, combinations thereof, oxides thereof, and any oxidation state thereof.
In one embodiment, the metal is high-spin. In one embodiment, the metal is low-spin. In one embodiment, the compound comprises a combination of high-spin metals and low-spin metals.
In one embodiment, the metal is redox-active. In one embodiment, the metal is redox-stable. In one embodiment, the metal undergoes oxidation in the presence of an oxidant. In one embodiment, the metal undergoes reduction in the presence of a reductant. In one embodiment, the metal does not undergo redox in the presence of an oxidant. In one embodiment, the metal does not undergo redox in the presence of a reductant.
In one embodiment, the metal is manganese. In one embodiment, the metal is iron. In one embodiment, the metal has a positive oxidation state. In one embodiment, the metal has an oxidation state of (I). In one embodiment, the metal has an oxidation state of (II). In one embodiment, the metal has an oxidation state of (III). In one embodiment, the compound has a positive formal charge. In one embodiment, the compound has a negative formal charge.
In one embodiment, the macrocyclic ligand is redox-active. In one embodiment, the macrocyclic ligand is redox-stable. In one embodiment, the macrocyclic ligand undergoes oxidation in the presence of an oxidant. In one embodiment, the macrocyclic ligand undergoes reduction in the presence of a reductant. In one embodiment, the macrocyclic ligand does not undergo redox in the presence of an oxidant. In one embodiment, the macrocyclic ligand does not undergo redox in the presence of a reductant.
In one embodiment, the composition further comprises a counterion. Exemplary counterions of the composition include but are not limited to halide, mesylate, acetate, fumarate, sulfate, maleate, citrate, tartrate, phosphate, acetate, hexafluoroantimonate, hexafluorophosphate, and triflate. In one embodiment, the counterion is triflate. In all embodiments, the number of counterions makes the overall complex neutral.
In some embodiments, the divalent linking group comprises an amide, ester, ether, thioether, carbamate, urea, amine, any alkyl chain, polyether chain, polymer chain, a cleavable bond, e.g. a bond that is unstable and/or is cleaved upon changes in certain environmental parameters (e.g., pH or redox potential) or upon exposure to certain reagents, chemicals, catalysts, or enzymes, a non-cleavable bond, any other linkage, or any combination thereof.
In one embodiment, each of X1 to X4 is NR1. In one embodiment, at least one of X1 to X4 is NR1. In one embodiment, at least two of X1 to X4 is NR1. In one embodiment, at least three of X1 to X4 is NR1.
In one embodiment, when each of X1 to X4 is NR1, at least one R1 is hydrogen. In one embodiment, when each of X1 to X4 is NR1, at least two R1 are hydrogen. In one embodiment, when each of X1 to X4 is NR1, at least one NR1 is an alkyl, which is optionally further substituted. In one embodiment, when each of X1 to X4 is NR1, at least two NR1 are an alkyl, which are optionally further substituted.
In one embodiment, each of X1 to X4 is NR1 wherein R1 is represented by Formula A. In one embodiment, at least one of X1 to X4 is NR1 wherein R1 is represented by Formula A. In one embodiment, at least two of X1 to X4 is NR1 wherein R1 is represented by Formula A. In one embodiment, at least three of X1 to X4 is NR1 wherein R1 is represented by Formula A. In one embodiment, X1 and X3 are both represented by NR1 wherein R1 is represented by Formula A. In one embodiment, X1 and X2 are both represented by NR1 wherein R1 is represented by Formula A. In one embodiment, X1 and X4 are both represented by NR1 wherein R1 is represented by Formula A. In one embodiment, X2 and X3 are both represented by NR1 wherein R1 is represented by Formula A. In one embodiment, X2 and X4 are both represented by NR1 wherein R1 is represented by Formula A. In one embodiment, X3 and X4 are both represented by NR1 wherein R1 is represented by Formula A.
In one embodiment, R1 is an aryl, which is optionally further substituted. In one embodiment, R2 is an aryl, which is optionally further substituted. In one embodiment, at least one of R1 and R2 are aryl, which are each independently further substituted.
In one embodiment, R1 is substituted with at least one hydroxyl. In one embodiment, R2 is substituted with at least one hydroxyl. In one embodiment, R1 and R2 are each substituted with at least one hydroxyl.
In one embodiment, x is 1. In one embodiment, x is 2. In one embodiment, x is 3. In one embodiment, x is 4. In one embodiment, x is 5.
In one embodiment, y is 1. In one embodiment, y is 2. In one embodiment, y is 3. In one embodiment, y is 4.
In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is 3.
In one embodiment, the compound is neutral. In one embodiment, the compound has a negative formal charge. In one embodiment, the compound has a positive formal charge.
In one embodiment, Formula A is represented by Formula B:
In one embodiment, the compound has at least one R3, wherein the at least one R3 is hydroxyl. In one embodiment, the compound R3 is hydrogen.
In one embodiment, the OH is bonded to the metal M. In one embodiment, the OH is not bonded to the metal M. In one embodiment, the OH is deprotonated.
In one embodiment, each of X1 to X4 is NR1 wherein R1 is represented by Formula B. In one embodiment, at least one of X1 to X4 is NR1 wherein R1 is represented by Formula B. In one embodiment, at least two of X1 to X4 is NR1 wherein R1 is represented by Formula B. In one embodiment, at least three of X1 to X4 is NR1 wherein R1 is represented by Formula B. In one embodiment, X1 and X3 are both represented by NR1 wherein R1 is represented by Formula B. In one embodiment, X1 and X2 are both represented by NR1 wherein R1 is represented by Formula B. In one embodiment, X1 and X4 are both represented by NR1 wherein R1 is represented by Formula B. In one embodiment, X2 and X3 are both represented by NR1 wherein R1 is represented by Formula B. In one embodiment, X2 and X4 are both represented by NR1 wherein R1 is represented by Formula B. In one embodiment, X3 and X4 are both represented by NR1 wherein R1 is represented by Formula B.
In one embodiment, at least two of X1 to X4 is NR1 wherein R1 is represented by Formula B, both OH are bonded to the metal M. In one embodiment, at least two of X1 to X4 is NR1 wherein R1 is represented by Formula B, one OH is bonded to the metal M. In one embodiment, at least two of X1 to X4 is NR1 wherein R1 is represented by Formula B, none of the OH are bonded to the metal M.
In one embodiment, the metal M is further bonded to at least one water molecule. In one embodiment, the metal M is further bonded to two water molecules.
In one embodiment, Formula A is represented by Formula C:
In one embodiment, the OH is bonded to the metal M. In one embodiment, the OH is not bonded to the metal M. In one embodiment, the OH is deprotonated.
In one embodiment, each of X1 to X4 is NR1 wherein R1 is represented by Formula C. In one embodiment, at least one of X1 to X4 is NR1 wherein R1 is represented by Formula C. In one embodiment, at least two of X1 to X4 is NR1 wherein R1 is represented by Formula C. In one embodiment, at least three of X1 to X4 is NR1 wherein R1 is represented by Formula C. In one embodiment, X1 and X3 are both represented by NR1 wherein R1 is represented by Formula C. In one embodiment, X1 and X2 are both represented by NR1 wherein R1 is represented by Formula C. In one embodiment, X1 and X4 are both represented by NR1 wherein R1 is represented by Formula C. In one embodiment, X2 and X3 are both represented by NR1 wherein R1 is represented by Formula C. In one embodiment, X2 and X4 are both represented by NR1 wherein R1 is represented by Formula C. In one embodiment, X3 and X4 are both represented by NR1 wherein R1 is represented by Formula C.
In one embodiment, the compounds of the present invention are stable to water. In one embodiment, the compounds of the present invention undergo ionization. In one embodiment, the compounds of the present invention undergo ionization in acidic conditions. In one embodiment, the compounds of the present invention undergo ionization in neutral conditions. In one embodiment, the compounds of the present invention undergo ionization in basic conditions.
In one embodiment, the compounds of the present invention undergo dissociation in acidic conditions. In one embodiment, the compounds of the present invention undergo dissociation in basic conditions. In one embodiment, the compounds of the present invention do not undergo dissociation.
In one embodiment, the compound reacts with a reactive oxygen species (ROS). Exemplary reactive oxygen species include but are not limited to peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha oxygen. In one embodiment, the compound reacts with a radical forming compound.
Imaging and/or Contrast Agents
The present invention also provides an imaging and/or contrast agent comprising a composition comprising at least one compound of the present invention. In various aspects, the imaging and/or contrast agent is a magnetic resonance imaging contrast agent, photoacoustic imaging contrast agent, ultrasound imaging contrast agent, optical imaging contrast agent, computed tomography contrast agent, thermal imaging contrast agent, nuclear imaging contrast agent, magnetomotive imaging enhancement contrast agent, fluorescence imaging contrast agent, and any combination thereof.
In various aspects, the composition of the present invention acts as an imaging and/or contrast agent for magnetic resonance imaging, continuous wave photoacoustic imaging, combined photoacoustic and ultrasound imaging, magnetomotive imaging, optical coherent tomography, computed tomography, nuclear imaging modalities, or any combination thereof.
In some embodiments, the composition is used as contrast enhancement for magnetic resonance imaging, optical imaging methods, such as optical coherence tomography, computed tomography, fluorescence imaging, and photoacoustic imaging.
In one aspect, the imaging and/or contrast agent comprises the composition of the present invention and a pharmaceutically acceptable excipient. In various embodiments, the imaging and/or contrast agent further comprises an additional organic component.
To obtain selectivity, the imaging and/or contrast agent may be passively or actively targeted to regions of diagnostic interest, such as organs, vessels, sites of disease, tumorous tissue, or a specific organism in a subject. In active targeting, the imaging and/or contrast agents may be attached to biological recognition agents to allow them to accumulate in or to be selectively retained by or to be slowly eliminated from certain parts of the body, such as specific organs, parts of organs, bodily structures and disease structures and lesions. Active targeting is defined as a modification of biodistribution using chemical groups that will associate with species present in the desired tissue or organism to effectively decrease the rate of loss of contrast agent from the specific tissue or organism.
Active targeting of an imaging and/or contrast agent can be considered as localization through modification of biodistribution of the imaging and/or contrast agent by means of a targeting domain that is attached to or incorporated into the imaging and/or contrast agent. The targeting domain can associate or bind with one or more receptor species present in the tissue or organism of diagnostic interest. This binding will effectively decrease the rate of loss of contrast agent from the specific tissue or organism of diagnostic interest. In such cases, the imaging and/or contrast agent can be modified synthetically to incorporate the targeting domain. Targeted contrast agents can localize because of binding between the ligand and the targeted receptor. Alternatively, contrast agents can distribute by passive biodistribution, i.e., by passive targeting, into diseased tissues of interest such as tumors. Thus, even without synthetic manipulation to incorporate a targeting domain that can bind to a receptor site, passively targeted contrast agents can accumulate in a diseased tissue or in specific locations in the subject, such as the liver. The present invention comprises use of an imaging and/or contrast agent that is linked to a targeting domain that has an affinity for binding to a receptor. Preferably the receptor is located on the surface of a diseased or disease-causing cell in a human or animal subject.
The imaging and/or contrast agents are formulated in a pharmaceutically acceptable excipient, such as wetting agents, buffers, disintegrants, binders, fillers, flavoring agents and liquid carrier media such as sterile water, water/ethanol etc. The imaging and/or contrast agent should be suitable for administration either by injection or inhalation or catheterization or instillation or transdermal introduction into any of the various body cavities including the alimentary canal, the vagina, the rectum, the bladder, the ureter, the urethra, the mouth, etc. For oral administration, the pH of the composition is preferably in the acid range (e.g., 2 to 7) and buffers or pH adjusting agents may be used. The contrast media may be formulated in conventional pharmaceutical administration forms, such as tablets, capsules, powders, solutions, dispersion, syrups, suppositories etc.
The present invention is further drawn to, in part, an imaging method comprising the steps of contacting a biological tissue with a composition comprising the compound of the present invention, applying energy to a biological tissue comprising the composition, and imaging the biological tissue comprising the composition.
In various embodiments, the method of imaging a biological tissue comprises application of at least one imaging technique. Exemplary imaging techniques include, but are not limited to, magnetic resonance imaging, photoacoustic imaging, ultrasound imaging, optical imaging, computed tomography, thermal imaging, nuclear imaging, magnetomotive imaging enhancement, and any combination thereof.
In some embodiments, the biological tissue is present in a mammal. In one embodiment, the biological tissue is present in mice. In one embodiment, the biological tissue is present in rats. In one embodiment, the biological tissue is present in humans.
In some embodiments, the method of imaging comprises applying energy to a biological tissue. In some embodiments, applying energy to a biological tissue comprises application of a radio frequency field. In some embodiments, applying energy to a biological tissue comprises application of a magnetic field. In some embodiments, applying energy to a biological tissue comprises irradiation of the biological tissue with a light source. In some embodiments, applying energy to a biological tissue comprises application of a magnetic field and a radio frequency field.
Contrast agents permit light absorption and sound generation in regions not otherwise possible. Contrast agents may also improve signal to noise ratio by increasing the amplitude of the sound wave. Increasing the sound wave amplitude allows an increase in the possible maximum depth of detection and thereby allows imaging of objects further below the surface of the body.
The use of contrast media provides significant amplification of the signal strength, and thus permits improved imaging. This may be achieved by selectively absorbing radiation in certain organs or healthy or diseased bodily structures or parts thereof, and/or by efficiently converting the radiation into heat, and/or by facilitating or improving heat-pressure conversion, and/or by scattering and diffusing the incident light so that it more uniformly illuminates the target organs.
Tissue of particular interest for imaging include, without limitation, tissues not shielded by bone, e.g., breast tissue, liver tissue: etc.; and blood vessels, which have been found to provide for unexpected amplification of signal. Subjects of interest for imaging include those suspected or know to have liver cancer, breast cancer, atherosclerosis, soft tissue sarcomas, and the like.
The preferred dosage of the contrast media will vary according to a number of factors, such as the administration route, the age, weight and species of the subject, but in general containing in the order of from 1 μmol/kg to 1 mmol/kg bodyweight of the contrast agent.
Administration may be parenteral (e.g., intravenously, intraarterially, intramuscularly, interstitially, subcutaneously, transdermally, or intrasternally) or into an externally voiding body cavity (e.g., the gastrointestinal tract, bladder, uterus, vagina, nose, ears or lungs), in an animate human or non-human (e.g., mammalian, reptilian or avian) body. Usually administration is accomplished by intravenous or intratumor injection.
Imaging of the desired area is performed by detection and appropriate analysis of the sound waves resulting from irradiation. Detection may be performed at the same surface of the sample as the source of incident radiation (reflection) or alternatively at another surface such as the surface diametrically opposed to the incident light, i.e., the sample's back surface (transmission). Suitable methods of detection include the use of a microphone, piezoelectric transducer, capacitance transducer, fiber-optic sensor, cMUT, or alternatively non-contact methods (see Tam, 1986, supra for a review). Techniques and equipment used in ultrasound imaging may be used.
The methods and uses described herein are especially useful for imaging blood-containing structures (e.g., blood vessels), which may be present in tumors, diseased tissue or particular organs, by the use of contrast agents with specificity for that region/structure, e.g., by use of biological recognition agents with the desired specificity.
Continuous wave radiation may be used with its amplitude or frequency modulated. When continuous wave radiation is used, the photoacoustic effects may be analyzed in the frequency domain by measuring amplitude and phase of one or several Fourier components. Alternatively, and preferably, short pulses (impulses) of radiation are employed which allow stress confinement. When pulses are used, analysis may be made in the time domain, i.e., on the basis of the time taken for the sound wave to reach the detector, thus simplifying analysis and aiding depth profiling.
Information obtained from the methods of the invention described herein can be used alone, or in combination with other information (e.g., age, family history, disease status, disease history, vital signs, blood chemistry, PSA level, Gleason score, lymph node staging, metastasis staging, expression of other gene signatures relevant to outcomes of a disease or disorder, such as cancer, etc.) from the subject or from the biological sample obtained from the subject. In some embodiments, the imaging data is combined or correlated with other data or test results that include, but are not limited to measurements or results from serologic testing methods, enzyme immunoassay (EIA), complement fixation (CF), immunodiffusion, clinical presentation, serology, radiography, histology, culture, and clinical parameters or other algorithms for developing or having a disease or disorder, such as cancer. In some embodiments, data include, but are not limited to age, ethnicity, PSA level, Gleason score, lymph node staging, metastasis staging, and other genomic data, and specific expression values of other gene signatures relevant to infection outcomes. In some embodiments, the data comprises subject information, such as medical history, travel history, and/or any relevant family history. Several serology techniques that can be used in combination with the compositions and methods of the present invention. Examples of serology techniques include, but are not limited to: ELISA, agglutination, precipitation, complement-fixation, fluorescent antibodies, and chemiluminescence.
The following are non-limiting examples of cancers that can be imaged, detected, and/or treated by the disclosed methods and compositions: acute lymphoblastic; acute myeloid leukemia; adrenocortical carcinoma; adrenocortical carcinoma, childhood; appendix cancer; basal cell carcinoma; bile duct cancer, extrahepatic; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; liposarcoma and anaplastic liposarcoma; brain stem glioma, childhood; brain tumor, adult; brain tumor, brain stem glioma, childhood; brain tumor, central nervous system atypical teratoid/rhabdoid tumor, childhood; central nervous system embryonal tumors; cerebellar astrocytoma; cerebral astrocytotna/malignant glioma; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medulloepithelioma; pineal parenchymal tumors of intermediate differentiation; supratentorial primitive neuroectodermal tumors and pineoblastoma; visual pathway and hypothalamic glioma; brain and spinal cord tumors; breast cancer; bronchial tumors; Burkitt lymphoma; carcinoid tumor; carcinoid tumor, gastrointestinal; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; central nervous system lymphoma; cerebellar astrocytoma cerebral astrocytoma/malignant glioma, childhood; cervical cancer; chordoma, childhood; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer; Ewing family of tumors; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; biliary track cancer, cholangiocarcinoma, anal cancer, neuroendocrine tumors, small bowel cancer, gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (gist); germ cell tumor, extracranial; germ cell tumor, extragonadal; germ cell tumor, ovarian; gestational trophoblastic tumor; glioma; glioma, childhood brain stem; glioma, childhood cerebral astrocytoma; glioma, childhood visual pathway and hypothalamic; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer; histiocytosis, langerhans cell; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell tumors; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, chronic lymphocytic; leukemia, chronic myelogenous; leukemia, hairy cell; lip and oral cavity cancer; liver cancer; lung cancer, non-small cell; lung cancer, small cell; lymphoma, aids-related; lymphoma, burkitt; lymphoma, cutaneous T-cell; lymphoma, non-Hodgkin lymphoma; lymphoma, primary central nervous system; macroglobulinemia, Waldenstrom; malignant fibrous histiocvtoma of bone and osteosarcoma; medulloblastoma; melanoma; melanoma, intraocular (eye); Merkel cell carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndrome, (childhood); multiple myeloma/plasma cell neoplasm; mycosis; fungoides; myelodysplastic syndromes; myelodysplastic/myeloproliferative diseases; myelogenous leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple; myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer, islet cell tumors; papillomatosis; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal parenchymal tumors of intermediate differentiation; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumor; plasma celt neoplasm/multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal pelvis and ureter, transitional cell cancer; respiratory tract carcinoma involving the nut gene on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma, ewing family of tumors; sarcoma, Kaposi; sarcoma, soft tissue; sarcoma, uterine; Sezary syndrome; skin cancer (nonmelanoma); skin cancer (melanoma); skin carcinoma, Merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma, squamous neck cancer with occult primary, metastatic; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma, cutaneous; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor, gestational; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; and Wilms tumor.
In one aspect, the amount of cells associated with cancer are used to monitor subjects undergoing treatments and therapies for a cancer, subjects who have had a cancer, and subjects who are in remission of a previously diagnosed and treated cancer. In some embodiments, the amount of cells associated with a cancer are used to select or modify treatments in subjects having a cancer, subjects who have had a cancer, and subjects who are in remission of a previously diagnosed and treated cancer.
In some embodiments, the method of treating further comprises the step of allowing the composition to accumulate in at least one cell of interest, wherein the targeting domain facilitates accumulation of the composition in the at least one cell of interest.
In some embodiments, the imaging method further comprises the step of allowing the composition to accumulate in a region of the biological tissue.
In various aspects, the composition of the present invention may be delivered to a cell or biological tissue of interest by a red-blood cell-hitchhiking methods that are well-known to those of skill in the art, and such methods are incorporated herein by reference. Examples of such red-blood cell-hitchhiking methods are described in Brenner et al., 2018, Nature Commun., 9:2684.
The present invention is further drawn to, in part, a method of enhancing magnetic resonance imaging (MRI) in a living subject, the method comprising: administering to a subject an effective amount of a composition imaging the patient by MRI, wherein the MRI image is enhanced as compared to an MR image obtained without said contrast agent, wherein the contrast agent comprises a metal M and a macrocyclic ligand represented by General Formula I, or a salt thereof:
In some embodiments, the contrast agent undergoes a reaction with a reactive oxygen species (ROS). Exemplary reactive oxygen species include but are not limited to peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha oxygen. In one embodiment, the compound reacts with a radical forming compound. In one embodiment, the reactive oxygen species is H2O2. In one embodiment, the reactive oxygen species is superoxide. In one embodiment, the reactive oxygen species is molecular oxygen.
In some embodiments, the contrast agent undergoes a reaction with a reactive oxygen species in a solution. In one embodiment, the solution is aqueous. In one embodiment, the solution is organic. In one embodiment, the solution is a biological fluid. Exemplary bodily fluids include, but are not limited to, sweat, whole blood, urine, stool, saliva, lymph fluid, cerebrospinal fluid, synovial fluid, cystic fluid, ascites, pleural effusion, fluid obtained from a pregnant woman in the first trimester, fluid obtained from a pregnant woman in the second trimester, fluid obtained from a pregnant woman in the third trimester, maternal blood, amniotic fluid, chorionic villus sample, fluid from a preimplantation embryo, maternal urine, maternal saliva, placental sample, fetal blood, lavage, cervical vaginal fluid, interstitial fluid, ocular fluid, blood, plasma, serum, sputum, feces, semen, mucous, lymph, nasal lavage, eye lavage, peritoneal cavity lavage, vaginal lavage, bladder lavage, rectal lavage, fine needle aspiration of spinal fluid, and synovial fluid aspiration.
In one embodiment, the composition comprises the contrast agent in solution. In one embodiment, the composition comprises the contrast agent in organic solution. In one embodiment, the composition comprises the contrast agent in aqueous solution. In one embodiment, the composition comprises the contrast agent in a suspension. In one embodiment, the composition comprises the contrast agent in a pharmaceutically acceptable carrier.
As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art.
In one embodiment, the contrast agent is administered in a concentration of 0.01 to 5.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 4.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 3.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 2.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.05 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.15 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.20 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.25 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.30 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.35 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.40 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.45 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.50 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.55 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.60 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.65 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.70 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.85 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.90 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.95 to 1.00 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.95 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.90 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.85 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.80 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.75 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.70 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.65 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.60 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.55 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.50 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.45 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.40 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.35 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.30 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.25 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.20 mM. In one embodiment, the contrast agent is administered in a concentration of 0.10 to 0.15 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.10 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.20 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.30 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.40 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.50 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.60 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.70 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.80 mM. In one embodiment, the contrast agent is administered in a concentration of 0.01 to 0.90 mM.
The present compositions and contrast agents can be adapted for administration using a wide variety of methods of delivery, including, but not limited to, e.g., subcutaneously, intravenously, peritoneally, orally, intramuscular, topical, nasally, intradermally, ocularly, rectally, vaginally, or combinations thereof.
EXPERIMENTAL EXAMPLES The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.
The present approach involves the preparation redox-responsive contrast agents for magnetic resonance imaging (MRI) that activate upon reaction with H2O2 (Karbalaei, S. et al., 2021, Inorg. Chem., 60:8368; Yu, M. et al., 2014, J. Am. Chem. Soc., 136:12836; Yu, M. et al., 2017, Inorg. Chem., 56:2812; Yu, M. et al., 2012, Inorg. Chem. 51:9153).
Recently reported sensors have consisted of Mn(II) ions complexed to quinol-containing polydentate ligands. Positive r1 responses to H2O2 are observed with N4O2 or N5O coordination spheres, where the N-donors come from either neutral pyridine or amine groups. With a weakly anionic coordination sphere, the Mn(III/II) reduction potential is high enough to discourage oxidation of the metal to the less paramagnetic+3 oxidation state. Terminal oxidants, such as H2O2, instead preferentially oxidize the ligand, differentiating the present sensors from redox-responsive MRI contrast agents reported by Gale, Caravan, and others (Wang, H. et al., 2019, J. Am. Chem. Soc., 141:5916; Loving, G. S. et al., 2013, J. Am. Chem. Soc., 135:4620; Gale, E. M. et al., 2014, Inorg. Chem., 53:10748). The oxidation of the quinol portions of the ligands to para-quinones is accompanied by an increase in r1. Water molecules are proposed to displace para-quinones from the metal center, enhancing r1 through better aquation of the Mn(II). Having a second quinol in the coordination sphere generally improves the r1 response to H2O2. The relaxivities of [Mn(H2qp1)(MeCN)]2+ and [Mn(H4qp2)Br2] increase by 10% and 30%, respectively, upon oxidation by excess H2O2.
This strategy necessarily relies on reducing the ligand's affinity for the Mn(II), and in the case of [Mn(H4qp2)Br2], a substantial portion of the metal ion is likely released upon oxidation by H2O2. Anionic groups and macrocycles are frequently used to stabilize Gd(III)- and Mn(II)-containing MRI contrast agents (Caravan, P. et al., 1999, Chem. Rev., 99:2293; Drahos, B. et al., 2012, Eur. J. Inorg. Chem., 2012:1975). H4qp2 pyridines were previously replaced with carboxylic acids in order to stabilize Mn(II) complexes; the carboxylic acids entirely deprotonate to carboxylates at pH 7. Although the more anionic H3qc13- and H2qc14-forms of the H6qc1 ligand do indeed allow it to coordinate more tightly to the dicationic metal center, the additional negative charges in the coordination sphere render the manganese more susceptible to oxidation. The oxidation of the metal center to the less paramagnetic Mn(III) eliminates the r1 response of this complex to H2O2. A Mn(II) complex with a fluorinated tetradentate ligand with nearly identical chelating groups likewise gets readily oxidized to a lower relaxivity Mn(III) species upon reaction with excess H2O2 (Chen, H. et al., 2020, Chem. Commun., 56:4106). The other strategy to discourage metal release would be to incorporate a macrocycle into the ligand framework to stabilize the resultant transition metal complexes both thermodynamically and kinetically (Hubin, T. J. 2003, Coord. Chem. Rev., 241:27).
In this work, a new macrocyclic ligand, 1,8-bis(2,5-dihydroxybenzyl)-1,4,8,11-tetraazacyclotetradecane (H4qp4), and its complex with Mn(II) were synthesized. It was found that the redox reactivity of the Mn(II) compound was similar to those of Mn(II) complexes with H2qp1 and H4qp2 in that 1) the reactivity with air is slight and 2) the reactivity with H2O2 primarily oxidizes the ligand rather than the metal ion. In addition to the anticipated stabilization of the Mn(II) complexes with the quinol- and para-quinone forms of the ligand, the inclusion of the macrocycle improves the maximal MRI response, with r1 increasing by 130% upon oxidation by H2O2.
Furthermore, a highly water- and air-stable Fe(II) complex with the quinol-containing macrocyclic ligand H4qp4 reacts with H2O2 to yield Fe(III) complexes with less highly chelating forms of the ligand that have either one or two para-quinones. The reaction increased the T1-weighted relaxivity over four-fold, enabling the complex to detect H2O2 using clinical MRI technology. The iron-containing sensor differed from its manganese analog, which also detects H2O2, in that it was the oxidation of the metal center, rather than the ligand, that primarily enhanced the relaxivity.
The H4qp4 ligand was synthesized in one step from cyclam, two equiv. of 2,5-dihydroxybenzaldehyde, and excess NaBH4—Al2O3(
A Mn(II) complex with H4qp4, [Mn(H3qp4)](OTf) (1), was prepared by refluxing the ligand and Mn(OTf)2 in 1:1 MeCN/THF for 2 d. The complexation reaction required a much higher temperature and a much longer reaction time than the syntheses of [Mn(H2qp1)(MeCN)](OTf)2 (2) and [Mn(H4qp2)Br2] (3). The incorporation of metal ions into macrocycles often requires such measures. Complex 1 differed from previously isolated Mn(II) complexes with polydentate quinol-containing ligands in that it featured a deprotonated quinol. Elemental analysis of powdered samples of 1 indicated that there is a single triflate per manganese. The EPR and the magnetic susceptibility measurements both indicated that the metal ion is high-spin Mn(II). Since there is only one counteranion, this thereby necessitates a −1 charge on the polydentate ligand (H3qp4−). The presence of a quinolate was further supported by the presence of two bands in the UV/vis spectrum of the isolated product in MeCN (
Complex 1 was stable to air in the solid and solution states for prolonged periods of time. Samples of 1 in MeCN displayed negligible changes to their UV/vis features over a 12 h exposure to air. If solutions of 1 in either aqueous or organic solvents were kept under air for 1-2 weeks, the compound eventually oxidized to [MnIII(H2qp4)](OTf) (4), where H2qp42− is the doubly deprotonated form of the ligand.
1,4,8,11-tetracyclotetradecane (cyclam) (1.00 g, 4.99 mmol) and 2,5-dihydroxybenzaldehyde (1.37 g, 9.91 mmol) were combined in 15 mL of dry MeOH. The mixture was heated at reflux for 4 h under N2. The reaction mixture was then cooled to 0° C. with an ice bath. Once the temperature reached 0° C., 20 mL of additional dry MeOH and NaBH4—Al2O3 (wt. 10%, 0.83 g, ˜0.02 mol) were gradually added to the solution. The resultant solution was heated at reflux for 6 h under N2 and then cooled to 0° C. The residual reductant was titrated with 1 M HCl until the solution reached pH 8, depositing the crude product as a solid which was collected via filtration. The solid was dissolved in acetone and filtered. The acetone was rotavapped to yield the product as a yellow powder (842 mg, 38% yield). Typical yields range from 38 to 42%. 1H NMR (400 MHz, DMSO-d6, 297 K): S 8.55 (s, 2H), 6.97 (s, 2H), 6.50-6.56 (m, 4H), 6.45 (s, 2H), 3.16 (s, 4H), 2.45-2.59 (m, overlap with solvent peak), 2.28 (s, 4H), 1.69 (s, 4H), 1.14 (s, 2H). 13C NMR (100 MHz, DMSO-d6, 297 K): S 149.98, 149.17, 124.98, 118.02, 117.10, 115.48, 53.80, 53.11, 50.50, 49.26, 46.38, 24.71. MS (ESI): calcd for MH, 445.2815; found, 445.2821.
H4qp4 (500 mg, 1.12 mmol) and Mn(OTf)2 (397 mg, 1.12 mmol) were dissolved in 5 mL of dried 1:1 MeCN-THF under N2. The mixture was stirred at 60° C. for 48 h; over this time, a green solid precipitated from the solution. The crude product was collected via filtration and washed with cold MeCN to yield the product as a green powder (577 mg, 76% yield). Typical yields range from 70 to 75%. MS (ESI): calcd for [Mn(H2qp4)]+, 497.1961 and [Mn(H2qp4)(OTf)]+, 646.1481; found, 497.1931 and 646.1470. Solid-state magnetic susceptibility (294 K): μeff=5.6μB. Optical spectroscopy (MeCN, 294 K): 300 nm (6800 M−1 cm−1), 388 nm (3500 M−1 cm−1). IR (cm−1): 3282 (m), 3069 (w), 2852 (w), 1611 (w), 1511 (m), 1483 (m), 1361 (m), 1279 (s), 1191 (s), 1238 (s), 1212 (s), 1180 (s), 1150 (s), 1090 (m), 1060 (m), 1026 (s), 992 (m), 916 (m), 868 (m), 815 (s), 751 (m), 631 (s), 572 (m), 510 (m). Elemental analysis (powder) calcd for C25H35N4O7F3S1Mn·1.5 H2O: C, 44.51%; H, 5.67%; N, 8.30%. Found: C, 44.27%; H, 5.14%; N, 8.09%.
Both H4qp4 and [Mn(H3qp4)](OTf) (1) were prepared in straightforward manners using techniques slightly modified from previously successful procedures. The synthesis for a closely related cyclam derivative with two phenols appended to two of the amines was done in two distinct steps (Luo, H. et al., 2001, Can. J. Chem., 79:1105), but the heightened air-sensitivity of the quinols under basic conditions led to the exploration of a one-pot reaction. Once the ligand was prepared, the installation of the Mn(II) into the macrocycle proceeded cleanly using protocols commonly used for other macrocycle complexation reactions (McGowan, P. C. et al., 2001, Inorg. Chem., 40:1445; Major, J. L. et al., 2007, Proc. Natl. Acad. Sci., 104:13881; Regueiro-Figueroa, M. et al., 2014, Chem. Eur. J. 20:17300; Wang, S. et al., 2009, Inorg. Chem., 48:719; Phukan, B. et al., 2018, Inorg. Chem. 57:2631). Unexpectedly, the Mn(II) was bound to the singly deprotonated ligand H3qp4−, rather than neutral H4qp4, in the solid isolated from precipitation of the Mn(II) complex from organic solvents. Analogous complexes with H2qp1 and H4qp2 (
As anticipated, 1 contained a high-spin Mn(II) metal center as evidenced by EPR, UV/Vis, and solid-state magnetic susceptibility data. The UV/Vis features corresponded to intraligand transitions for the quinol and quinolate groups; these provided convenient spectroscopic signatures to follow the oxidation state of the ligand.
Structures of [H6qp4](OTf)2 and 4
The crystal structure of the doubly protonated H4qp4 ligand was generated (
Attempts to crystallize 1 were unsuccessful, but 4 was crystallized by slowly diffusing CH2Cl2 into a saturated solution of 1 in MeCN under air over 2 weeks (
The speciation of the free H4qp4 ligand was investigated from pH 3 to 9 (
The inclusion of a macrocycle into the ligand framework greatly stabilized 1 in water relative to previously prepared contrast agents. The stability and speciation of 1 were assessed using potentiometric pH titration data acquired from pH 3 to pH 9. The best-fitting model for the data suggested that there was negligible Mn(II) release from the ligand even at pH 3 (
The stability of the Mn(II)-H4qp4 complex in water was confirmed by high performance liquid chromatography (HPLC) (
Complex 1 was electrochemically characterized by cyclic voltammetry (CV) in an aqueous 50 mM phosphate solution buffered to pH 7.2. An irreversible feature with Epa=1.25 V vs. Ag/AgCl was observed and assigned to the oxidation of the metal to Mn(III). In addition, a redox feature with E1/2 of 100 mV vs. Ag/AgCl was detected (295 mV vs. NHE,
The ability of 1 to interact with water was studied using variable temperature 17O NMR using the methodology pioneered by Gale et al (
Complex 1 did not display any noticeable short-term reactivity to air. The UV/vis spectrum of 1 in MeCN did not appreciably change over the course of a 12 h exposure to air (
Complex 1 differed from 2 and 3 in that it strongly resisted metal ion exchange. The reaction between 0.1 mM Fe(ClO4)2 and 0.1 mM 1 in either MeCN or buffered water did not yield UV/vis-detectable quantities of [Fe(H3qp4)]+, even at 18 h (
Complex 1 was much more thermodynamically stable in water than the previously characterized 3, which had a similar coordination sphere around the metal center in aqueous solution. The pMn value measured at pH 7.4 with 1.0 mM concentrations of ligand and Mn(II) was 9.81, which represented over four orders of magnitude of improvement over the 5.36 value measured for 3. The gains in stability could be attributed largely to macrocyclic effects since H4qp2 and H4qp4 provide similar coordination spheres: four neutral N-donors in addition to the two quinols/quinolates. Complex 1 was also more stable than 2, which had a pMn of 7.25 under the standard conditions (33).
The stability of 1 also exceeded those of most other reported mononuclear Mn(II)-containing MRI contrast agents (Phukan, B. et al., 2018, Inorg. Chem., 57:2631; Gale, E. M. et al., 2016, J. Am. Chem. Soc., 138:15861; Geraldes, C. F. G. C. et al., 1986, Magn. Reson. Med. 3:242). The Gale and Caravan groups have recently reported a series of linear ligands with multiple carboxylate donors that coordinate tightly to Mn(II). One representative example, PyC3A3−, formed a Mn(II) complex with a log KML of 14.14 (
The inclusion of the cyclam into the ligand also endowed 1 with a high level of kinetic stability as assessed by metal competition experiments. Once in the macrocycle, the Mn(II) could not be facilely displaced by either Fe(II) or Zn(II), two of the most common transition metal ions in biology. Complexes 2 and 3, conversely, gradually reacted with equimolar amounts of free Fe(II) and quickly reacted with Zn(II). The inertness of 1 towards metal ion exchange compared well to those of other Mn(II)-containing MRI contrast agents with macrocyclic organic components. The kinetic stability extended to Mn(II) complexes with oxidized forms of the H4qp4 ligand. Oxidation by H2O2 yielded a mixture of Mn(II) complexes with the monoquinol/monoquinone and diquinone ligands H2qp4 and qp4 (
Like 2 and 3, 1 is a redox-active T1-weighted MRI contrast agent. Curiously, the pre-oxidation relaxivity of 1 (3.15 mM−1 s−1) was much lower than that of 3 (5.46 mM−1 s−), despite the two compounds having similar coordination spheres and acid/base behavior. Both r1 values are the weighted averages of the relaxivities of the individual Mn(II) species present at pH 7.00: [Mn(H3qp4)(H2O)]+ and [Mn(H2qp4)(H2O)] in the case of 1. The average aquation number measured for 1 was slightly higher than that of 3, but this would be anticipated to raise rather than lower the T1-weighted relaxivity, r1. The pKa values for the two Mn(II)-bound quinols in 1 and 3 were also similar: 5.82 and 7.14 for 3 versus 5.09 and 7.39 for 1. The higher pKa value in each pair would be anticipated to modulate the rate of proton transfer (Aime, S. et al., 2018, Inorg. Chem., 57:5567). The slightly more basic quinolate in 1 could slow the rate of proton transfer between the Mn(II)-quinols and the bulk water enough to substantially reduce its contribution to the measured r1, but further studies are needed to fully elucidate that possible relationship. The rate constants for water exchange at 298 K differed the most, with the kex value measured for 3 (4.9×106 s−1) being approximately a third of that of 1 (1.7×107 s−1). Complex 1, unlike 3, appeared to exchange water molecules through a dissociative mechanism, as evidenced by the highly positive entropy of activation (
The relaxivity measured for non-oxidized 1, like that of 3, was higher under acidic conditions (
Investigation of Reactivity Between 1 and H2O2
Upon reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), the quinols in 1 appeared to be partially oxidized to para-quinones as assessed by mass spectrometry (MS), ultraviolet-visible spectrometry (UV/Vis), and infrared spectroscopy (IR) (
When 1 reacted with a large 10 mM excess of H2O2, MS revealed similar m/z features to those seen for the oxidation of the Mn(II) complex by DDQ, suggesting that the quinols were likewise converted to para-quinones by this oxidant (
When the reaction between 1.0 mM 1 and 10 mM H2O2 in pH 7.0 HEPES buffer was monitored by electron paramagnetic resonance spectroscopy (EPR), the spectrum taken before the oxidant was added looked nearly identical to those taken at 60 min and 90 min, suggesting that the manganese largely remained in the +2 oxidation state throughout the reaction (
Upon oxidation by H2O2, the aquation number of the Mn(II) center in 1 increased by approximately one water molecule, resulting in an average of 2.2 water molecules bound to each metal center (
In addition to the higher stability, the inclusion of the cyclam into the ligand framework had the non-intuitive benefit of improving the relaxivity response to H2O2. The 130% increase in r1 was approximately quadruple the percentile increase observed for 3. Complex 3 rapidly destabilizes as the pH drops below 7.0 due to the protonation of the Mn(II)-quinolates, and it was reasonable to assume that the oxidation of the quinols to para-quinones likewise weakens the binding affinity of the ligand enough to trigger release of the Mn(II). The H2O2-enhanced r1 for 3, however, could not be attributed to the release of the metal. The T1-weighted relaxivity of [Mn(H2O)6]2+ was measured independently (5.26 mM−1 s−) and found to be approximately equal to that of the pre-activated form of 3 (5.46 mM−1 s−). Although one may expect [Mn(H2O)6]2+ to have a higher r1 due to its higher aquation number, this was not observed. Proton exchange between water molecules and the Mn(II)-bound quinols/quinolates would be anticipated to markedly increase the r1 values of 1, 2, and 3 relative to q=1 systems that lack metal-bound hydroxyl groups from their organic ligands. Counter-intuitively, Mn(II) ion release from the oxidized forms of H4qp2 may actually curtail the response of 3 to H2O2.
If 1 were to release free Mn(II) upon oxidation, an increase in r1 due to the lower starting value would be anticipated, but this mechanism was not consistent with the data. NMR measurements indicated that the oxidation of 1 by H2O2 did not release significant amounts of Mn(II). Further, the maximum r1 exceeds that of [Mn(H2O)6]2+. Instead, it was believed that the increase in relaxivity results from the formation of more highly aquated Mn(II) species with the oxidized forms of the ligand. Since the H2O2 reaction did not make either [Mn(Hqp4)(H20)x]+ or [Mn(qp4)(H2O)x]2+ cleanly, the individual r1 values of these two species could not be measured to ascertain their contributions to the overall relaxivity. Oxidation by H2O2 did improve the aquation of the metal center, as evidenced by variable temperature 17O NMR measurements (
The relaxivities of complex 1 before and during its reaction with H2O2 were assessed using a 3 T MRI scanner (
Relaxivities (r1) were obtained from the slopes of plots of 1/T1 versus manganese concentration. Prior to oxidation by excess H2O2, the r1 was 3.16 mM−1 s−. The data taken for samples kept under air for 1 h and 2 h overlaid well, suggesting that the short-term reactivity with air was slight. After oxidation by H2O2, the relaxivity rose, reaching 5.09 mM−1 s− by 1 h and peaking at 7.35 mM−1 s−1 as assessed by measurements taken at 2 h (
Complex 1 differed substantially from 2 and 3 in that its response to H2O2 depends on the manner in which the oxidant is administered. When a large excess of H2O2 was added to 1 in a single portion, the oxidation of the quinols occurs after an induction period. The oxidation by 10 mM of H2O2 took approximately 90 min to complete, as assessed by changes to both the relaxivity and the UV/vis spectrum. When a smaller portion of H2O2 is added, the quinols are oxidized more quickly and without a noticeable induction period. Based on these observations, it was proposed that the initial reaction between H2O2 and 1 generated an oxidant that can react either unimolecularly to oxidize one of the quinols to a para-quinone or bimolecularly with another equiv. of H2O2 to yield 02 and regenerate 1 (
Preliminary data suggested that 1 can indeed catalyze the degradation of H2O2. When reactions between 100 nM 1 and 10 mM H2O2 were followed spectrophotometrically, the absorbance of the H2O2 peak at 240 nm decreased quickly (
Biological environments generally provide low but rapidly replenishing concentrations of H2O2 that are closer to 0.6 mM than 10 mM (Kinnula, V. L. et al., 1991, Am. J. Physiol., 261:L84; Lyublinskaya, O. et al., 2019, Redox Biol., 24:101200; Sies, H. 2014, J. Biol. Chem., 289:8735). It was therefore anticipated that 1 would provide a fast r1 response to physiologically generated H2O2 (
A Mn(II) complex with a cyclam macrocycle derivatized with two quinols is a highly water-stable MRI contrast agent that displays a positive r1 response to H2O2, one of the most prevalent reactive oxygen species in biology. The percentile relaxivity response was approximately four times that of a Mn(II) complex with a linear ligand that provided a similar set of donor atoms: two quinols/quinolates and four neutral N-donors. In addition to boosting the thermodynamic stability, the macrocycle also provided a kinetic barrier for metal ion dissociation, and the Mn(II) complex appeared to retain the metal after the oxidation of the quinols to more poorly metal-binding para-quinones. The greater stability of the current complex addresses a key concern about other quinol-containing MRI contrast agent sensors that were previously prepared and should smooth the pathway towards using such complexes to monitor biologically relevant oxidative stress.
The reactivity with H2O2 appeared to proceed through two competing pathways. With low H2O2 levels, intramolecular oxidation of the quinols to para-quinones occurs. This process appeared to be slow relative to analogous reactions seen with manganese complexes with linear quinol-containing ligands. With high—and physiologically unrealistic—H2O2 concentrations, catalase activity was observed. Under such conditions, the quinol oxidation and the concomitant increase in r1 only occurred after much of the H2O2 has been consumed.
The previously prepared probes exhibit changes to their T1-weighted relaxivity (r1) upon oxidation by H2O2. The r1 of a 1H-based contrast agent is influenced by the nature and extent of its interactions with bulk water molecules and the electronic properties of the metal ion component. With respect to the interactions with water molecules, groups that can exchange protons with bulk water and sites for the direct coordination of water to the metal ion both improve the relaxivity. The number of water coordination sites on the metal is commonly referred to as the aquation number (q). The electronic structure of the metal ion is connected to two important parameters: the paramagnetism and the electron spin relaxation time. Highly paramagnetic metal ions with slow electron spin relaxation times, such as high-spin Mn(II) and Gd(III), enable high r1 values. Conversely, metal ions with fast electron spin relaxation times, such as Fe(II), are associated with low r1 values and near negligible impact on MRI contrast even when they are highly paramagnetic.
Most of the prior sensors were air-stable Mn(II) complexes with polydentate quinol-containing ligands (Karbalaei, S. et al., 2021, Inorg. Chem., 60:8368; Yu, M. et al., 2014, J. Am. Chem. Soc., 136:12836; Yu, M. et al., 2017, Inorg. Chem., 56:2812). Upon reacting with H2O2, the quinols oxidize to para-quinones. Water molecules displace the para-quinone portions of the ligand, and the higher aquation thereby both increases the r1 of the complex and improves MRI contrast. Although the manganese may be transiently oxidized, it mostly reverted back to the +2 oxidation state. The Mn(II) complexes could be modified to be exceptionally water-stable and could respond to H2O2 with a 130% increase in r1, but the high background relaxivities of the pre-activated sensors complicated analysis of biological redox environments. Enhanced contrast in a region could have resulted from a combination of two factors: oxidation of the sensor to its more emissive form and the mere accumulation of the sensor in its pre-activated and/or oxidized forms in the area of interest.
In order to decrease the background signal provided by the pre-activated form of the sensor, the Mn(II) in the most successful sensor, [Mn(H3qp4)](OTf), was replaced with Fe(II). Relative to Mn(II), Fe(II) complexes had a much lesser impact on the T1 of water molecule 1H nuclei due to their much faster electron spin relaxation (Wang, H. et al., 2019, J. Am. Chem. Soc., 141:5916). Fe(II) also differed from Mn(II) in that it could be oxidized to the +3 oxidation state much more facilely (Cotton, F. A. 1988, Advanced Inorganic Chemistry, 5 ed., John Wiley & Sons). When the metal centers are high-spin, the resultant Fe(III) complexes are more paramagnetic and exhibit slower electron spin relaxation; as such, they can interact with water molecules to substantially improve T1-weighted MRI contrast (Hoener, B.-A. et al., 1991, J. Magn. Reson., 1:357; Schwert, D. D. et al., 2002, Top. Curr. Chem., 221:165). Gale and Caravan recently designed a redox-responsive MRI contrast agent that relied on the oxidation of Fe(II) to Fe(III) to detect biologically relevant oxidants. Although the percentile increase in r1 was large and the pre-activated ferrous complex had little impact on the MR image, the activated form of the sensor had a relatively small r1. The r1 of the presently described iron-containing sensor, conversely, could potentially be enhanced by both ligand and metal oxidation (
[FeII(H3qp4)](OTf) (1) was prepared by mixing the H4qp4 ligand (
H4qp4 (500 mg, 1.12 mmol) and Fe(OTf)2 (400 mg, 1.13 mmol) were dissolved in mL of dry MeCN under N2. The mixture was stirred at 60° C. for 48 h. The slow addition of CH2Cl2 deposited the product as a dark blue powder, which was collected via filtration (628 mg, 83% yield). MS (ESI): calcd for [Fe(H2qp4)]+, 498.1929; found, 498.1888. Solid-state magnetic susceptibility (294 K): μeff=4.8μB. Solution-state magnetic susceptibility (CD3CN, 298 K): μeff=4.7μB. Optical spectroscopy (MeCN, 294 K): 299 nm (ε=10800 M−1 cm−1), 595 nm (ε=2200 M−1 cm−1). IR (KBr, cm−1): 3378 (s), 3229 (s), 3070 (m), 2855 (w), 2323 (w), 1612 (w), 1512 (w), 1485 (s), 1469 (w), 1449 (m), 1364 (w), 1219 (s), 1151 (s), 1115 (w), 1079 (w), 1024 (s), 992 (w), 916 (w), 869 (m), 814 (s), 792 (w), 762 (m), 633 (s), 600 (w), 542 (w), 514 (m), 478 (w), 421 (w). 1H NMR (500 MHz, CD3CN, 298 K): S 16.54, 6.75, 6.65, 5.0-2.0 (m), 1.13, 0.65, −2.20, −9.77, −14.55. Elemental analysis (powder) calcd for C25H35N4O7F3S1Fe·1.5H2O·1CH2Cl2: C, 42.70%; H, 5.30%; N, 7.38%. Found: C, 42.82%; H, 4.83%; N, 7.88%. HPLC (method 1): tR=7.50 (min).
H4qp4 (500 mg, 1.12 mmol) and Fe(OTf)3 (637 mg, 1.13 mmol) were dissolved in 5 mL of dry MeCN under N2. The mixture stirred at 60° C. for 48 h. Upon the slow addition of ether to the MeCN solution, the product deposited as a dark brown powder that could be isolated by filtration (780 mg, 83% yield). MS (ESI): calcd for [Fe(H2qp4)]+, m/z 498.1929; found, m/z 498.1921. Solid-state magnetic susceptibility (294 K): μeff=5.7μB. Optical spectroscopy (MeCN, 294 K): 299 nm (ε=7900 M−1 cm−1). IR (KBr, cm−1): 3458 (s), 3302 (s), 3182 (s), 3098 (w), 2957 (w), 2890 (w), 2825 (m), 1655 (w), 1549 (w), 1520 (m), 1479 (w), 1448 (s), 1390 (w), 1342 (w), 1280 (w), 1247 (w), 1223 (w), 1192 (w), 1156 (s), 1131 (w), 1091 (m), 1052 (w), 1025 (s), 992 (w), 934 (w), 920 (w), 892 (w), 868 (w), 823 (m), 755 (m), 636 (m), 583 (w), 511 (w), 470 (w). Elemental analysis (powder) calcd for C26H3SN4O10F6S2Fe·0.5 (C2H5)2O (powder): C, 40.29%; H, 4.83%; N, 6.71%. Found: C, 40.16%; H, 4.91%; N, 7.05%. HPLC (method 1): tR=8.02 (min).
Complex 1, like its Mn(II) analog, was found to be exceptionally stable in water (
The titration data were consistent with negligible metal ion dissociation from 1, even under highly acidic conditions (
Cyclic voltammetry (CV) analysis of 1 in an aqueous solution containing 0.10 M phosphate buffered to pH 7.2 revealed two redox events (
As with the Mn(II) analog, prolonged (>1 week) exposure to air did eventually oxidize the metal, but not the ligand, in 1 to yield [FeIII(H2qp4)](OTf) (2,
Investigation of Reactivity of 1 with H2O2
Complex 1 reacted with H2O2 to yield Fe(III) species with oxidized ligands. Evidence for the oxidation of the quinols in the ligand to para-quinones was provided by UV/vis and IR (
The oxidation of the metal to Fe(III) was confirmed with EPR (
The oxidation of 1 by H2O2 appeared to be partly reversible in that the ligand could be reduced back to the diquinol form but the metal remained in the +3 oxidation state. When 1 was oxidized by 4 equiv. of H2O2 for 60 min, the subsequent reaction with 4 equiv. of either cysteine or dithionite yielded a species with a m/z feature at 498.19, which was consistent with an Fe(III) complex with the reduced and doubly deprotonated form of the ligand (
The MRI properties of 1 were assessed using methods that were previously employed in studies of manganese-containing MRI contrast agents. The relaxivity of 1 was measured before and after its reaction with excess H2O2 using a 3 T MRI scanner (
The r1 value of pre-activated 1 was found to be 0.22 (±0.09) mM−1 s−1, which was similar to values measured for other Fe(II) complexes. The relaxivity of pre-activated 1 was also measured at various pH values between 2 and 12 (
The quinol- and Mn(II)-containing H2O2 sensors previously reported displayed more drastic drops in their relaxivity upon basification. The decrease in R1 was attributed to the weakly metal-binding quinols getting deprotonated to much more strongly binding quinolates. Under acidic conditions, water molecules can displace the quinols, leading to higher aquation numbers and thereby higher relaxivities. It was confirmed that water exchange occurred on the Fe(II) metal center at pH 7.4 with 17O NMR (
Upon oxidation by H2O2, the r1 increased over 4-fold to 0.91 (±0.26) mM−1 s−1. Gale's Fe(II)-PyC3A complex, by comparison, exhibited a 10-fold increase in relaxivity at 1.4 T as the complex is oxidized to Fe(III), with even larger responses under stronger magnetic fields. As with the Mn(II) complex with H4qp4, the enhancement in r1 occurred gradually when a large excess of H2O2 was added in a single portion. The r1 required 60 min to reach its maximum value after 10 mM H2O2 was added. The maximum r1 was in the middle of the range for Fe(III)-containing MRI contrast agents (Snyder, E. M. et al., 2020, Angew. Chem. Int. Ed., 59:2414). The relatively low value of r1 for the activated sensor may suggest that the Fe(III) may lack an inner-sphere water. When mixtures of 1 and H2O2 were analyzed by 17O NMR, it was found that the iron did not alter the line-width of the water peak, confirming negligible water exchange on the NMR timescale for the oxidized product(s) (
Complex 2, which featured Fe(III) bound to the reduced form of the H4qp4 ligand, was prepared through aerobic oxidation of 1 in trace quantities. The conjugate acid of 2, [FeIII(H3qp4)](OTf)2 (3), could also be prepared by directly reacting H4qp4 with FeIII(OTf)3 in MeCN. Although it was not structurally characterized, 3 was characterized by elemental analysis, MS, IR, UV/vis, and EPR (
The speciation of Fe(III)/H4qp4 mixtures was analyzed between pH 3.0 and 8.0 using potentiometric pH titrations (
The stability of 3 was assessed in the presence of a competing metal ion. When the reaction was monitored between 10 mM 3 and 20 mM Zn(ClO4)2 by 1H NMR, only trace amounts of the Zn(II) complex were observed with H4qp4 at 48 h (
The relaxivity of 3, which will spontaneously deprotonate to 2 at pH 7.0, was assessed in order to determine how much metal oxidation by itself contributes to the H2O2 response of 1. The measured r1 of 0.87 mM−1 s− was within error of the 0.91 mM−1 s− value (
The Mn(II) complex with H4qp4 likewise displayed a delayed response to H2O2. Counterintuitively, adding a smaller concentration of H2O2 accelerated the oxidation of the Mn(II) complex to its higher-relaxivity form. Based on these observations, it was previously proposed that the oxidation of the H4qp4 complex proceeds through a transient higher-valent metal complex that can either oxidize the quinols through a relatively slow intramolecular reaction or react with a second equiv. of H2O2. The intramolecular reaction would become more prominent as the H2O2 is depleted. With high concentrations of H2O2, conversely, the compound acts as a functional mimic of catalase.
When a lower amount of H2O2 was added to oxidize 1, the quinol-derived features in the UV/vis vanished more quickly (
The lipophilicity of MRI contrast agents can impact how quickly they clear the body, with more lipophilic compounds possibly getting indefinitely trapped within cells (Carney, C. E. et al., 2015, J. Biol. Inorg. Chem., 20:971). The lipophilicity of 1 was assessed by first measuring the n-butanol/water partition coefficients and then correlating it to a n-octanol/water value using an established calibration curve (Ran, Y. et al., 2002, Chemosphere, 48:487; Engelmann, F. M. et al., 2007, Int. J. Pharm., 329:12). The log PBW value was calculated to be −0.00047 (Eq. 1), which translated to a log POW value of −0.5407 (Eq. 2). The results indicated that 1 was relatively hydrophilic and should not be cell-permeant.
The cytotoxicity of 1 was investigated using rat cardiac cells (H9c2). Although uncomplexed iron is highly toxic, it was found that H9c2 cells can tolerate high doses of 1 (
To conclude, it was found that substituting Fe(II) for Mn(II) in the H4qp4 framework resulted in a highly water- and kinetically air-stable complex that exhibited a higher percentile increase in r1 upon reaction with H2O2. Despite the similar aqueous speciations of the Fe(II) and Mn(II) compounds, the responses to H2O2 relied on fundamentally different mechanisms. Whereas the higher r1 of the manganese sensor was connected to an increase in q enabled by oxidation of the organic component, that of the iron system appeared to result almost entirely from changes to the characteristics of the oxidized metal center.
These data suggest the compounds may provide improved detection of H2O2 and other ROS molecules for diagnostic purposes. However, overall the invention is very early in development and has yet to be tested within animals or compared to existing FDA-approved contrasting agents. Hence, significant R&D and financial resources will be required to enter clinical trials and achieve FDA approval. Nevertheless, further development of this technology could lead to a new class of contrast agents and better visualization of tissues and disease diagnosis. Key benefits include a novel class of compounds that improve the imaging and analysis of ROS activity within the body, several prototype compounds have been developed with data showing the ability to be used as contrast agents in a concentration-dependent manner in vitro, and the potential to improve the diagnostic accuracy of MRI and potentially other imaging modalities for many diseases.
All chemicals and solvents were purchased from Sigma-Aldrich and used as received unless otherwise noted. All deuterated solvents were bought from Cambridge Isotopes. Diethyl ether (ether) and methanol (MeOH) were bought from Fisher. Methylene chloride (CH2Cl2) was purchased from Mallinckrodt Baker. 1,8-Bis(2,5-dihydroxybenzyl)-1,4,8,11-tetraazacyclotetradecane (H4qp4) was prepared through a previously described procedure (Karbalaei, S. et al., 2021, Inorg. Chem., 60:8368).
All 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on either a 400 MHz or a 600 mHz AV Bruker NMR spectrometer. All reported NMR resonance peak frequencies were referenced to internal standards. 17O NMR data were collected on a Bruker AVANCE DRX 400WB spectrometer with a superconducting wide-bore magnet operating at a 54.24 MHz resonance frequency and a 9.4 T magnetic induction. A Varian Cary 50 spectrophotometer was used to collect optical data, which were then processed using software from the WinUV Analysis Suite. Electron paramagnetic resonance (EPR) spectra were collected using a Bruker EMX-6/1 X-band EPR spectrometer operated in the perpendicular mode and subsequently analyzed with the program EasySpin. All EPR samples were run as frozen solutions in quartz tubes. A Johnson Matthey magnetic susceptibility balance (model MK I #7967) was used to measure the magnetic moments of solid samples of the metal complexes and estimated the diamagnetic component of the susceptibility using Pascal's constants (Bain, G. A. et al., 2008, J. Chem. Educ. 85:532). Solution-state magnetic moments were obtained using the Evans method (Evans, D. F. 1959, J. Chem. Soc., 2003). Cyclic voltammetry (CV) was performed under N2 at 294 K with an Epsilon electrochemistry workstation (Bioanalytical System, Inc.). The working, auxiliary, and reference electrodes were gold, platinum wire, and silver/silver(I) chloride, respectively. High-resolution mass spectrometry (HR-MS) data were collected at the Mass Spectrometer Center at Auburn University on a Bruker microflex LT MALDI-TOF mass spectrometer via direct probe analysis operated in the positive ion mode. Solid samples of the Mn(II) complex were dried, stored under N2, and sent to Atlantic Microlabs (Norcross, GA) for elemental analysis.
Crystallographic data for (H6qp4)(OTf)2 and the oxidized product [Mn(H2qp4)](OTf) were collected using a Bruker D8 VENTURE κ-geometry diffractometer system equipped with a Incoatec IμS 3.0 microfocus sealed tube and a multilayer mirror monochromator (Mo Kα, λ=0.71073 Å). Diffraction data were integrated with the Bruker SAINT software package using a narrow-frame algorithm. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The structure was solved and refined using the Bruker SHELXTL Software Package. Selected crystallographic data is presented in
The aqueous speciations of H4qp4 and its Mn(II) complex were assessed using a METROHM 765 Dosimat with a jacketed, airtight glass titration vessel. A Fisher Scientific Accumet Research AR15 pH meter was used to monitor the pH of the sample solutions during the titrations. The electrode was calibrated before each titration using commercially available standard solutions buffered to pH 4.0, 7.0, and 10.0. All samples were purged with argon prior to analysis and subsequently analyzed under an argon atmosphere at 25° C. to prevent carbonate contamination. All solution samples were prepared in solutions of 100 mM KCl in deionized Millipore water. The titrations investigating metal-ligand speciation were run with solutions that contained a 1:1 molar mixture of the ligand and MnCl2·(4H2O). Carbonate-free solutions of 0.10 M KOH and 0.10 M HCl were prepared using argon-saturated deionized Millipore water. The titration data were analyzed and fit to speciation models using the Hyperquad2006 program (Gans, P. et al., 1996, Talanta, 43:1739).
The aqueous speciation of H4qp4 was previously determined. Those for its Fe(II) and Fe(III) complexes were assessed using a METROHM 765 Dosimat with a jacketed, airtight glass titration vessel. A Fisher Scientific Accumet Research AR15 pH meter was used to monitor the pH of the sample solutions during the titrations. The electrode was calibrated before each titration using commercially available standard solutions buffered to pH 4.0, 7.0, and 10.0. All samples were purged with argon prior to analysis and subsequently analyzed under an argon atmosphere at 25° C. to prevent carbonate contamination. All solution samples were prepared in solutions of 100 mM KCl in deionized Millipore water. The titrations investigating metal-ligand speciation were run with solutions that contained a 1:1 molar mixture of the ligand and FeCl2·(4H2O). Carbonate-free solutions of 0.10 M KOH and 0.10 M HCl were prepared using argon-saturated deionized Millipore water. The titration data were analyzed and fit to speciation models using the Hyperquad2006 program.
HPLC was performed with UV detection at 254 nm using an Agilent 1100 series apparatus and an Agilent Zorbax SB-C18 column (4.6×150 mm, 5 μm pore size). The following eluents were used: A) 99.9% water with 0.1% trifluoroacetic acid (TFA) and B) 99.9% MeCN with 0.1% TFA. The following method was used:
Gradient 90% A and 10% B to 100% B over 20 min. Flow rate=0.20 mL/min, injection volume=25.0 μL, column temperature=37.0° C. Before each run, the HPLC instrument was flushed with eluent 100% A to 100% B over 16 min with a flow rate of 0.49 ml/min and an injection volume of 25.0 μL.
Aquation numbers (q) were calculated from the maximum 17O transverse relaxivity, r2max°, and the equation: q=r2max°/510; Gale, Zhu, and Caravan previously used this relationship to estimate the inner-sphere hydration state of Mn(II) in coordination complexes (Gale, E. M. et al., 2013, J. Am. Chem. Soc., 135:18600). Relaxation rates were measured both for aqueous solutions containing Mn(II) complexes and for metal-free solutions buffered to pH 7.4. The linewidths at half-height of the signal were determined by a deconvolution procedure on the real part of the Fourier transformed spectra with a Lorentzian shape function in the data analysis module of Bruker Topspin 1.3® software. The measurements were performed with a commercial 5 mm Bruker broadband probe thermostat with a Bruker B-VT 3000 variable temperature unit. Samples were prepared by adding a solution of solid dissolved in a minimal amount of MeCN to an aqueous solution containing either 60 mM HEPES or 60 mM MOPS buffered to pH 7.4. 100 (v/v) of 17O-labeled water (10%, D-Chem Ltd. Tel Aviv, Israel) was added to these solutions resulting in a total enrichment of 1% 170 in the studied samples. The resultant mixtures contained either 6.0 mM or 2.5 mM of the Mn(II) complex. The 2.5 mM sample was oxidized by 15 equiv. of H2O2 for 15 min prior to data acquisition. The temperature-dependence of 17O-line broadening was studied from 274.2 to 338.2 K.
All MRI data were collected at the Auburn University MRI Research Center on a Siemens Verio open-bore 3-T MRI clinical scanner. A 15-channel knee coil was used to simultaneously image 12-15 samples. The imaging procedure was identical to those used for similar studies (Zhang, Q. et al., 2011, Inorg. Chem. 50, 9365; Yu, M. et al., 2014, J. Am. Chem. Soc., 136:12836; Yu, M. et al., 2017, Inorg. Chem., 56:2812; Yu, M. et al., 2012, Inorg. Chem. 51:9153). An inversion recovery (IR) sequence was used that featured a non-selective adiabatic inversion pulse followed by a slice-selective gradient recalled echo (GRE) readout after a delay period corresponding to the inversion time (TI) (Bernstein, M. A. et al., 2004, Handbook of MRI Pulse Sequences; Haacke, E. M. et al., 1999, Magnetic Resonance Imaging: Physical Principles and Sequence Design). The GRE was a saturation readout, such that only one line of k-space was acquired per repetition time (TR), in order to maximize both signal strength and the accuracy of the T1 estimates. The specific imaging parameters were as follows: TR was set to 10 s, TI was varied from 10 to 2600 ms over 20 steps, the echo time (TE) was set to 2.75 ms, the flip angle equaled 90°, averages=1, slice thickness=10 mm, field of view=64×64 mm, matrix=64×64, resulting in a pixel size of 1.0×1.0×10.0 mm. All samples were run in 50 mM solutions of HEPES in water, buffered to pH 7.0 and kept at 22° C. The manganese content or iron content was systematically varied from 0.10 to 1.00 mM. The inverses of the T1 values from two separate batches of contrast agent were plotted versus the concentration of Mn(II) to obtain r1 values.
Image analysis was performed using custom Matlab programs (Mathworks, Natick, MA). The initial TI=4.8 ms image was used as a baseline to determine circular region of interest (ROI) boundaries for each sample; from these, the mean pixel magnitudes for each ROI were calculated. For each of the 36 subsequent TI images, the same ROI boundaries were applied, and the mean pixel magnitude calculations were repeated. This gave consistent ROI spatial definitions and a corresponding time course of magnitudes for each of the samples over all the TI time points. Each sample's complex phase was used to correct the magnitude polarity to produce a complete exponential T1 inversion recovery curve. The Nelder-Mead simplex algorithm (Nelder, J. A. et al., 1965, Comput. J., 7:308) was applied to each sample's exponential curve to estimate its corresponding T1 value.
In order to assess the ability of the Mn(II) complex to catalyze the degradation of H2O2, we reacted 100 nM 1 with 10 mM H2O2 in 100 mM phosphate solution buffered to pH 7.0 and monitored the absorbance at 240 nm over time. H2O2 has a molar extinction coefficient of 39.4 M−1 cm−at this wavelength (Nelson, D. P. et al., 1972, Anal. Biochem. 49:474). The consumption of H2O2 was evaluated using a UV-1601 Shimadzu spectrophotometer.
The aquation numbers (qH2O), rate constants and activation parameters for the water exchange at Fe(II) and Fe(III) centers were determined from the temperature dependence of the 17O-line broadening measured for aqueous solutions containing Fe(II) or Fe(III) complexes and for metal-free solutions buffered to pH 7.4 over the temperature range 274.2-343.2 K. The line widths at half-height of the signal were determined by a deconvolution procedure on the real part of the Fourier-transformed spectra with a Lorentzian shape function in the data analysis module of Bruker Topspin 1.3 software. The measurements were performed with a commercial 5 mm Bruker broadband probe thermostated with a Bruker B-VT 3000 variable-temperature unit. Samples were prepared by adding a solution of solid dissolved in a minimal amount of MeCN (20% (v/v)) to an aqueous solution containing 60 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffered to pH 7.4. 17O-labeled water (10%; D-Chem Ltd., Tel Aviv, Israel) was added to this solution resulting in a total enrichment of 1% 170 in the studied samples. The resultant mixture contained 9.8 mM of the Fe(II) complex. The Fe(III) complex was obtained by the oxidation of 9.8 mM Fe(II) using 10 equiv. of H2O2 in 60 mM MOPS buffered to pH 7.4. The reaction was allowed to proceed for 15 min prior to data acquisition. The NMR data were fit to the Swift-Connick equation (Swift, T. J. 1962, J. Chem. Phys. 37:307). The qH2O variable was set as free parameter, and the B value was fixed. The number of exchanging water molecules was optimized at qH2O=1.6.
The partition coefficients were obtained in n-butanol/water (log PBW), in which the Fe(II) complex was more soluble, and then correlated to a corresponding n-octanol/water value by using a calibration curve prepared with well-known standards (Ran, Y. et al., 2002, Chemosphere, 48:487). The partition between water and n-butanol was determined experimentally with the Fe(II) complex using a variation of the shake-flask method (Engelmann, F. M. et al., 2007, Int. J. Pharm., 329:12). Distilled water and n-butanol were mixed vigorously for 24 h at 25° C., to promote solvent saturation in both phases, and the solvents were separated. Then, 0.1 mL of a solution of the Fe(II)-H4qp4 complex in water saturated with n-butanol was shaken with 1 mL of n-butanol saturated with water in a plastic tube for 3 min using a Vortex-Genie. The biphasic mixture was centrifuged (3 min at 6000 rpm), and the layers were separated. The concentration of the iron complex in each layer was measured spectrophotometrically. If dilution was needed for the UV/vis measurements, the aqueous layer was diluted with water; whereas for the organic layer, water-saturated n-butanol was used instead. The spectrum baseline was chosen accordingly. The log PBW was calculated using Eq. 1:
log PBW=log(CnBuOH/CH2O) (1)
log POW=1.55(log PBW)−0.54 (2)
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application claims priority to and the benefit of U.S. Provisional Application No. 63/346,641, filed May 27, 2022, the disclosure of which is incorporated herein by reference herewith in its entirety.
This invention was made with government support under Award Nos. 1662875 and 1954336 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63346641 | May 2022 | US |
