Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
  • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
  • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
  • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
  • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
  • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• This disclosure relates to fibrin- and collagen-specific imaging agents comprising metal oxide nanoparticles.
• Diagnostic imaging techniques such as magnetic resonance imaging (MRI), X- ray, nuclear radiopharmaceutical imaging, ultraviolet-visible-infrared light imaging, and ultrasound, are often used in medical diagnosis.
• Complexes of paramagnetic metal oxide ions e.g., gadolinium oxide, iron oxide, manganese oxide
• ligands are widely used to enhance and improve imaging contrast.
• Small metal oxide particles can be used to aid kidney elimination and interstitial tissue extravasation of the metal oxide-ligand complexes following acquisition of the diagnostic image.
• the present application describes fibrin- and collagen-specific imaging agents comprising extremely small metal oxide nanoparticles, as well as pharmaceutical compositions comprising the imaging agents described herein. Also provided herein are methods for imaging fibrin and collagen in a mammal.
• FIG. 1 shows the structure of SNIO-CBP.
• FIG. 2 shows SAXS profile of SNIO-CBP in the q range corresponding to singlenanometer range in the real space; scattering profile was fitted into spherical particles with a log-normal distribution at a mean diameter of 1 .47 nm.
• FIG. 3 shows gel filtration chromatogram of SNIO-CBP showing its size purity and hydrodynamic diameter.
• FIG. 4 shows gel filtration chromatograms of SNIO-CBP incubated with FBS and FBS alone, showing minimal degree of non-specific binding of SNIO-CBP in plasma. Red and orange peaks: binding products; bottom peaks: native species in FBS.
• FIG. 5 shows binding affinity of SNIO-CBP towards human type I collagen.
• FIG. 6 shows T1 -weighted MRI study of SNIO-CBP clearance in normal mice (i.v. injection, 2 nmol/g based on CBP concentration).
• FIG. 8 shows MR imaging of liver fibrosis in a mouse model of non-alcoholic steatohepatitis using SNIO-CBP (2 nmol/g CBP).
• Mice fed with choline-deficient, lamino acid-defined, high-fat diet (CDAHFD) for 14 weeks (n 6) act as a disease group.
• Mice that received 14 weeks of standard chow (SD, n 6) were used as control.
• Significant enhanced liver signal was observed in the CDAHFD group imaged with SNIO-CBP.
• Corresponding liver histology revealed the fibrosis and retention of the probe in the disease group.
• FIG. 9 shows a schematic representation of nanoparticle synthesis.
• FIG. 10 shows zeta potential of SNIO-CBP.
• FIG. 11 shows the stability of SNIO-CBP.
• FIG. 12 shows the binding affinity of SNIO-CBP to type I collagen.
• FIG. 13 shows longitudinal relaxivity (n).
• FIG. 14 shows SNIO-CBP pharmacokinetics in normal mice.
• FIG. 15 shows CCh induced liver fibrosis model.
• FIG. 16 shows ex vivo characterizations (CCh model).
• FIG. 17 shows diet-induced liver fibrosis.
• Collagens are a class of extracellular matrix proteins that represent about 30% of total body protein and contribute to the structure of tendons, bones, and connective tissues. Abnormal accumulation of collagen in various organs can lead to fibrosis, for example, myocardial fibrosis, heart failure, nonalcoholic steatohepatitis (NASH), liver cirrhosis, and primary biliary cirrhosis; lesions in the vasculature or breasts; collagen- induced arthritis; muscular dystrophy; scleroderma; Dupuytren's disease; and rheumatoid arthritis, among other debilitating conditions.
• Fibrin is a fibrillary protein derived from the soluble plasma protein fibrinogen.
• Fibrin is a major component of blood clots and is present in all thrombi, regardless of thrombus age or bodily location. Valued in part for high specificity and sensitivity, both fibrin- and collagen-binding compounds can be used for diagnostic imaging.
• protein-binding imaging agents having paramagnetic metal oxide ions and ligands to enhance image contrast and aid analysis. These imaging agents are readily eliminated from the patient by utilizing paramagnetic metal oxide ions having smaller particle sizes while still producing high resolution images.
• peptide refers to a chain of amino acids that is about 2 to about 25 amino acid residues in length. All peptide sequences herein are written from the N- to C-terminus. For any of the peptides described herein that contain two or more cysteine residues, it is understood that the cysteine residues can form one or more disulfide bonds under non-reducing conditions. Formation of a disulfide bond can result in the formulation of a cyclic peptide.
• natural or “naturally occurring” amino acid refers to one of the twenty most common amino acids occurring in nature. Natural amino acids modified to provide a label for detection purposes (e.g., radioactive labels, optical labels, or dyes) are considered to be natural amino acids. Natural L amino acids are referred to by their standard one- or three-letter abbreviations. D amino acids are referred to using the lower-case convention for standard one-letter abbreviations, and the “D-” prefix convention for standard three-letter abbreviations.
• target binding and “binding” refer to non-covalent interactions of a peptide or composition within a target. These non-covalent interactions are independent from one another and may be, inter alia, hydrophobic, hydrophilic, dipole-dipole, pi-stacking, hydrogen bonding, electrostatic associations, and/or Lewis acid-base interactions.
• the binding affinity for a target is expressed in terms of the equilibrium dissociation constant “Kd” to the target under a defined set of conditions.
• the term “relaxivity” refers to the increase in either of the magnetic resonance imaging (MRI) quantities 1/T1 or 1/T2 per millmolar (mM) concentration of paramagnetic ion, contrast agent, or compound, wherein T1 is the longitudinal or spin-lattice, relaxation time, and T2 is the transverse or spin-spin relaxation time of water protons or other imaging or spectroscopic nuclei, including protons found in molecules other than water. Relaxivity is expressed in units of mM’ 1 s’ 1 .
• the term “purified” refers to a peptide or compound that has been separated from either naturally occurring organic molecules with which it normally associates or, for a chemically-synthesized molecule, separated from other organic molecules present in the chemical synthesis.
• the polypeptide or compound is considered “purified” when it is at least 70% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%), by dry weight, free from any other proteins or organic molecules.
• the terms “purified” and isolated” are used interchangeably herein.
• imaging agents of Formula I are provided herein.
• NP is a nanoparticle metal oxide core comprising iron oxide, manganese oxide, or gadolinium oxide;
• C is a metal oxide-binding ligand
• L is a linker moiety
• CP is a collagen-binding peptide
• n is an integer selected from 0 to 10
• m and o are each integers independently selected from 1 to 5.
• n is 0, n is 1, n is 2, n is
• n is 4, or n is 5.
• m is 1 or m is 2.
• o is 1 or o is 2.
• NP is a nanoparticle metal oxide comprising iron oxide. In some embodiments of the imaging agent of Formula I, NP is a nanoparticle metal oxide comprising manganese oxide. In some embodiments of the imaging agents of Formula I, NP is a nanoparticle metal oxide core comprising gadolinium oxide. In some embodiments of the imaging agents of Formula I, NP is a nanoparticle metal oxide core comprising iron oxide, manganese oxide, gadolinium oxide, or a combination thereof.
• the nanoparticle core can comprise iron oxide and manganese oxide.
• the nanoparticle core can comprise iron oxide and gadolinium oxide.
• the nanoparticle core can comprise manganese oxide and gadolinium oxide.
• the nanoparticle core can comprise iron oxide, manganese oxide, and gadolinium oxide.
• the nanoparticle metal oxide core comprises about 5 to about 200 metal ions.
• the nanoparticle metal oxide core comprises about 10 to about 200, about 25 to about 200, about 50 to about 200, about 75 to about 200, about 100 to about 200, about 125 to about 200, about 150 to about 200, or about 175 to about 200 metal ions.
• the nanoparticle metal oxide core comprises about 5 to about 15, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 10 to about 35, about 10 to about 40, about 10 to about 45, about 10 to about 50, about 25 to about 50, about 50 to about 100, about 75 to about 100, about 100 to about 125, about 125 to about 150, or about 150 to about 175 metal ions.
• the nanoparticle metal oxide core is about 2.0 to about 10.0 nm in diameter.
• the nanoparticle metal oxide core is about 2.5 to about 10.0, about 3.0 to about 10.0, about 3.5 to about 10.0, about 4.0 to about 10.0, about 4.5 to about 10.0, about 5.0 to about 10.0, about 5.5 to about 10.0, about 6.0 to about 10.0, about 6.5 to about 10.0, about 7.0 to about 10.0, about 7.5 to about 10.0, about 8.0 to about 10.0, about 8.5 to about 10.0, or about 9.0 to about 10.0 nm in diameter. In some embodiments, the nanoparticle metal oxide core is about 3.0 to about 5.0 nm in diameter.
• the nanoparticle metal oxide core is about 3.4 to about 4.5 nm in diameter. In some embodiments, the nanoparticle metal oxide core is about 0.5 to 1.5 nm in diameter. In some embodiments, the nanoparticle metal oxide core is about 1.0 nm in diameter.
• the hydrodynamic size of the coated nanoparticle metal oxide core is about 2.0 to about 25 nm in diameter.
• the hydrodynamic size of the coated nanoparticle metal oxide core is about 2.5 to about 25, about 3.0 to about 25, about 3.5 to about 25, about 4.0 to about 25, about 4.5 to about 25, about 5.0 to about 25, about 5.0 to about 25, about 5.5 to about 25, about 6.0 to about 25, about 6.5 to about 25, about 7.0 to about 25, about 8.0 to about 25, about 8.5 to about 25, about 9.0 to about 25, about 9.5 to about 25, about 10 to about 25, about 12 to about 25, about 15 to about 25, about 17 to about 25, about 20 to about 25, or about 22 to about 25 nm in diameter. In some embodiments, the hydrodynamic size of the coated nanoparticle metal oxide core is about 2.9 to about 21 nm in diameter.
• the metal oxidebinding ligand is a benzenediol or a hydroxy carboxylic acid. In some embodiments of the imaging agents of Formula I, the metal oxide-binding ligand is a benzenediol. In some embodiments of the imaging agents of Formula I, the metal oxide-binding ligand is a hydroxy carboxylic acid.
• the benzenediol has the formula: wherein X and Y are independently selected from CR 8 and N;
• R 1 is selected from the group consisting of-CFFCFbCOOH, -CH2CH2NH2,
• x is an integer selected from 1-9;
• R 2 is H or an electron withdrawing group;
• R 8 is selected from the group consisting of H and Ci-Ce alkyl.
• the benzenediol has the formula:
• x is an integer selected from 1-9;
• R 2 is H or an electron withdrawing group
• R 8 is selected from the group consisting of H and Ci-Ce alkyl wherein indicates the points of attachment of the specified group to the nanoparticle metal oxide core.
• the benzenediol is indicates the points of attachment of the specified group to the linker.
• X is CR 8 , wherein R 8 is selected from the group consisting of H and Ci-Ce alkyl.
• R 8 is selected from the group consisting of H and Ci-Ce alkyl.
• X can be CH, CMe, and CEt.
• X is N.
• R 2 is H. In some embodiments of the imaging agents of Formula I, R 2 is an electron withdrawing group.
• R 2 can be -NO2, -SO3H, -SOiNa, -CF3, -SO2CF3, or - CN.
• Y is CR 8 , wherein R 8 is selected from the group consisting of H and Ci-Ce alkyl.
• R 8 is selected from the group consisting of H and Ci-Ce alkyl.
• Y can be CH, CMe, and CEt.
• Y is N.
• the hydroxy carboxylic acid is an a-hydroxy carboxylic acid.
• the hydroxy carboxylic acid can be lactic acid or glycolic acid.
• the hydroxy carboxylic acid is a -hydroxy carboxylic acid.
• the hydroxy carboxylic acid can be tropic acid or citric acid.
• the hydroxy carboxylic acid has the formula: wherein p is an integer selected from 1-4;
• R 3 is selected from the group consisting of H, -(CH2) q C(0)-, and -(CH2) q NH-; q is an integer selected from 1-5; or a pharmaceutically acceptable salt thereof.
• the hydroxy carboxylic acid has the formula: wherein indicates the points of attachment of the specified group to the nanoparticle metal oxide core.
• R 3 is -(CH 2 ) q C(O)- or -(CH 2 ) q NH-.
• the hydroxy carboxylic wherein ⁇ indicates the points of attachment of the specified group to the linker.
• the linker moiety has a molecular weight of about 200 to about 800 amu.
• the linker has a molecular weight of about 250 to about 800, about 300 to about 800, about 350 to about 800, about 400 to about 800, about 450 to about 800, about 500 to about 800, about 550 to about 800, about 600 to about 800, about 650 to about 800, about 700 to about 800, or about 750 to about 800 amu.
• the linker moiety has a molecular weight of about 250 to about 600, about 300 to about 650, about 350 to about 700, or about 400 to about 750 amu.
• the linker moiety is selected from the group consisting of -CH2CH2O-, -CH2CH2OCH2CH2O-, - CH2CH2O(CH 2 CH 2 O)2-, -CH2CH 2 O(CH 2 CH2O)3-, -CH 2 CH2O(CH 2 CH2O)4-, and - CH2CH2O(CH2CH 2 O) 5 -.
• the linker moiety is n some embodiments of the imaging agents of Formula I.
• the collagen-binding peptide is about 2 to about 25 amino acid residues in length. For example, about 2 to about 23, about 2 to about 20, about 2 to about 18, about 2 to about 15, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 5, or about 2 to about 4 amino acid residues in length. In some embodiments of the imaging agents of Formula I, the collagen-binding peptide is about 5 to about 25, about 8 to about 25, about 10 to about 25, about 12 to about 25, about 15 to about 25, about 18 to about 25, about 20 to about 25, or about 22 to about 25 amino acid residues in length.
• the collagen-binding peptide is about 5 to about 10, about 5 to about 15, about 10 to about 12, about 10 to about 15, about 10 to about 20, about 15 to about 18, or about 15 to about 20 amino acid residues in length.
• the collagen-binding peptide has the formula:
• the imaging agents of Formula I has the formula:
• nanoparticle metal oxide core comprising iron oxide, manganese oxide, gadolinium oxide, or a combination thereof
• X and Y are independently selected from CH and N;
• R 2 is selected from the group consisting of H, -NO2, -CF3, -SO3H, SChNa, -CHCHSOiNa; and n is an integer selected from 1-6.
• the imaging agents further comprises an additional metal oxide-binding ligand.
• the additional metal oxide-binding ligand wherein indicates the points of attachment of the specified group to the nanoparticle metal oxide core.
• the metal oxide-binding ligand and the additional metal oxide-binding ligand cover about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of the surface of the nanoparticle metal oxide core. In some embodiments, the metal oxide-binding ligand covers about 1-10%, 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of the surface of the nanoparticle metal oxide core.
• the additional metal oxide-binding ligand cover about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of the surface of the nanoparticle metal oxide core.
• the ratio of the metal oxide-binding ligand to the additional metal oxide-binding ligand on the surface of the nanoparticle metal oxide core of about 1 : 1, 1 :5, 1 : 10, 1 :15, 1 :20, 20: 1, 15: 1, 10: 1, or 5: 1.
• the ratio is about 1.1.
• the ratio is about 1.5.
• the ratio is about 1.10.
• the ratio is about 1.15.
• the ratio is about 1.20.
• the ratio is about 20: 1.
• the ratio is about 15: 1.
• the ratio is about 10: 1.
• the ratio is about 5: 1.
• the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is selected from the group consisting of -SO3H and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, X is N, Y is CH, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is N, Y is CH, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is N, Y is CH, and R 2 is selected from the group consisting of -SO3H and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is N, Y is CH, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, X is CH, Y is N, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is N, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is N, and R 2 is selected from the group consisting of -SO3H and -SC Na. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is N, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, X is CH, Y is CH, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is CH, Y is CH, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is CH, Y is CH, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is CH, Y is CH, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, X is N, Y is CH, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is N, Y is CH, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is N, Y is CH, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is N, Y is CH, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, X is CH, Y is N, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is CH, Y is N, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is CH, Y is N, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, X is CH, Y is N, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, X is CH, Y is CH, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, X is N, Y is CH, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, X is N, Y is CH, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, X is N, Y is CH, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, X is N, Y is CH, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, X is CH, Y is N, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, X is CH, Y is N, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, X is CH, Y is N, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, X is CH, Y is N, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is lactic acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is selected from the group consisting of- SO3H and -SOsNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is glycolic acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is selected from the group consisting of-SC H and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is citric acid, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is citric acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is citric acid, and R 2 is selected from the group consisting of -SO3H and -SC Na. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is citric acid, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is H, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid i integer selected from 1-4, R 3 is H, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is
• R 2 is selected from the group consisting of -SO3H and -SChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is H, and R 2 is
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is - (CH2)qC(O)- , q is an integer selected from 0-5, and R 2 is -NO2.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is -CF3.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is selected from the group consisting of -SChH and -SOaNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is -CHCHSChNa
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 3 is -(CH2) q NH-, q is an integer selected from 0-5, and R 2 is -CF3.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2) q NH-, q is an integer selected from 0-5, and R 2 is selected from the group consisting of -SChH and -SOaNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2) q NH-, q is an integer selected from 0-5, and R 2 is -CHCHSOiNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is -NO2.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is lactic acid, and R 2 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is - CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is glycolic acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is selected from the group consisting of -SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is citric acid, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is citric acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is citric acid, and R 2 is selected from the group consisting of-SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is citric acid, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is H, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid i integer selected from 1-4,
• R 3 is H, and R 2 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is H, and R 2 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is H, and R 2 is -
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is - (CH2)qC(O)- , q is an integer selected from 0-5, and R 2 is -NO2.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is integer selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2) q NH-, q is an integer selected from 0-5, and R 2 is -NO2.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 3 is -(CH2) q NH- q is an integer selected from 0-5, and R 2 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2) q NH-, q is an integer selected from 0-5, and R 2 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is integer selected from 1-4, R 3 is -(CH2) q NH-, q is an integer selected from 0-5, and R 2 is -CHCHSC Na.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is -NO2.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is lactic acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is lactic acid, and R 2 is -CHCHSOiNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is glycolic acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is selected from the group consisting of -SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is glycolic acid, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is citric acid, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is citric acid, and R 2 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is citric acid, and R 2 is selected from the group consisting of-SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is citric acid, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is H, and R 2 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid i integer selected from 1-4,
• R 3 is H, and R 2 is -CF3.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is H, and R 2 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is
• R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2)qC(O)-, q is an integer selected from 0-5, and R 2 is -NO2.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is -CF3.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is integer selected from 1-4, R 3 is -(CH2) q C(O)-, q is an integer selected from 0-5, and R 2 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is
• the nanoparticle metal oxide core is gadolinium oxide
• the hydroxy carboxlic acid is integer selected from 1-4
• R 3 is -(CH2) q NH- q is an integer selected from 0-5, and R 2 is -CF3.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 3 is -(CH2) q NH-, q is an integer selected from 0-5, and R 2 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is gadolinium oxide
• the hydroxy carboxylic acid is integer selected from 1-4
• R 3 is -(CH2) q NH-
• q is an integer selected from 0-5
• R 2 is -CHCHSC Na.
• Collagen-binding peptides as described herein have an affinity for the extracellular matrix protein collagen, including human and other animal Collagen Type I. Collagens are particularly useful extracellular matrix proteins to target.
• Collagens I and III are the most abundant components of the extracellular matrix of myocardial tissue, representing over 90% of total myocardial collagen and about 5% of dry myocardial weight. The ratio of collagen I to collagen III in the myocardium in approximately 2:1, and their total concentration is approximately 100 pM in the extracellular matrix.
• Human collagen type l is a trimer of two chains with an [al(I)]2 [a2(I)] stoichiometry characterized by a repeating G-X-Y sequence motif, where X is most frequently proline and Y is frequently hydroxyproline.
• a compound described herein can have an affinity for human, rat, and/or dog collagen type I.
• Peptides can be assayed for affinity to the appropriate extracellular matrix protein by methods as disclosed in WO 01/09188 and WO 01/08712, and as described below.
• peptides can be screened for binding to an extracellular matrix protein by methods well known in the art, including pull-down assays, equilibrium dialysis, affinity chromatography, and inhibition or displacement of probes bound to the matrix protein.
• peptides can be evaluated for their ability to bind to collagen, such as dried human, rat or dog collagen type I.
• a collagen binding peptide can bind human collagen with a dissociation constant of less than 25 pM, less than 10 pM, less than 5 pM, less than 1 pM, or less than 100 nM. In some embodiments the collagen binding peptide can bind rat collagen with a dissociation constant of less than 25 pM, less than 10 pM, less than 5 pM, less than 1 pM, or less than 100 nM. In some embodiments the collagen binding peptide can bind dog collagen with a dissociation constant of less than 25 pM, less than 10 pM, less than 5 pM, less than 1 pM, or less than 100 nM.
• Peptides may be synthesized directly using conventional techniques, including solid-phase peptide synthesis, solution-phase synthesis, etc. See, for example, Stewart et al., Solid-Phase peptide Synthesis (1989), W.H. Freeman Co., San Francisco; Merrifield, J. Am. Chem. Soc., 1963 85:2149-2145; and Bodanszky and Bodanszky, The Practice of Peptide Synthesis (1984), Springer-Verlag, New York. Peptides may also be prepared or purchased commercially. Automated peptide synthesis machines, such as manufactured by Perkin-Elmer Applied Biosystems, may also be used.
• the collagen binding peptide can be purified once it has been isolated or synthesized by either chemical or recombinant techniques.
• purification purposes there are many standard methods that may be employed including reversed-phase high- pressure liquid chromatography (RP-HPLC) using an alkylated silica column such as C4-, Cs- or Cis-silica.
• RP-HPLC reversed-phase high- pressure liquid chromatography
• a gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer.
• the mobile phase can also include a small amount of trifluoroacetic acid.
• Ion-exchange chromatography can also be used to separate peptides based on their charge.
• the degree of purity of the collagen binding peptide may be determined by various methods, including identification of a major large peak on HPLC.
• the peptide produces a single peak that is at least 95% of the input material on an HPLC column.
• the peptide produces a single peak that is at least 97%, at least 98%, at least 99% or even 99.5% of the input material on an HPLC column.
• MR compounds can exhibit high relaxivity as a result of binding to collagen, which can lead to better image resolution.
• the increase in relaxivity upon binding is 1.5-fold or more (e.g., at least a 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold increase in relaxivity).
• targeted MR compounds can increase the relaxivity upon binding by 7-8 fold, 9-10 fold, or even greater than 10 fold.
• relaxivity is measured using an NMR spectrometer.
• the relaxivity of an MRI compound at 20 MHz and 37 °C is at least 8 mM’ 1 s' 1 per paramagnetic metal ion (e.g., at least 10, 15, 20, 25, 30, 35, 40, or 60 mM' 1 s' 1 per paramagnetic metal ion).
• the MR compounds have a relaxivity greater than 60 mM' l s' 1 at 20 MHz and 37 °C.
• targeted MR compounds can be taken up selectively by areas in the body having higher concentrations of collagen relative to other areas.
• Selectivity of uptake of targeted agents can be determined by comparing the uptake of the agents by myocardium as compared to the uptake by blood.
• the selectivity of targeted compounds also can be demonstrated using MRI and observing enhancement of myocardial signal as compared to blood signal.
• imaging agents of Formula II are provided herein.
• NP is a nanoparticle metal oxide core comprising iron oxide, manganese oxide, or gadolinium oxide;
• C is a metal oxide-binding ligand
• L is a linker moiety
• FP is a fibrin-binding peptide
• b is an integer selected from 0 to 10
• a and c are each integers independently selected from 1 to 5.
• a is 0, a is 1, a is 2, a is 3, a is 4, or a is 5.
• b is 1 or b is 2.
• c is 1 or c is 2.
• NP is a nanoparticle metal oxide comprising iron oxide. In some embodiments of the imaging agents of Formula II, NP is a nanoparticle metal oxide comprising manganese oxide. In some embodiments of the imaging agents of Formula II, NP is a nanoparticle metal oxide core comprising gadolinium oxide. In some embodiments of the imaging agents of Formula II, NP is a nanoparticle metal oxide core comprising iron oxide, manganese oxide, gadaolinium oxide, or a combination thereof.
• the nanoparticle core can comprise iron oxide and manganese oxide.
• the nanoparticle core can comprise iron oxide and gadolinium oxide.
• the nanoparticle core can comprise manganese oxide and gadolinium oxide.
• the nanoparticle core can comprise iron oxide, manganese oxide, and gadolinium oxide.
• the nanoparticle metal oxide core comprises about 5 to about 200 metal ions.
• the nanoparticle metal oxide core comprises about 10 to about 200, about 25 to about 200, about 50 to about 200, about 75 to about 200, about 100 to about 200, about 125 to about 200, about 150 to about 200, or about 175 to about 200 metal ions.
• the nanoparticle metal oxide core comprises about 5 and about 15, about 10 and about 15, about 10 and about 20, about 10 and about 25, about 10 and about 30, about 10 and about 35, about 10 and about 40, about 10 and about 45, about 10 and about 50, about 25 and about 50, about 50 and about 100, about 75 and about 100, about 100 and about 125, about 125 to about 150, or about 150 to about 175 metal ions.
• the nanoparticle metal oxide core is about 2.0 to about 10.0 nm in diameter.
• the nanoparticle metal oxide core is about 2.5 to about 10.0, about 3.0 to about 10.0, about 3.5 to about 10.0, about 4.0 to about 10.0, about 4.5 to about 10.0, about 5.0 to about 10.0, about 5.5 to about 10.0, about 6.0 to about 10.0, about 6.5 to about 10.0, about 7.0 to about 10.0, about 7.5 to about 10.0, about 8.0 to about 10.0, about 8.5 to about 10.0, or about 9.0 to about 10.0 nm in diameter. In some embodiments, the nanoparticle metal oxide core is about 3.0 to about 5.0 nm in diameter.
• the nanoparticle metal oxide core is about 3.4 to about 4.5 nm in diameter.
• the hydrodynamic size of the coated nanoparticle metal oxide core is about 2.0 to about 25 nm in diameter.
• the hydrodynamic size of the coated nanoparticle metal oxide core is about 2.5 to about 25, about 3.0 to about 25, about 3.5 to about 25, about 4.0 to about 25, about 4.5 to about 25, about 5.0 to about 25, about 5.0 to about 25, about 5.5 to about 25, about 6.0 to about 25, about 6.5 to about 25, about 7.0 to about 25, about 8.0 to about 25, about 8.5 to about 25, about 9.0 to about 25, about 9.5 to about 25, about 10 to about 25, about 12 to about 25, about 15 to about 25, about 17 to about 25, about 20 to about 25, or about 22 to about 25 nm in diameter. In some embodiments, the hydrodynamic size of the coated nanoparticle metal oxide core is about 2.9 to about 21 nm in diameter.
• the metal oxi debinding ligand is a benzenediol or a hydroxy carboxylic acid. In some embodiments of the imaging agents of Formula II, the metal oxide-binding ligand is a benzenediol. In some embodiments of the imaging agents of Formula II, the metal oxide-binding ligand is a hydroxy carboxylic acid.
• the benzenediol has the formula: wherein A and B are independently selected from CR 9 and N;
• R 6 is H or an electron withdrawing group;
• R 9 is selected from the group consisting of H and Ci-Ce alkyl.
• the benzenediol has the formula:
• d is an integer selected from 1-9;
• R 6 is H or an electron withdrawing group
• R 9 is selected from the group consisting of H and Ci-Ce alkyl wherein indicates the points of attachment of the specified group to the nanoparticle metal oxide core.
• the benzenediol is indicates the points of attachment of the specified group to the linker.
• X is CR 9 , wherein R 9 is selected from the group consisting of H and Ci-Ce alkyl.
• R 9 is selected from the group consisting of H and Ci-Ce alkyl.
• X can be CH, CMe, and CEt.
• X is N.
• R 6 is H. In some embodiments of the imaging agents of Formula II, R 6 is an electron withdrawing group.
• R 6 can be -NO2, -SO3H, -SChNa, -CF3, -SO2CF3, or -CN.
• the hydroxy carboxylic acid is an a-hydroxy carboxylic acid.
• the hydroxy carboxylic acid can be lactic acid or glycolic acid.
• the hydroxy carboxylic acid is a P-hydroxy carboxylic acid.
• the hydroxy carboxylic acid can be tropic acid or citric acid.
• the hydroxy carboxylic acid has the formula: wherein e is an integer selected from 1-4;
• R 7 is selected from the group consisting of H, -(CH 2 )IC(O)-, and -(CH 2 )fNH-; f is an integer selected from 0-5; or a pharmaceutically acceptable salt thereof.
• the hydroxy carboxylic acid has the formula: wherein '''’1 indicates the points of attachment of the specified group to the nanoparticle metal oxide core.
• R 7 is -(CH 2 )fC(O)- or -(CH 2 )fNH-.
• the hydroxy carboxylic acid wherein indicates the points of attachment of the specified group to the linker.
• the linker moiety has a molecular weight of about 200 to about 800 amu.
• the linker has a molecular weight of about 250 to about 800, about 300 to about 800, about 350 to about 800, about 400 to about 800, about 450 to about 800, about 500 to about 800, about 550 to about 800, about 600 to about 800, about 650 to about 800, about 700 to about 800, or about 750 to about
• the linker moiety has a molecular weight of about 250 to about 600, about 300 to about 650, about 350 to about 700, or about 400 to about 750 amu.
• the linker moiety is selected from the group consisting of-NHCH(R 4 )CO-, -NH(CH2) g C(O)-, NHCH2CH 2 OCH2CH2CH 2 C(O)-, -NHCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 C(O)-, NHCH2C6H4CH2NH-, -NH(CH 2 )hNH- -NHCH2OCH2NH-, -NHCH 2 CH2OCH2CH 2 NH-, -NHCH2CH 2 OCH 2 CH2OCH 2 CH2NH-
• R 4 is an amino acid side chain; h is an integer selected from 2-6; i is an integer selected from 2-10; and the linker can be read either right-to-left or left-to-right; wherein the indicates the points of attachment of the specified group to the fibrin-binding peptide.
• the linker moiety is selected from the group consisting of -CH 2 CH 2 O-, -CH 2 CH 2 OCH 2 CH 2 O-, - CH 2 CH 2 O(CH 2 CH 2 O) 2 -, -CH 2 CH 2 O(CH 2 CH 2 O) 3 -, -CH 2 CH 2 O(CH 2 CH 2 O)4-, and - CH 2 CH 2 O(CH 2 CH 2 O)s-.
• the linker moiety is
• the fibrin-binding peptide is about 2 to about 25 amino acid residues in length. For example, about 2 to about 23, about 2 to about 20, about 2 to about 18, about 2 to about 15, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 5, or about 2 to about 4 amino acid residues in length. In some embodiments of the imaging agents of Formula II, the fibrin-binding peptide is about 5 to about 25, about 8 to about 25, about 10 to about 25, about 12 to about 25, about 15 to about 25, about 18 to about 25, about 20 to about 25, or about 22 to about 25 amino acid residues in length.
• the fibrin-binding peptide is about 5 to about 10, about 5 to about 15, about 10 to about 12, about 10 to about 15, about 10 to about 20, about 15 to about 18, or about 15 to about 20 amino acid residues in length.
• the fibrin-binding peptide has the formula:
• X 1 is selected from the group consisting of:
• X 2 is selected from the group consisting of:
• X 3 is selected from the group consisting of H and OH;
• X 4 is selected from the group consisting of H, I, Br, and Cl;
• X 5 is selected from the group consisting of H and CH2COOH;
• X 6 is selected from the group consisting of:
• X 7 is selected from the group consisting of CH2CH2C(O)NH2 and CH2CH(CH3)2; wherein the indicates the point of attachment of the specified group to the fibrin- binding peptide.
• the imaging agents of Formula II has the formula:
• a and B are independently selected from CH and N;
• R 6 is selected from the group consisting of -H, -NO2, -CF3, -SO3H, -SChNa,
• n is an integer selected from 1-6.
• the imaging agents further comprises an additional metal oxide-binding ligand.
• the additional metal oxide-binding ligand indicates the points of attachment of the specified group to the nanoparticle metal oxide core.
• the metal oxide-binding ligand and the additional metal oxide-binding ligand cover about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of the surface of the nanoparticle metal oxide core. In some embodiments, the metal oxide-binding ligand covers about 1-10%, 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of the surface of the nanoparticle metal oxide core.
• the additional metal oxide-binding ligand cover about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of the surface of the nanoparticle metal oxide core.
• the ratio of the metal oxide-binding ligand to the additional metal oxide-binding ligand on the surface of the nanoparticle metal oxide core of about 1 : 1, 1 :5, 1 : 10, 1 :15, 1 :20, 20: 1, 15: 1, 10: 1, or 5: 1.
• the ratio is about 1.1.
• the ratio is about 1.5.
• the ratio is about 1.10.
• the ratio is about 1.15.
• the ratio is about 1.20.
• the ratio is about 20: 1.
• the ratio is about 15: 1.
• the ratio is about 10: 1.
• the ratio is about 5: 1.
• the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is selected from the group consisting of-SC H and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, A is N, B is CH, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is N, B is CH, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is N, B is CH, and R 6 is selected from the group consisting of -SO3H and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is N, B is CH, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, A is CH, B is N, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is N, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is N, and R 6 is selected from the group consisting of -SO3H and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is N, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, A is CH, B is CH, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is CH, B is CH, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is CH, B is CH, and R 6 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is CH, B is CH, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, A is N, B is CH, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is N, B is CH, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is N, B is CH, and R 6 is selected from the group consisting of-SOiH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is N, B is CH, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, A is CH, B is N, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is CH, B is N, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is CH, B is N, and R 6 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, A is CH, B is N, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is selected from the group consisting of-SChH and -SChNa In some embodiments, the nanoparticle metal oxide core is iron oxide, A is CH, B is CH, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, A is N, B is CH, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, A is N, B is CH, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, A is N, B is CH, and R 6 is selected from the group consisting of-SOsH and -SChNa. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, A is N, B is CH, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, A is CH, B is N, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, A is CH, B is N, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, A is CH, B is N, and R 6 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, A is CH, B is N, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is lactic acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is selected from the group consisting of-
• the nanoparticle metal oxide core is iron oxide
• the hydroxy carboxylic acid is lactic acid
• R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is glycolic acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is selected from the group consisting of-SC H and -SChNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is citric acid, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is citric acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is citric acid, and R 6 is selected from the group consisting of- SO3H and -SOsNa. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid is citric acid, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is H, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid i integer selected from 1-4, R 7 is H, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is
• R 6 is selected from the group consisting of -SO3H and -SChNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is H, and R 6 is
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -NO2.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -CF3.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is selected from the group consisting of -SChH and -SOaNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -CHCHSChNa
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 7 is -(CH2)fNH- f is an integer selected from 0-5, and R 6 is -CF3.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fNH-, f is an integer selected from 0-5, and R 6 is selected from the group consisting of -SChH and -SOaNa.
• the nanoparticle metal oxide core is iron oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)rNH- f is an integer selected from 0-5, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is -NO2.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is lactic acid, and R 6 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is -CHCHSOiNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is glycolic acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is selected from the group consisting of -SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is citric acid, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is citric acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is citric acid, and R 6 is selected from the group consisting of-SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is citric acid, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is H, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid i integer selected from 1-4,
• R 7 is H, and R 6 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is H, and R 6 is selected from the group consisting of -SOaH and -SChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is H, and R 6 is -CHCHSC Na.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -NO2.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid is integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fNH- f is an integer selected from 0-5, and R 6 is -NO2.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 7 is -(CH2)fNH-, f is an integer selected from 0-5, and R 6 is -CF3.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fNH- f is an integer selected from 0-5, and R 6 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is manganese oxide, the hydroxy carboxylic acid i is an integer selected from 1-4, R 7 is -(CH2)rNH- f is an integer selected from 0-5, and R 6 is -CHCHSOiNa
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is -NO2.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is lactic acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is selected from the group consisting of-SChH and -SChNa. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is lactic acid, and R 6 is -CHCHSOiNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is glycolic acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is selected from the group consisting of -SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is glycolic acid, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is citric acid, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is citric acid, and R 6 is -CF3. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is citric acid, and R 6 is selected from the group consisting of-SChH and -SC Na. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is citric acid, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is H, and R 6 is -NO2. In some embodiments, the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid i integer selected from 1-4,
• R 7 is H, and R 6 is -CF3.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is H, and R 6 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is
• R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -NO2.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxlic acid is integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -CF3.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid is integer selected from 1-4, R 7 is -(CH2)fC(O)-, f is an integer selected from 0-5, and R 6 is -CHCHSChNa.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is
• the nanoparticle metal oxide core is gadolinium oxide
• the hydroxy carboxlic acid is integer selected from 1-4
• R 7 is -(CH2)fNH-
• f is an integer selected from 0-5
• R 6 is -CF3.
• the nanoparticle metal oxide core is gadolinium oxide, the hydroxy carboxylic acid i integer selected from 1-4, R 7 is -(CH2)fNH- f is an integer selected from 0-5, and R 6 is selected from the group consisting of-SChH and -SChNa.
• the nanoparticle metal oxide core is gadolinium oxide
• the hydroxy carboxylic acid i is an integer selected from 1-4
• R 7 is -(CH2)rNH- f is an integer selected from 0-5
• R 6 is -CHCHSOiNa Fibrin-Binding Imaging Agents
• Fibrin-binding peptides as described herein have an affinity for fibrin (e.g., thrombi, solid tumors, and atherosclerotic plaques) within a mammal. Any peptide capable of binding fibrin may be used.
• fibrin e.g., thrombi, solid tumors, and atherosclerotic plaques
• Any peptide capable of binding fibrin may be used.
• the peptides disclosed in WO 2008/071679, U.S. Patent Nos. 6,984,373; 6,991,775; and 7,238,341 and U.S. Patent Application No. 2005/0261472 may be used.
• the ability of peptides to bind fibrin can be assessed by known methodology.
• affinity of the peptide for fibrin can be assessed using the DD(E) fragment of fibrin, which contains subunits of 55 kD (Fragment E) and 190 kD (Fragment DD).
• the DD(E) fragment can be biotinylated and immobilized via avidin to a solid substrate (e.g., a multi-well plate). Peptides can be incubated with the immobilized DD(E) fragment in a suitable buffer and biding detected using known methodology. See, for example, WO 2001/09188.
• Binding can also be assessed in a blood plasma-derived clot assay (see e.g., Overoye-Chan et al. J. Am. Chem. Soc. 2008 130:6025-39).
• known concentrations of peptide are incubated in blood plasma (human or other species), and thrombin is added to induce clot formation.
• the clot is separated from the serum, and the concentration of the peptide in the serum ([peptide]free) is determined (e.g., by HPLC or if the peptide is labeled with a fluorophore by fluorescence, or if labeled by a radionuclide concentration is determined by radioactivity).
• Binding can also be assessed by a dried fibrin assay.
• purified fibrinogen 2.5 mg/mL; 7 /M fibrin
• thrombin a dried fibrin assay.
• the resulting clots bind to the plate without loss of protein.
• the clots are rehydrated with buffer containing known concentrations of peptide.
• the concentration of peptide in the supernatant ([peptide]free) is determined (e.g., by HPLC or if the peptide is labeled with a fluorophore by fluorescence, or if labeled by a radionuclide concentration is determined by radioactivity).
• a dissociation constant (Kd) for fibrin binding can be determined by fitting a plot of [peptide]bound versus [peptide]free to either a stoichiometric (see e.g.
• Peptides may be synthesized directly using conventional techniques, including solid-phase peptide synthesis, solution-phase synthesis, etc. See, for example, Stewart et al., Solid-Phase peptide Synthesis (1989), W.H. Freeman Co., San Francisco; Merrifield, J. Am. Chem. Soc., 1963 85:2149-2145; Bodanszky and Bodanszky, The Practice of Peptide Synthesis (1984), Springer-Verlag, New York. Peptides may also be prepared or purchased commercially. Automated peptide synthesis machines, such as manufactured by Perkin-Elmer Applied Biosystems, may also be used.
• the fibrin binding peptide is purified once it has been isolated or synthesized by either chemical or recombinant techniques.
• purification purposes there are many standard methods that may be employed including reversed- phase high-pressure liquid chromatography (RP-HPLC) using an alkylated silica column such as C4-, Cs- or Cis-silica.
• RP-HPLC reversed- phase high-pressure liquid chromatography
• a gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid.
• Ion-exchange chromatography can also be used to separate peptides based on their charge.
• the degree of purity of the fibrin binding peptide may be determined by various methods, including identification of a major large peak on HPLC.
• the peptide produces a single peak that is at least 95% of the input material on an HPLC column.
• the peptide produces a single peak that is at least 97%, at least 98%, at least 99% or at least 99.5% of the input material on an HPLC column.
• the imaging agents of the present disclosure can be formulated as a pharmaceutical composition in accordance with routine procedures.
• a pharmaceutical composition comprises a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
• a pharmaceutical composition comprises a compound of Formula II, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
• the imaging agents can include pharmaceutically acceptable derivatives thereof.
• “Pharmaceutically acceptable” means that the compound or composition can be administered to an animal without unacceptable adverse effects.
• a “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of the imaging agents of the present disclosure that, upon administration to a recipient, is capable of providing (directly or indirectly) the imaging agents or an active metabolite or residue thereof.
• Other derivatives are those that increase the bioavailability of the imaging agents when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood), or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) thereby increasing the exposure relative to the parent species.
• Pharmaceutically acceptable salts of the imaging agents of the present disclosure include counter ions derived from pharmaceutically acceptable inorganic and organic acids and bases known in the art. For example, alkali and alkaline earth metal cations; sodium; primary, secondary and tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethylglucamine, N-methylglucamine; and amino acids such as lysine, arginine and ornithine.
• compositions described herein can be administered by any route, including both oral and parenteral administration.
• Parenteral administration includes, but is not limited to, subcutaneous, intravenous, intraarterial, interstitial, intrathecal, and intracavity administration.
• pharmaceutical compositions may be given as a bolus, as two or more closes separated in time, or as a constant or non-linear flow infusion.
• compositions of the present disclosure can be formulated for any route of administration.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent, a stabilizing agent, and a local anesthetic such as lidocaine to ease pain at the site of the injection.
• the composition for intravenous administration may include 80 millimolar sucrose.
• the ingredients will be supplied either separately, e.g. in a kit, or mixed together in a unit dosage form, for example, as a dry lyophilized powder or water free concentrate.
• the composition may be stored in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent in activity units.
• compositions of the present disclosure comprise the imaging agents described herein and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable ingredient, excipient, carrier, adjuvant or vehicle.
• compositions of the present disclosure are administered to the patient in the form of an injectable composition.
• the method of administering a compound is parenterally, meaning intravenously, intra-arterially, intrathecally, interstitially or intracavitarilly.
• Pharmaceutical compositions of this disclosure can be administered to animals including humans in a manner similar to other diagnostic or therapeutic agents.
• the dosage to be administered, and the mode of administration will depend on a variety of factors including age, weight, sex, condition of the patient and genetic factors, and will ultimately be decided by medical personnel subsequent to experimental determinations of varying dosage followed by imaging as described herein.
• a method for imaging collagen in a mammal comprises administering to the mammal an effective amount of an imaging agents of Formula I, or a pharmaceutically acceptable salt thereof; acquiring an image of the collagen of the mammal using a nuclear imaging technique and acquiring an image of the mammal using magnetic resonance imaging; and overlaying said images to localize the collagen within the anatomical image of the mammal.
• the image of the collagen of the mammal using a nuclear imaging technique and the image of the mammal using magnetic resonance imaging are acquired simultaneously.
• the image of the collagen of the mammal using a nuclear imaging technique is acquired first, and the image of the mammal using magnetic resonance imaging is acquired second.
• the imagine of the mammal using magnetic resonance imaging is acquired first, and the image of the collagen of the mammal using a nuclear imaging technique is acquired second.
• a method for imaging collagen in a mammal comprises administering to the mammal an effective amount of an imaging agents of Formula I, or a pharmaceutically acceptable salt thereof; then acquiring an image of the collagen of the mammal using a nuclear imaging technique and acquiring an image of the mammal using computed tomography; then overlaying said images to localize the collagen within the anatomical image of the mammal.
• the image of the collagen of the mammal using a nuclear imaging technique and the image of the mammal using computed tomography are acquired simultaneously.
• the image of the collagen of the mammal using a nuclear imaging technique is acquired first, and the image of the mammal using computed tomography is acquired second.
• the image of the mammal using computed tomography is acquired first, and the image of the collagen of the mammal using a nuclear imaging technique is acquired second.
• the nuclear imaging technique is single photon emission, computed tomography, or a combination thereof.
• the mammal is a human.
• the mammal is a rat.
• the mammal is a dog.
• the method further comprises administering to the mammal an effective amount of a second imaging agents.
• the second imaging agents does not target collagen.
• the second imaging agents comprises an MRI imaging agents.
• the second imaging agents is an MRI imaging agents.
• gadoteridol, gadopentetate, gadobenate, gadoxetic acid, gadodiamide, gadoversetamide, and gadofosveset or a CT imaging agents selected from the group consisting of iopamidol, iohexol, ioxilan, iopromide, iodixanol, ioxaglate, metrizoate, and diatrizoate.
• a method for imaging fibrin in a mammal comprises administering to the mammal an effective amount of an imaging agents of Formula I, or a pharmaceutically acceptable salt thereof; acquiring an image of the fibrin of the mammal using a nuclear imaging technique and acquiring an image of the mammal using magnetic resonance imaging; and overlaying said images to localize the fibrin within the anatomical image of the mammal.
• the image of the fibrin of the mammal using a nuclear imaging technique and the image of the mammal using magnetic resonance imaging are acquired simultaneously.
• the image of the fibrin of the mammal using a nuclear imaging technique is acquired first, and the image of the mammal using magnetic resonance imaging is acquired second.
• the image of the mammal using magnetic resonance imaging is acquired first, and the image of the fibrin of the mammal using a nuclear imaging technique is acquired second.
• a method for imaging fibrin in a mammal comprises administering to the mammal an effective amount of an imaging agents of Formula I, or a pharmaceutically acceptable salt thereof; then acquiring an image of the fibrin of the mammal using a nuclear imaging technique and acquiring an image of the mammal using computed tomography; then overlaying said images to localize the fibrin within the anatomical image of the mammal.
• the image of the fibrin of the mammal using a nuclear imaging technique and the image of the mammal using computed tomography are acquired simultaneously.
• the image of the fibrin of the mammal using a nuclear imaging technique is acquired first, and the image of the mammal using computed tomography is acquired second.
• the image of the mammal using computed tomography is acquired first, and the image of the fibrin of the mammal using a nuclear imaging technique is acquired second.
• the nuclear imaging technique is single photon emission, computed tomography, or a combination thereof.
• the mammal is a human. In some embodiments of the method for imaging fibrin, the mammal is a rat. In some embodiments of the method for imaging fibrin, the mammal is a dog.
• the method further comprises administering to the mammal an effective amount of a second imaging agents.
• the second imaging agents does not target fibrin.
• the second imaging agents comprises an MRI imaging agents.
• the second imaging agents is an MRI imaging agents.
• gadoteridol, gadopentetate, gadobenate, gadoxetic acid, gadodiamide, gadoversetamide, and gadofosveset or a CT imaging agents selected from the group consisting of iopamidol, iohexol, ioxilan, iopromide, iodixanol, ioxaglate, metrizoate, and diatrizoate.
• the first and second image data sets can be overlaid to determine the presence of the collagen or fibrin within the mammal.
• the first and second image data sets can be combined to produce a third data set that includes an image of the collagen or the fibrin target and an image of anatomical region where the collagen or the fibrin is located.
• the third data set is capable of indicating the location of the collagen or the fibrin, if present, within the mammal.
• the third data set may be displayed on a display device in order to indicate the location of the stationary target within the vascular system.
• the third data set may also indicate the size of the stationary target within the mammal.
• EXAMPLE 1 Synthesis of exceedingly small Fe x O y nanoparticles with citrate coating using microwave irradiation
• Iron oxide nanoparticles coated with citrate were synthesized according to a known procedure (Pellico et al., Langmuir 2017, 33, 10239-10247). The size of the nanoparticle and the thickness of the coating was controlled by varying the reaction temperature. The size of the iron oxide core varies from 3.4-4.5 nm in diameter. The hydrodynamic size of the citrate-coated nanoparticle varies from 2.9-21 nm in diameter.
• Iron oxide nanoparticles coated with catechol -based metal oxide-binding ligands were synthesized according to a known procedure (Wei et al. Nano Lett. 2012, 22, 22- 25). The iron oxide particles were first synthesized with an oleic acid surface and the catechol derivatives were synthesized separately.
• MEAA 2-[2-(2-methoxyethoxy)ethoxy]acetic acid
• SPION Superparamagnetic iron oxide nanoparticles
• SNIOCBP single-nanometer iron oxide
• SNIO-CBP was prepared via conjugation of alkyne-functionalized SNIO (SNIO- alkyne) with type I collagen binding peptide CBP-azide via copper-catalyzed alkyneazide reaction (FIG. 1).
• SAXS Small-angle X-ray scattering
• FIG. 3 Gel filtration chromatography showed a mean hydrodynamic diameter of 3.8 nm (FIG. 3).
• SNIO-CBP has near-zero zeta potential and exhibited minimal non-specific binding in plasma after incubation with fetal bovine serum (FBS) (FIG. 4).
• the SNIO/CBP ratio was determined to be 1/1.2, according to 56 Fe ICP-MS and7-amino acid quantification.
• SNIO-CBP shows a dissociation constant of 23.2 /M in binding with type-I collagen (FIG. 5).
• Longitudinal relaxivity (rl) of SNIO-CBP was measured to be 4.5 s' 1 (mM Fe)' 1 at 1.41 T and 37 °C, or 145 s -1 (mM peptide) 1 .

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