WO2022006007A1 - Procédés d'imagerie d'infections bactériennes - Google Patents

Procédés d'imagerie d'infections bactériennes Download PDF

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WO2022006007A1
WO2022006007A1 PCT/US2021/039436 US2021039436W WO2022006007A1 WO 2022006007 A1 WO2022006007 A1 WO 2022006007A1 US 2021039436 W US2021039436 W US 2021039436W WO 2022006007 A1 WO2022006007 A1 WO 2022006007A1
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compound
imaging
tissue
organ
subject
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PCT/US2021/039436
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English (en)
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Matthias Nahrendorf
Edmund J. Keliher
Peter Panizzi
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The General Hospital Corporation
Auburn University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/041—Heterocyclic compounds
    • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K51/0455—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the conjugates are useful to diagnose bacterial endocarditis in the heart of the patient.
  • Bacterial endocarditis is an often lethal infection of the heart valves and surrounding endocardium.
  • Major challenges to clinically managing endocarditis include identifying the causal pathogens, timing surgical interventions and rising bacterial resistance to antibiotics.
  • Staphylococcus aureus (S. aureus) is the most common cause of acute endocarditis in patients, likely because these bacteria produce potent factors promoting bacterial virulence and survival.
  • S. aureus has been differentiated from other pathogens, such as S. epidermidis, through its distinct ability to clot blood. This directly results from S.
  • SC and vWBp both bind active prothrombin through N-terminal interactions and stay locked into the vegetation through independent C-terminal binding interactions.
  • This anchoring mechanism essentially paints vegetations with layers of thrombin-like proteolytic activity.
  • a fibrin-rich wall shields the bacterial colony against drugs and immune cells.
  • dabigatran conjugates labeled with either a near-infrared fluorochrome or a positron emission tomography (PET) isotope (e.g., fluorine-18).
  • PET positron emission tomography
  • the dabigatran conjugates of this disclosure are advantageously useful in integrated PET/magnetic resonance (MR) imaging for detecting of S. aureus endocarditis in mice and piglets.
  • MR magnetic resonance
  • the compounds are useful for monitoring treatment of endocarditis using, for example, an immunotherapy that neutralizes SC and vWBp in mice that have the condition.
  • the imaging with the conjugates of this disclosure showed that the immunotherapy reduced thrombin deposition, boosted innate immune cell defense and impeded vegetation formation.
  • the present disclosure provides a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein L 1 , n, and R 1 are as described herein.
  • the present disclosure provides a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of imaging an organ or tissue of a subject, the method comprising (i) administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same; (ii) waiting a time sufficient to allow the compound to accumulate in the organ or tissue to be imaged; and (iii) imaging the imaging the organ or tissue with an imaging technique.
  • the present disclosure provides a method of diagnosing a bacterial infection in an organ or tissue of a subject, the method comprising (i) administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same; (ii) waiting a time sufficient to allow the compound to accumulate in the organ or tissue to be imaged; and (iii) imaging the imaging the organ or tissue with an imaging technique, wherein observing an image attributable to the fluorophore or the radioisotope within the compound of Formula (I) is indicative of the bacterial infection in the organ or tissue of the subject.
  • the present disclosure provides a method of monitoring treatment of a bacterial infection in a subject, the method comprising (i) administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same; (ii) waiting a time sufficient to allow the compound to accumulate in an organ or tissue of the subject; (iii) imaging the organ or tissue of the subject with an imaging technique; (iv) administering to the subject a therapeutic agent in an effective amount to treat the bacterial infection; (v) after iv), administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same; (vi) waiting a time sufficient to allow the compound to accumulate in the organ or tissue of the subject; (vii) imaging the organ or tissue of the subject with an imaging technique; and (viii) comparing the image of step iii) and the image of step vii).
  • A Synthesis scheme of a near infrared fluorescent dabigatran derivative (DAB-VT680XL) and a positron emission tomography fluorine-18 tracer 18 F-dabigatran ( 18 F-DAB), both prepared in 2 steps from dabigatran.
  • B LC-MS analysis of DAB-VT680XL showing the [M - 2H + ] 2- /2 (917.66 m/z) and [M - 3H + ] 3- /3 (611.41 m/z) ions and
  • C 19 F-DAB showing the [M + H + ] + (724.60 m/z).
  • FIG.3 DAB-VT680XL targeting of endocarditic vegetations in mice.
  • A Gram stain of aortic root after inducing endocarditis in mice shows S. aureus in dark purple. Arrow heads indicate aortic valve leaflet.
  • B High magnification view of boxed area in (A).
  • L Bioluminescence imaging demonstrates bacterial infection, with signal arising from vegetations (arrows) identified on autopsy (M) and FRI (N).
  • B PET/MR images illustrating the imaging signal in aortic valve with endocarditis lesion (arrow). Color scale depicts becquerels/mL.
  • D PET/MR images illustrating the imaging signal in tricuspid valve endocarditis lesion (arrow).
  • Color scale depicts becquerels/mL.
  • E Ex vivo autoradiography of aortic valve indicates radioactive signal in the aortic valve vegetations, which were verified on autopsy (F) and a source of bioluminescence (G) arising from S. aureus (arrows). F IG. 7. Immunotherapy neutralizing virulence factors disrupts vegetations and improves survival.
  • A-C Specificity of monoclonal antibodies by immunoblotting against vWBp-(1-263) in lane 1; vWBp-(1-474) in lane 2; SC-(1- 325) in lane 3; SC-(1-660) in lane 4. Lane 5 contains protein standards with the indicated molecular weights.
  • the indicated antibodies in panel A GMA-2510 monoclonal [anti-von Willebrand factor-binding protein (anti-vWBp)] and in panel B GMA-2105 monoclonal [anti-staphylocoagulase (anti-SC)], are specific for their respective targets.
  • Panel C shows probing for total mouse IgG (anti-IgG polyclonal against both the heavy and light chains of murine IgG) and reflects the bound antibodies shown in panels A and B.
  • FIG.9. DAB-VT680XL does not enrich in acute myocardial infarcts.
  • A FMT/CT images obtained in mice 5 days after coronary ligation show no signal in the apical infarct (right panel) compared to endocarditis (left panel).
  • FIG.10 FMT in mice with endocarditis induced by S. epidermidis Xen43.
  • FIG.11 Fluorescence imaging of renal S. aureus infection with DAB- VT680XL.
  • A Bioluminescent imaging shows hot spots in the lower back of the mouse, consistent with bacterial accumulation.
  • B These areas were fluorescent after intravenous injection of DAB-VT680XL.
  • C, D Same as (A, B) with ex vivo opened situs, confirming that signal arises from kidneys.
  • aureus relies on virulence factors to evade therapeutics and the host’s immune response, usurping the host’s clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor-binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance.
  • small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies.
  • PET positron emission tomography
  • At least one L 1 is (-O-isopropylene-)x.
  • n is an integer from 2 to 10.
  • n is an integer from 2 to 8.
  • n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • x is an integer from 1 to 5.
  • x is 1, 2, or 3.
  • the moiety (L 1 )n has formula: .
  • the moiety (L 1 )n has formula: .
  • the compound of Formula (I) has formula: , or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) has formula: , or a pharmaceutically acceptable salt thereof.
  • R 1 is a fluorophore.
  • fluorophores include any fluorescent chemical compound that can re-emit light upon light excitation.
  • the fluorophores can by excited by a light of a wavelength form about 300 nm to about 800 nm, and then emit light of a wavelength from about 350 nm to about 770 nm (e.g., violet, blue, cyan, green, yellow, orange or red light), which can be detected by fluorescent imaging devices, including the ability to measure the intensity of the fluorescence.
  • R 1 is a near-infrared fluorophore (e.g., absorbing and/or emitting light from about 400 nm to about 1700 nm, from about 700 nm to about 1700 nm, or from about 1000 nm to about 1700 nm).
  • a near-infrared fluorophore e.g., absorbing and/or emitting light from about 400 nm to about 1700 nm, from about 700 nm to about 1700 nm, or from about 1000 nm to about 1700 nm.
  • fluorophores include VT680XL, 680XL, MB, indocyanine green, IRDye800CW, cyanine dyes (including non-sulfonated, zwitterionic, phosphonated, and quaternium ammonium dyes), BODIPY, CH1055, AF488, hydroxycoumarin blue, methoxycoumarin blue, Alexa fluor blue, aminocoumarin blue, Cy2 green (dark), FAM green (dark), alexa fluor 488 green (light), Fluorescein FITC green (light), Alexa fluor 430 green (light), Alexa fluor 532 green (light), HEX green (light), Cy3 yellow, TRITC yellow, alexa fluor 546 yellow, Alexa fluor 5553 yellow, R- phycoerythrin (PE) 480; yellow, rhodamine red-X orange, tamara red, Cy3.5581 red, rox red, alexa fluor 568 red, Red
  • R 1 is selected from C 6-10 aryl, C 3-10 cycloalkyl, 5-14 membered heteroaryl, and 4-10 membered heterocycloalkyl, each of which is optionally substituted with at least one substituent selected from halo, C1-4 haloalkyl, C 1-6 alkoxy, and C 1-6 haloalkoxy, wherein said halo, C1-4 haloalkyl, C 1-6 alkoxy, and C1- 6 haloalkoxy comprise at least one radioisotope.
  • R 1 is C 6-10 aryl, substituted with at least one substituent selected from halo, C1-4 haloalkyl, C 1-6 alkoxy, and C 1-6 haloalkoxy, wherein said halo, C1-4 haloalkyl, C 1-6 alkoxy, and C 1-6 haloalkoxy comprise at least one radioisotope.
  • said C 6-10 aryl is phenyl.
  • said C 6-10 aryl is substituted with at least one halo.
  • said C 6-10 aryl is substituted with at least one C 1-4 haloalkyl.
  • said C 6-10 aryl is substituted with at least one C 1-6 alkoxy. In some embodiments, said C 6-10 aryl is substituted with at least one C 1-6 haloalkoxy. In some embodiments, the at least one radioisotope is a positron emitter.
  • the positron emitter is selected from the group consisting of 11 C, 13 N, 15 O, 18 F, 34m Cl, 38 K, 45 Ti, 51 Mn, 52m Mn, 52 Fe, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 71 As, 72 As, 74 As, 75 Br, 76 Br, 82 Rb, 86 Y, 89 Zr, 90 Nb, 94m Tc, 110m In, 118 Sb, 120 I, 121 I, 122 I, and 124 I.
  • the radioisotope is 11 C or 18 F. In some embodiments, the radioisotope is 11 C.
  • the radioisotope is 18 F.
  • the compound of Formula (I) is selected from any one of the following compounds: , , or a pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) is selected from any one of the following compounds: , , or a pharmaceutically acceptable salt thereof.
  • Pharmaceutically acceptable salts In some embodiments, a salt of any one of the compounds of the present disclosure is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.
  • acids commonly employed to form pharmaceutically acceptable salts of the compounds include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids.
  • inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phospho
  • Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionat
  • pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.
  • bases commonly employed to form pharmaceutically acceptable salts of the compounds include hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri- alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamine), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)
  • the compounds of the present disclosure are useful in imaging techniques (e.g., as described below), and more specifically in diagnosing and/or monitoring treatment of bacterial infections and associated inflammation, as well as other conditions where fibronectin is implicated.
  • imaging techniques e.g., as described below
  • bacterial infections e.g., the fibronectin-associated conditions
  • other aspects of these diagnostic and monitoring methods are described below. It is estimated that about 65% of all bacterial infections are associated with bacterial biofilms. These include both, device- and non-device-associated infections.
  • Native valve endocarditis is an inflammation caused by interaction of bacteria with the vascular endothelium and pulmonic valves of the heart.
  • NBTE non- bacterial thrombotic endocarditis
  • Biofilms may also occur on or within indwelling medical devices such as contact lenses, central venous catheters, mechanical heart valves, peritoneal dialysis catheters, prosthetic joints, pacemakers, urinary catheters. These biofilms may be composed of only a single or of different types of microbial species. In this case, microbial cells attach and produce biofilm on mechanical heart valves and surrounding tissues, a condition known as prosthetic valve endocarditis. Examples of bacteria responsible for this unpleasant condition are Streptococcus species, S. aureus, S. epidermidis, gram-negative Bacillus, Enterococcus and Candida spp.
  • micro-organisms may be from the skin or from other indwelling devices like central venous catheters or dental work.
  • tissue damage may occur as a result of accumulation of platelets and fibrin at the location of suture and on the devices. Microbial cells have better ability to colonize these locations.
  • bacteria that form biofilms including fibrin-containing biofilms
  • Bactococcus faecalis Enterococcus faecalis
  • Staphylococcus aureus Staphylococcus aureus
  • Streptococcus mutans a bacteria that is known to be responsible for dental caries and may enter blood stream and lead to endocarditis
  • Staphylococcus epidermidis Staphylococcus epidermidis
  • Streptococcus viridans E. coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa.
  • the compounds of this disclosure can be used for diagnosing or monitoring treatment of a bacterial infection and associated inflammation of the heart.
  • infections include endocarditis (e.g., native valve endocarditis or non-bacterial thrombotic endocarditis).
  • the infection may be located in a particular heart segment, such as right atrium, pulmonary valve, right ventricle, tricuspid valve, left ventricle, mitral valve, aortic valve, or left atrium.
  • the compounds are particularly useful to detect inflammation of the heart’s valves, such as pulmonary valve, aortic valve, mitral valve, or tricuspid valve.
  • the compounds of this disclosure can be used for diagnosing or monitoring treatment of a bacterial infection and associated inflammation of the kidney.
  • a non-limiting example of such a condition includes renal S. aureus lesions.
  • the compounds of this disclosure can be used for diagnosing or monitoring treatment of a bacterial infection of an organ or tissue selected from: artery (e.g., aorta, pulmonary artery, umbilical artery, brachiocephalic artery, carotid artery, subclavian artery), vein (e.g., inferior vena cava, abdominal vena cava, subclavian vein), lung, liver, kidney, skin, brain, eye, bone, intestine, gallbladder, pancreas, trachea, bladder, uterus, spleen, cartilage, muscle (e.g., smooth muscle, cardiac muscle, skeletal muscle), cartilage, epithelium, tendon, and ligament.
  • artery e.g., aorta, pulmonary artery, umbilical artery, brachiocephalic artery, carotid artery, subclavian artery
  • vein e.g., inferior vena cava, abdominal vena cava, sub
  • the organ or tissue is a heart. In some embodiments, the organ or tissue is kidney.
  • the bacterial infection is selected from nosocomial infection, skin infection, respiratory infection, wound infection, endovascular infection, CNS infection, abdominal infection, blood stream infection, urinary tract infection, pelvic infection, invasive systemic infection, gastrointestinal infection, dental infection, and connective tissue infection. Any of these infections can be caused by one or more of the biofilm-forming bacteria described herein (e.g., S. aureus including MRSA).
  • Suitable examples of specific bacterial infections include pneumonia, tuberculosis, obstructive pulmonary disease, sinusitis, gastroenteritis, meningitis, arthritis, osteomyelitis, endocarditis, and conjunctivitis.
  • osteomyelitis is a disease of bones, which may be caused by bacterial cells or fungi. Bacteria enter the bones through the bloodstream, trauma or through previous infections. When microbes enter through the bloodstream and the metaphysis of the bone becomes infected, this leads to the recruitment of white blood cells (WBCs) to the site. These WBCs attempt to phagocytose or lyse the pathogens by secreting enzymes.
  • WBCs white blood cells
  • the compounds and compositions described here are useful for diagnosing any disease where fibrosis, blood clotting, and/or thrombosis may be implicated, such as cancers (e.g., lung, brain, pancreatic, melanoma, prostate, colon cancers), cardiovascular disease (e.g., myocardial infarction, atherosclerosis, arterial thrombosis), autoimmune diseases (e.g., multiple sclerosis, diabetes, irritable bowel syndrome, Celiac disease, Crohn’s disease), and pelvic inflammatory disease.
  • cancers e.g., lung, brain, pancreatic, melanoma, prostate, colon cancers
  • cardiovascular disease e.g., myocardial infarction, atherosclerosis, arterial thrombosis
  • autoimmune diseases e.g., multiple sclerosis, diabetes, irritable bowel syndrome, Celiac disease, Crohn’s disease
  • pelvic inflammatory disease e.g., multiple sclerosis, diabetes, irritable
  • the present disclosure provides a method of diagnosing (e.g., detecting) a bacterial infection and/or a condition in which thrombin is implicated in an organ or a tissue of a subject (e.g., the subject in need thereof).
  • the subject in need of diagnosis and/or treatment is determined by a treating physician.
  • the condition can be suspected by the physician, for example, by detecting specific biomarkers of the disease in a blood or a serum of the subject, or as a result of a physical exam.
  • the diagnosing can be attained, for example, by imaging the organ or tissue of the subject by an imaging technique, for example, as described herein.
  • a method of imaging the organ or tissue comprises (i) administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same; (ii) waiting a time sufficient to allow the compound to accumulate in the organ or tissue to be imaged (e.g., 1 min, 5 min, 10 min, 15 min, or 30 min), and (iii) imaging the organ or tissue with an imaging technique.
  • the compound of Formula (I) comprises 18 F or 11 C radioisotope
  • the suitable imaging techniques include positron emission tomography (PET) and its modifications.
  • the imaging technique may be selected from positron emission tomography (PET) imaging, positron emission tomography with computer tomography (PET/CT) imaging, and positron emission tomography with magnetic resonance (PET/MRI) imaging, as well as other suitable methods.
  • PET positron emission tomography
  • PET/CT computer tomography
  • PET/MRI positron emission tomography with magnetic resonance
  • observing an image attributable to the fluorophore or the radioisotope within the compound of Formula (I) is indicative of the condition being diagnosed (e.g., bacterial infection) in the organ or tissue of the subject.
  • the present disclosure provides a method of monitoring treatment of a bacterial infection and/or a condition in which thrombin is implicated in a subject, the method comprising (i) administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, (ii) waiting a time sufficient to allow the compound of Formula (I) to accumulate in an organ or tissue of the subject (e.g., 5 min, 15 min, or 30 min); (iii) imaging the organ or tissue of the subject with an imaging technique; (iv) administering to the subject a therapeutic agent in an effective amount to treat the disease or condition (e.g., an FDA-approved or an experimental drug substance for treating a bacterial infection); (v) after (iv), administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof; (vi) waiting a time sufficient to allow the compound of Formula (I) to accumulate in the organ or tissue of the subject (e.
  • the compounds of this disclosure are useful to monitor treatment of a bacterial infection using one or more antibiotic agents or antibiotic therapies.
  • the antibiotic therapy is an immunotherapy which includes administering to a patient antibodies specific to virulence factors that bacteria (e.g., S. aureus) employs to activate the clotting cascade resulting in a biofilm with a fibrin wall protecting the bacterial colony from the host’s immune system.
  • the antibiotic is a cationic antimicrobial peptide (CAMP).
  • the cationic antimicrobial peptide is a defensin peptide (e.g., defensin 1 such as beta-defensin 1 or alpha-defensin 1), or cecropin, andropin, moricin, ceratotoxin, melittin, magainin, dermaseptin, bombinin, brevinin (e.g., brevinin-1), esculentin, buforin II (e.g., from amphibians), CAP18 (e.g., from rabbits), LL37 (e.g., from humans), abaecin, apidaecins (e.g., from honeybees), prophenin (e.g., from pigs), indolicidin (e.g., from cattle), brevinins, protegrin (e.g., from pig), tachyplesins (e.g., from horseshoe crabs), or drosin peptide (e.
  • the antibiotic is selected from the quinolone class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of levofloxacin, norfloxacin, ofloxacin, ciprofloxacin, perfloxacin, lomefloxacin, fleroxacin, sparfloxacin, grepafloxacin, trovafloxacin, clinafloxacin, gemifloxacin, enoxacin, sitafloxacin, nadifloxacin, tosulfloxacin, cinnoxacin, rosoxacin, miloxacin, moxifloxacin, gatifloxacin, cinnoxacin, enoxacin, fleroxacin, lomafloxacin, lomefloxacin, miloxacin, nalidixic acid, nadifloxacin, oxolinic acid, pefloxacin, pirimidic
  • the antibiotic is selected from a ⁇ -lactam, a monobactam, oxazolidinone, and lipopeptide.
  • the antibiotic is selected from the cephalosporin class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of cefazolin, cefuroxime, ceftazidime, cephalexin, cephaloridine, cefamandole, cefsulodin, cefonicid, cefoperazine, cefoprozil, and ceftriaxone.
  • the antibiotic is selected from the penicillin class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of penicillin G, penicillin V, procaine penicillin, and benzathine penicillin, ampicillin, and amoxicillin, benzylpenicillin, phenoxymethylpenicillin, oxacillin, methicillin, dicloxacillin, flucloxacillin, temocillin, azlocillin, carbenicillin, ricarcillin, mezlocillin, piperacillin, apalcillin, hetacillin, bacampicillin, sulbenicillin, mecicilam, pevmecillinam, ciclacillin, talapicillin, aspoxicillin, cloxacillin, nafcillin, and pivampicillin.
  • the antibiotic is selected from the carbapenem class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of thienamycin, tomopenem, lenapenem, tebipenem, razupenem, imipenem, meropenem, ertapenem, doripenem, panipenem (betamipron), and biapenem.
  • the antibiotic is selected from the lipopeptide class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of polymyxin B, colistin (polymyxin E), and daptomycin.
  • the antibiotic is selected from the aminoglycoside class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of gentamicin, amikacin, tobramycin, debekacin, kanamycin, neomycin, netilmicin, paromomycin, sisomycin, spectinomycin, and streptomycin.
  • the antibiotic is selected from the glycopeptide class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of vancomycin, teicoplanin, telavancin, ramoplanin, daptomycin, decaplanin, and bleomycin.
  • the antibiotic is selected from the macrolide class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin/midecamycinacetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin/tylocine, roxithromycin, dirithromycin, troleandomycin, spectinomycin, methymycin, neomethymycin, erythronolid, megalomycin, picromycin, narbomycin, oleandomycin, triacetyl-oleandomycin, laukamycin, kujimycin A, albocyclin and cineromycin B.
  • the antibiotic is selected from the ansamycin class of antibiotic compounds. In some aspects of these embodiments, the antibiotic is selected from the group consisting of streptovaricin, geldanamycin, herbimycin, rifamycin, rifampin, rifabutin, rifapentine and rifamixin. In some embodiments, the antibiotic is selected from the sulfonamide class of antibiotic compounds.
  • the antibiotic is selected from the group consisting of sulfanilamide, sulfacetarnide, sulfapyridine, sulfathiazole, sulfadiazine, sulfamerazine, sulfadimidine, sulfasomidine, sulfasalazine, mafenide, sulfamethoxazole, sulfamethoxypyridazine, sulfadimethoxine, sulfasymazine, sulfadoxine, sulfametopyrazine, sulfaguanidine, succinylsulfathiazole and phthalylsulfathiazole.
  • the antibiotic is selected from the group consisting of quinolones, fluoroquinolones, ⁇ -lactams, cephalosporins, penicillins, carbapenems, lipopeptide antibiotics, glycopeptides, macrolides, ansamycins, sulfonamides, and combinations of two or more thereof.
  • Positron emission tomography PET has become an important clinical diagnostic and research modality, and also a valuable technology in drug discovery and development. PET offers picomolar sensitivity and is a fully translational technique that requires specific probes radiolabeled with a usually short-lived positron-emitting radionuclide.
  • PET has provided the capability of measuring biological processes at the molecular and metabolic levels in vivo by the detection of the photons formed as a result of the annihilation of the emitted positrons.
  • PET offers the possibility of visualizing and analyzing complex physiological and pathophysiological conditions. PET has often been used to detect disease-related biochemical changes before the disease-associated anatomical changes can be found using standard medical imaging modalities.
  • PET/CT Positron emission tomography–computed tomography
  • PET/CT is a medical imaging technique using a device that combines in a single gantry system both a positron emission tomography (PET) scanner and an x-ray computed tomography (CT) scanner.
  • PET positron emission tomography
  • CT x-ray computed tomography
  • the compounds of this disclosure, appropriately labeled, can be used to generate PET images of in a diseased or injured tissue, such as a tissue or an organ infected with bacteria producing a biofilm.
  • Useful reporter groups that may be introduced to the compound of the present disclosure include radioactive isotopes, such as 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 81 mKr, 82 Rb, 86 Y, 89 Zr, 111 In, 123 I, 124 I, 133 Xe, 201 Tl, 125 I, 35 S 14 C, 3 H. Images acquired from both devices can be taken sequentially, in the same session, and combined into a single superposed (co-registered) image. Thus, functional imaging obtained by PET, which depicts the spatial distribution of metabolic or biochemical activity in the body can be more precisely aligned or correlated with anatomic imaging obtained by CT scanning.
  • radioactive isotopes such as 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 81 mKr, 82 Rb, 86 Y, 89 Zr, 111 In, 123 I, 124 I, 133 Xe, 201 Tl, 125
  • Two- and three- dimensional image reconstruction may be rendered as a function of a common software and control system.
  • PET/CT scans can be used to diagnose a health condition in human and animal subjects.
  • the animals are anesthetized, e.g., by isoflurane, prior to imaging, and anesthesia is maintained during the process.
  • CT acquisition precedes PET and lasts approximately 4 minutes, acquiring 360 cone beam projections with a source power and current of 80 keV and 500 ⁇ A, respectively. Projections are reconstructed into three-dimensional volumes.
  • the imaging bed then moves into the PET gantry.
  • imaging is carried out in about 1-4 hours after the compound is administered to the patient.
  • a high-resolution Fourier re-binning algorithm is used to re-bin sinograms, followed by a filtered back-projection algorithm for reconstruction.
  • the reconstructed PET image, through dynamic framing of the sinograms, is composed of a series of 1, 3, and 5 minute frames.
  • PET and CT reconstructed images are then fused using Inveon Research Workplace (IRW) software (Siemens).
  • IRW Inveon Research Workplace
  • PET-MRI Positron Emission Tomography–Magnetic Resonance Imaging
  • PET-MRI Positron emission tomography–magnetic resonance imaging
  • PET-MRI is a hybrid imaging technology that incorporates magnetic resonance imaging (MRI) soft tissue morphological imaging and positron PET functional imaging.
  • PET/MRI scans can be used to diagnose a health condition in humans and animals, e.g., for research and agricultural purposes.
  • the compounds and compositions of this disclosure when appropriately labelled with a radioisotope, can be used in PET/MRI.
  • a fusion approach is implemented using external fiducial landmarks provided by a “vest” optimized for the particular organ, e.g., for cardiac imaging.
  • the vest surrounds the subject’s chest to create a frame that follows minor movements due to transfer between scanners or light anesthesia.
  • the tubes are filled with 15% iodine in water, rendering them visible in MRI. Subject motion is minimized with an imaging bed that can be used in both imaging systems.
  • FMT-CT imaging is performed at 680/700 nm excitation/emission wavelength at specified time (e.g., 2, 4, 8, 24, or 48 hours) after injection of the compound of the present disclosure comprising a fluorochrome.
  • Total imaging time for FMT acquisition is typically 5 to 8 minutes.
  • Data are post-processed using a normalized Born forward equation to calculate three- dimensional fluorochrome concentration distribution.
  • CT angiography can immediately follow FMT to robustly identify the aortic root.
  • Pharmaceutical compositions also provides pharmaceutical compositions comprising an effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also comprise any one of the additional therapeutic agents described herein.
  • the application also provides pharmaceutical compositions and dosage forms comprising any one the additional therapeutic agents described herein.
  • the carrier(s) are “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in an amount used in the medicament.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of the present application include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as
  • compositions or dosage forms may contain any one of the compounds and therapeutic agents described herein in the range of 0.005% to 100% with the balance made up from the suitable pharmaceutically acceptable excipients.
  • the contemplated compositions may contain 0.001%-100% of any one of the compounds and therapeutic agents provided herein, in one embodiment 0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%, wherein the balance may be made up of any pharmaceutically acceptable excipient described herein, or any combination of these excipients.
  • Routes of administration and dosage forms The pharmaceutical compositions of the present application include those suitable for any acceptable route of administration.
  • Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intra-abdominal, intra- arterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intranasal, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous,
  • compositions and formulations described herein may conveniently be presented in a unit dosage form, e.g., tablets, sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore, MD (20th ed.2000). Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions of the present application suitable for oral administration may be presented as discrete units such as capsules, sachets, granules or tablets each containing a predetermined amount (e.g., effective amount) of the active ingredient; a powder or granules; a solution or a suspension in an aqueous liquid or a non-aqueous liquid; an oil-in-water liquid emulsion; a water-in- oil liquid emulsion; packed in liposomes; or as a bolus, etc.
  • Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption.
  • carriers that are commonly used include lactose, sucrose, glucose, mannitol, and silicic acid and starches.
  • Other acceptable excipients may include: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as ka
  • useful diluents include lactose and dried corn starch.
  • the active ingredient is combined with emulsifying and suspending agents.
  • certain sweetening and/or flavoring and/or coloring agents may be added.
  • Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.
  • compositions suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions or infusion solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, saline (e.g., 0.9% saline solution) or 5% dextrose solution, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • the injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
  • the pharmaceutical compositions of the present application may be administered in the form of suppositories for rectal administration.
  • compositions can be prepared by mixing a compound of the present application with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
  • the pharmaceutical compositions of the present application may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, U.S.
  • Topical compositions of the present disclosure can be prepared and used in the form of an aerosol spray, cream, emulsion, solid, liquid, dispersion, foam, oil, gel, hydrogel, lotion, mousse, ointment, powder, patch, pomade, solution, pump spray, stick, towelette, soap, or other forms commonly employed in the art of topical administration and/or cosmetic and skin care formulation.
  • the topical compositions can be in an emulsion form.
  • Topical administration of the pharmaceutical compositions of the present application is especially useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the topical composition comprises a combination of any one of the compounds and therapeutic agents disclosed herein, and one or more additional ingredients, carriers, excipients, or diluents including, but not limited to, absorbents, anti-irritants, anti-acne agents, preservatives, antioxidants, coloring agents/pigments, emollients (moisturizers), emulsifiers, film-forming/holding agents, fragrances, leave- on exfoliants, prescription drugs, preservatives, scrub agents, silicones, skin- identical/repairing agents, slip agents, sunscreen actives, surfactants/detergent cleansing agents, penetration enhancers, and thickeners.
  • the compounds and therapeutic agents of the present application may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, or catheters.
  • Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in U.S. Patent Nos.6,099,562; 5,886,026; and 5,304,121.
  • the coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof.
  • the coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition.
  • Coatings for invasive devices are to be included within the definition of pharmaceutically acceptable carrier, adjuvant or vehicle, as those terms are used herein.
  • the present application provides an implantable drug release device impregnated with or containing a compound or a therapeutic agent, or a composition comprising a compound of the present application or a therapeutic agent, such that said compound or therapeutic agent is released from said device and is therapeutically active.
  • a compound of the present disclosure is present in an effective amount (e.g., a therapeutically effective amount).
  • Effective doses may vary, depending on the diseases treated, the severity of the disease, the route of administration, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician.
  • an effective amount of the compound can range, for example, from about 0.001 mg/kg to about 500 mg/kg (e.g., from about 0.001 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 150 mg/kg; from about 0.01 mg/kg to about 100 mg/kg; from about 0.01 mg/kg to about 50 mg/kg; from about 0.01 mg/kg to about 10 mg/kg; from about 0.01 mg/kg to about 5 mg/kg; from about 0.01 mg/kg to about 1 mg/kg; from about 0.01 mg/kg to about 0.5 mg/kg; from about 0.01 mg/kg to about 0.1 mg/kg; from about 0.1 mg/kg to about 200 mg/kg; from about 0.1 mg/kg to about 150 mg/kg; from about 0.1 mg/kg to about 100 mg/kg; from about 0.1 mg/kg to about 50 mg/kg; from about 0.1 mg/kg to about 10 mg/kg; from about 0.1 mg/kg; from
  • an effective amount of a compound is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, or about 5 mg/kg.
  • the foregoing dosages can be administered on a daily basis (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, thrice daily) or non-daily basis (e.g., every other day, every two days, every three days, once weekly, twice weekly, once every two weeks, once a month).
  • Kits The present invention also includes pharmaceutical kits useful, for example, in the treatment of disorders, diseases and conditions referred to herein, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure.
  • kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc.
  • Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
  • the kit may optionally include an additional therapeutic agent as described herein.
  • substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the members of such groups and ranges.
  • C 1-6 alkyl is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.
  • aryl, heteroaryl, cycloalkyl, and heterocycloalkyl rings are described.
  • a pyridine ring or “pyridinyl” may refer to a pyridin-2-yl, pyridin-3-yl, or pyridin-4-yl ring.
  • aromatic refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i.e., having (4n + 2) delocalized ⁇ (pi) electrons where n is an integer).
  • n-membered where n is an integer typically describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n.
  • piperidinyl is an example of a 6-membered heterocycloalkyl ring
  • pyrazolyl is an example of a 5-membered heteroaryl ring
  • pyridyl is an example of a 6- membered heteroaryl ring
  • 1,2,3,4-tetrahydro-naphthalene is an example of a 10- membered cycloalkyl group.
  • the phrase “optionally substituted” means unsubstituted or substituted. The substituents are independently selected, and substitution may be at any chemically accessible position.
  • substituted means that a hydrogen atom is removed and replaced by a substituent.
  • a single divalent substituent e.g., oxo
  • oxo can replace two hydrogen atoms. It is to be understood that substitution at a given atom is limited by valency.
  • Cn-m indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C 1-4 , C 1-6 , and the like.
  • Cn-m alkyl employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons.
  • alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert- butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3- pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like.
  • the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.
  • Cn-m haloalkyl refers to an alkyl group having from one halogen atom to 2s+1 halogen atoms which may be the same or different, where “s” is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms.
  • the haloalkyl group is fluorinated only.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • Cn-m alkylene employed alone or in combination with other terms, refers to a divalent alkyl linking group having n to m carbons.
  • alkylene groups include, but are not limited to, ethan-1,1-diyl, ethan-1,2- diyl, propan-1,1,-diyl, propan-1,3-diyl, propan-1,2-diyl, butan-1,4-diyl, butan-1,3- diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl, and the like.
  • the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.
  • Cn-m alkoxy refers to a group of formula -O-alkyl, wherein the alkyl group has n to m carbons.
  • Example alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • Cn-m haloalkoxy refers to a group of formula –O-haloalkyl having n to m carbon atoms.
  • An example haloalkoxy group is OCF3.
  • the haloalkoxy group is fluorinated only.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • the term “amino” refers to a group of formula –NH2.
  • Cn-m alkylamino refers to a group of formula -NH(alkyl), wherein the alkyl group has n to m carbon atoms.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • alkylamino groups include, but are not limited to, N-methylamino, N-ethylamino, N- propylamino (e.g., N-(n-propyl)amino and N-isopropylamino), N-butylamino (e.g., N- (n-butyl)amino and N-(tert-butyl)amino), and the like.
  • di(Cn-m-alkyl)amino refers to a group of formula - N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • Cn-m alkoxycarbonyl refers to a group of formula -C(O)O-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl (e.g., n-propoxycarbonyl and isopropoxycarbonyl), butoxycarbonyl (e.g., n-butoxycarbonyl and tert-butoxycarbonyl), and the like.
  • carboxycarbonyl to a group of formula –C(O)NH2.
  • Cn-m alkylcarbamyl refers to a group of formula -C(O)-NH(alkyl), wherein the alkyl group has n to m carbon atoms.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • di(Cn-m-alkyl)carbamyl refers to a group of formula –C(O)N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • carboxy refers to a -C(O)OH group.
  • halo refers to F, Cl, Br, or I. In some embodiments, a halo is F, Cl, or Br.
  • aryl employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings).
  • Cn-m aryl refers to an aryl group having from n to m ring carbon atoms.
  • Aryl groups include, e.g., phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to 10 carbon atoms. In some embodiments, the aryl group is phenyl or naphtyl.
  • cycloalkyl refers to non-aromatic cyclic hydrocarbons including cyclized alkyl and/or alkenyl groups.
  • Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Ring- forming carbon atoms of a cycloalkyl group can be optionally substituted by 1 or 2 independently selected oxo or sulfide groups (e.g., C(O) or C(S)).
  • cycloalkyl moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like.
  • a cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring.
  • Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbons (C 3-10 ).
  • the cycloalkyl is a C 3-10 monocyclic or bicyclic cyclocalkyl.
  • the cycloalkyl is a C3-7 monocyclic cyclocalkyl.
  • Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like.
  • cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
  • heteroaryl refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen.
  • the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.
  • any ring-forming N in a heteroaryl moiety can be an N-oxide.
  • the heteroaryl is a 5-10 membered monocyclic or bicyclic heteroaryl having 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.
  • the heteroaryl is a 5-6 monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.
  • the heteroaryl is a five- membered or six-membereted heteroaryl ring.
  • a five-membered heteroaryl ring is a heteroaryl with a ring having five ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S.
  • Exemplary five-membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.
  • a six-membered heteroaryl ring is a heteroaryl with a ring having six ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S.
  • Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.
  • heterocycloalkyl refers to non-aromatic monocyclic or polycyclic heterocycles having one or more ring-forming heteroatoms selected from O, N, or S.
  • heterocycloalkyl monocyclic 4-, 5-, 6-, 7-, 8-, 9- or 10- membered heterocycloalkyl groups.
  • Heterocycloalkyl groups can also include spirocycles.
  • Example heterocycloalkyl groups include pyrrolidin-2-one, 1,3- isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like.
  • Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by 1 or 2 independently selected oxo or sulfido groups (e.g., C(O), S(O), C(S), or S(O)2, etc.).
  • the heterocycloalkyl group can be attached through a ring- forming carbon atom or a ring-forming heteroatom.
  • the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds.
  • heterocycloalkyl moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc.
  • a heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring.
  • the heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.
  • the heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.
  • the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, an azetidine ring may be attached at any position of the ring, whereas a pyridin-3-yl ring is attached at the 3-position.
  • compound as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
  • Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.
  • the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
  • Compounds of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Example prototropic tautomers include ketone – enol pairs, amide - imidic acid pairs, lactam – lactim pairs, enamine – imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo.
  • an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal.
  • an in vitro cell can be a cell in a cell culture.
  • an in vivo cell is a cell living in an organism such as a mammal.
  • the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system.
  • “contacting” a cell with a compound of the invention includes the administration of a compound of the present invention to an individual or patient, such as a human, having the cell, as well as, for example, introducing a compound of the invention into a sample containing a cellular or purified preparation containing the cell.
  • the term “individual”, “patient”, or “subject” used interchangeably refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
  • the phrase “effective amount” or “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • the term “radioisotope” refers to an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring).
  • isotopic enrichment factor refers to the ratio between the isotopic abundance and the natural abundance of a specified isotope.
  • 18 F refers to the radioisotope of fluorine having 9 protons and 9 neutrons.
  • F refers to the stable isotope of fluorine having 9 protons and 10 neutrons (i.e., the “ 19 F isotope”).
  • a compound of the present disclosure has an isotopic enrichment factor for each designated 18 F atom of at least 3500 (52.5% 18 F incorporation at each designated 18 F atom), at least 4000 (60% 18 F incorporation), at least 4500 (67.5% 18 F incorporation), at least 5000 (75% 18 F), at least 5500 (82.5% 18 F incorporation), at least 6000 (90% 18 F incorporation), at least 6333.3 (95% 18 F incorporation), at least 6466.7 (97% 18 F incorporation), at least 6600 (99% 18 F incorporation), or at least 6633.3 (99.5% 18 F incorporation).
  • 11 C refers to the radioisotope of carbon having 6 protons and 5 neutrons.
  • C refers to the stable isotope of carbon having 6 protons and 6 neutrons (i.e., the “ 12 C isotope”).
  • a compound of the present disclosure has an isotopic enrichment factor for each designated 11 C atom of at least 3500 (52.5% 11 C incorporation at each designated 11 C atom), at least 4000 (60% 11 C incorporation), at least 4500 (67.5% 11 C incorporation), at least 5000 (75% 11 C), at least 5500 (82.5% 11 C incorporation), at least 6000 (90% 11 C incorporation), at least 6333.3 (95% 11 C incorporation), at least 6466.7 (97% 11 C incorporation), at least 6600 (99% 11 C incorporation), or at least 6633.3 (99.5% 11 C incorporation).
  • treating refers to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), or 2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
  • MSSA methicillin-susceptible Staphylococcus aureus
  • Xen 43 is S. epidermidis strain. All Xen strains were purchased from PerkinElmer Inc.. S. aureus RFP+ is a fluorescence version of methicillin-resistant S. aureus USA 300, NE1260R JE2 pckA::rfp that was obtained from Dr. J. Bose of the University of Kansas Medical Center. Briefly, strains were cultured in liquid brain heart infusion broth under constant shaking at 150-200 rpm at 37 °C. For injecting the animals, overnight cultures were diluted 20-fold in sterile Dulbecco’s phosphate buffer saline without calcium or magnesium (PBS, Lonza).
  • PBS sterile Dulbecco’s phosphate buffer saline without calcium or magnesium
  • the injections’ approximate CFU counts were assessed by light scattering at 600 nm using a Shimadzu UV-2101PC spectrophotometer according to the manufacturer’s guidelines.
  • bioluminescence production was confirmed using a bioluminescence imager (FujiFilm LAS-1000) set to 10 min integration time.
  • true CFU numbers were verified by serial plating on 5% sheep blood agar (Hardy Diagnostics) and expression of either the bioluminescent or fluorescent reporter gene was confirmed by imaging the agar plates.
  • the Xen strain with strongest bioluminescence in the piglets was selected after a limited screen, and the porcine-passaged strain was cultured from a port abscess.
  • mice endocarditis to induce mouse endocarditis, protocols for artery isolation surgery were followed, 4.0 suture material insertion and S. aureus infection as previously reported. A 4-0 suture was advanced through the surgically exposed right carotid artery into the left ventricular outflow tract and fixed in place. After a 24 hour recovery period, 1 ⁇ 10 6 CFU S. aureus in 100 ⁇ L PBS were injected through the tail vein. Continual endothelial damage to the aortic valve caused by the indwelling suture allowed the bacteria to attach and form vegetations. All animal experimentation and cohort size determination were approved in advance by the Massachusetts General Hospital’s Subcommittee on Research Animal Care.
  • Piglet endocarditis a total of 36 newly weaned piglets (16-20 days old, weighing 10-15 lb) were purchased from the Swine Research and Education Center at Auburn University for use in model development (bioluminescence imaging (BLI) only), fluorescence probe co-localization, and clinical PET/MRI studies. The animals were acclimated for 5-7 days prior to central-line implantation surgery. Piglets were sedated with dexmedetomidine (Dexdomitor; Zoetis) and butorphanol.
  • ketamine 10 mg/kg; Ketaset; Zoetis
  • dexmedetomidine 20 mcg/kg
  • butorphanol 0.4 mg/kg
  • a line block of 0.5 % lidocaine Xylocaine-MPF; Fresenius Kabi USA
  • a combination of sharp and blunt dissection was used to identify and isolate the left jugular vein and then to create a subcutaneous pocket for the vascular access port (VAP; 5Fr ClearPort; Access Technologies).
  • the vascular port consisted of a titanium outlet with a silicone septum and catheter.
  • a small jugular venotomy was made and a 0.025 guide-wire was introduced into the vascular lumen.
  • the polyurethane VAP catheter was placed over the guide wire and advanced into the right ventricle under fluoroscopic guidance. Correct positioning of the catheter was confirmed using multiple injections of radiopaque contrast under fluoroscopic observation. Ports were implanted in the front right region of the neck. Once the desired catheter positioning was confirmed, the VAP catheter was secured within the jugular vein with several circumferential sutures of 3-0 polypropylene (Prolene; Ethicon). The catheter tubing was cut to an appropriate length and connected to the VAP that was then secured within the subcutaneous pocket with multiple polypropylene sutures.
  • the surgical site was lavaged with saline and closed with 3-0 poliglecaprone 25 (Monocryl; Ethicon) in the subcutaneous and intra-dermal layers.
  • a 22-gauge Posi-grip Huber point needle was placed into the VAP, continued patency was confirmed and the VAP was heparin- locked.
  • the VAP site was marked with a permanent skin marker for ease of injection.
  • Analgesia was provided with carprofen (2.2 mg/kg per os every 12 hrs; Rimadyl; Zoetis) and butorphanol (0.2-0.4 mg/kg, intramuscularly every 4-6 hrs).
  • piglets were injected with 4-8 ⁇ 10 8 CFU of S. aureus Xen 36 (PerkinElmer Inc.) through the VAP using a Huber needle. Thereafter the port was flushed with 5 mL sterile PBS.
  • aortic valve endocarditis piglets were similarly prepared but the aorta was accessed via the left carotid artery. Aortic valve damage was induced by repeated passing of a 2.5 mm diameter cytology brush (Endoscopy Support Services) through the valve. The brush was positioned under fluoroscopy guidance aided by repeated contrast injection. A venous leg catheter was used to administer anesthesia and the S.
  • aureus Xen 36 inoculum (5-8 ⁇ 10 8 CFU) followed by a bolus 60 mL sterile saline flush.
  • piglets were injected with 0.4 ⁇ mol DAB-VT680XL in 2 mL sterile PBS via the ear vein using a 25-gauge butterfly. Animals were euthanized 10-12 hours later and BLI and fluorescence reflectance imaging (FRI) performed immediately following necropsy using an IVIS Lumina XRMS imaging system (PerkinElmer). For PET/MRI, piglets were transferred to Mt. Sinai Hospital. All experimentation and the transport were approved by the Institutional Animal Care and Use Committee for Auburn University under protocol AU# 2016-2860.
  • Synthesizing fluorescent and fluorine-18 labeled dabigatran the fluorescent and nuclear thrombin-specific imaging agents are derived from the FDA-approved thrombin inhibitor dabigatran. Synthesizing both agents requires converting the parent compound’s carboxylic acid functionality to an amine, which can be further modified with either a fluorochrome or 18 F-prosthetic group.
  • Thrombin activity assay to confirm that modification did not inhibit binding activity, VT680XL and 19 F-labeled dabigatran were examined with the SensoLyte AFC Thrombin Assay Kit (AnaSpec).
  • 1 8 F-DAB blood half-life To determine the blood half-life of the fluorine-18 ( 18 F)-labeled imaging agent derived from the thrombin inhibitor dabigatran ( 18 F- DAB), blood from 18 F-DAB-injected mice was collected by retro-orbital bleeding and sampled with gamma-counting. Under isoflurane (1.5-3 %) anesthesia, 18 F-DAB was injected via tail vein (approximately 250 ⁇ Ci in 100 ⁇ l PBS) in 12-week-old 6 C57BL/6 mice.
  • Intravital microscopy of thrombi in the femoral artery intravital microscopy was used to visualize DAB-VT680XL binding to freshly formed thrombi.
  • Arterial thrombosis was induced by applying ferric chloride solution (500 mM concentration; Sigma) on the exposed femoral artery of mice.
  • Fluorescently conjugated anti-CD41 mAb (Biolegend) was injected via tail vein to label platelets in vivo before thrombosis induction.
  • DAB-VT680XL and control fluorochrome VT680XL were injected intravenously 5 min after thrombosis induction. Images were acquired with IVM (Olympus) in vivo.
  • Intravital microscopy of vegetation in the femoral artery a 12-0 Ethicon suture material was inserted into the saphenous artery, advanced into the femoral artery and fixed in position while maintaining sufficient blood flow. Mice were allowed to recover for 6 hours before injection of 10 6 CFU S.
  • Mice were anesthetized using 1-2 % isoflurane, then placed on a heated (37 °C) stage for imaging, and the wound was reopened. Imaging was done using an Olympus (IV100) microscope with a water- immersion objective (UMPlanFL N 20 ⁇ NA 0.50, Olympus). Three channels were recorded (Ly-6G FITC, 488 nm excitation; RFP, 561 nm excitation; DAB-VT680XL, 647 nm excitation) to generate z-stacks at 2 ⁇ m steps. Image post-processing was performed using ImageJ software. FMT/CT: on day 3 after suture insertion and 48 hours after injection of either 1 ⁇ 10 6 CFU S.
  • FMT/CT imaging was performed. To this end, mice were injected with 2 nmol of the fluorescent imaging probe and imaged 2 hours later using an FMT-2500 LX Quantitative Tomography Imaging System (PerkinElmer). After excitation at 680 nm and emission collection at 700 nm, a three-dimensional dataset containing fluorescence concentration per voxel was reconstructed. FMT imaging was accompanied by hybrid X-ray CT angiography (Inveon PET-CT, Siemens). Image fusion was achieved using Osirix software and fiducial markers on a dedicated multimodal imaging cassette frame, as described previously.
  • IsoVue 370 was infused at 50 ⁇ l/min through a tail vein catheter.
  • the CT was reconstructed using a modified Feldkamp cone beam reconstruction algorithm (COBRA, Exxim Inc.), bilinear interpolation and a Shepp-Logan reconstruction filter. Voxels were scaled to Hounsfield units.
  • the isotropic spatial resolution was 110 ⁇ m for CT and 1 mm for FMT.
  • Fused data sets were used to place regions of interest in the left ventricular outflow tract and the aortic valve region.
  • FMT/CT underwent ex vivo fluorescence imaging of excised aortas on an OV-110 epifluorescence microscope (Olympus).
  • Hematoxylin and eosin (H&E), Gram staining (Sigma-Aldrich) and immunofluorescence staining for CD11b were performed to verify the presence of S. aureus bacteria and myeloid cells on the aortic valve.
  • Fluorescence microscopy (Eclipse 80i, Nikon) was performed to investigate microscopic DAB-VT680XL localization in the vegetation, and bright field images were scanned and analyzed using a Nanozoomer 2.0RS (Hamamamatsu).
  • aortic root was excised, counted on a Wallac wizard 3 gamma counter to obtain percent injected dose per gram tissue (%IDGT) and imaged for bioluminescent signal. This was followed by overnight exposure on an autoradiography cassette. Plates were read on a Typhoon 9400 Variable Mode Imager (GE Healthcare). Target to background of both the bioluminescent signal and autoradiography were quantified using manual ROI’s of the aorta and background in Amira software (ThermoFisher Scientific).
  • the murine monoclonal antibodies against synthetic peptides corresponded to the N-terminal residues 1 through 10 of either SC or vWBp from S. aureus Newman D2 Tager 104 strain.
  • Corresponding peptides were synthesized with an additional C-terminal cysteine that conjugated to keyhole limpet hemocyanin (KLH) and ovalbumin (OA) using m-maleimidobenzoyl-N-hydroxysuccinimide ester.
  • KLH keyhole limpet hemocyanin
  • OA ovalbumin
  • mice were injected with KLH-peptide conjugate (50 ⁇ g) in incomplete Freund’s adjuvant.
  • Serum titers from each mouse were determined by solid-phase ELISA, and spleen cells from the mouse with the highest serum titer were fused to NS1 myeloma cells on day 162, as described using polyethylene glycol. Hybridoma were selected using hypoxanthine, azaserine and thymidine. Fusion clones were screened by solid-phase ELISA with peptide-OA coated microtiter plates. Selected clones showing signal above ⁇ 2 ⁇ - background were expanded, re-screened, sub-cloned three times by limiting dilution and stored in liquid nitrogen.
  • SC-specific antibodies were designated GMA-2105 and others specific for vWBp were designated GMA-2510.
  • Hybridoma cells were grown in Hybridoma-SFM media (Gibco) and antibodies purified by protein G affinity chromatography. Purified antibody was sterile-filtered and stored at 4 oC.
  • Antibody aggregation was ruled out by size exclusion chromatography on an S-300 column and dynamic light scattering with a Zetasizer Nano-S instrument (Malvern Panalytical). The isotype of each respective antibody was independently verified using/via goat anti-mouse isotype-specific antibody (Bethyl Laboratories) using a MagPix (Luminex).
  • Antibody specificity for SC and vWBp Western blot confirmed specificity of prothrombin activation-specific monoclonal antibodies. Previously characterized recombinant proteins were subjected to SDS gel electrophoresis with lanes corresponding to (1) vWBp-(1-263), (2) vWBp-(1-474), (3) SC-(1-325),(4) SC-(1- 660) and (5) protein standards with indicated molecular weights. The elaborated proteins were transferred to PVDF membrane for western blot analysis to probe the specificity and cross-reactivity of the monoclonal antibodies targeting the critical N- termini of either SC or vWBp.
  • the blot was probed with both SC and vWBp neutralizing antibodies and then stained for total mouse IgG content using an anti-mouse IgG (H+L)-FITC polyclonal antibody.
  • the blot was imaged for fluorescence using a Fuji-films FLA5100 with the 473 laser and LBP channel.
  • Fibrinogen turbidity assays cleavage of fibrinogen by either prothrombin ⁇ vWBp-(1-263) or prothrombin ⁇ SC-(1-325) complexes was monitored from the increase in turbidity at 450 nm at 25 °C in 50 mM Hepes, 110 mM NaCl, 5 mM CaCl2, 1 mg/mL polyethylene glycol (PEG) 8000 (pH 7.4) buffer by using a SpectraMax 340 PC 384 plate reader (Molecular Devices Inc.). Individual reaction conditions were tested to determine the effect of the respective antibodies on the ability of either vWBp or SC to activate prothrombin and subsequently cleave of fibrinogen.
  • PEG polyethylene glycol
  • GMA-2510 (anti-vWBp Ab) was incubated with vWBp-(1-263) and GMA-2105 (anti-SC Ab) was incubated with SC-(1-325) for 25 minutes at 25 °C prior to addition of prothrombin. The 3 components were then incubated together for an additional 25 minutes at 25 °C prior sub-sampling into the turbidity assay.
  • the vWBp assays had final concentrations of 75 nM prothrombin ⁇ vWBp(1-263) complex with either 0 nM, 300 nM or 1.5 ⁇ M anti-vWBp ab.
  • the SC assays had 15 nM prothrombin ⁇ SC(1-325) complex with either 0 nM, 50 nM, or 300 nM anti-SC ab.
  • Fibrinogen 1.5 mg/mL was added simultaneously to initiate all reactions. Progress curves were collected over time ranges necessary to observe total substrate depletion under the positive control conditions. Survival study: to determine the potentially beneficial impact of eliminating prothrombin activation by bacteria, we simultaneously administered either both GMA-2105 and 2510 mAbs or an isotype control mAb (Green Mountain Antibodies, Burlington, VT). Endocarditis was induced in 30 mice, which were randomized to treatment groups.
  • mice Six hours post surgery, the mice received GMA-2105, GMA-2510 or isotype-labeled mAbs by intraperitoneal injection. Mice were kept under normal husbandry without further treatment except for pain management with buprenorphine as needed until death occurred, humane endpoints were reached or up to day 7 after injection of the 1 ⁇ 10 6 CFU S. aureus.
  • MRI of piglets left ventricular ejection fraction was quantified from retrospectively gated short-axis cardiac cine MR images (Siemens 3T Biograph mMR).
  • Acquisition parameters for cine short axis stacks were as follows: repetition time (TR) 56.24 ms, echo time (TE) 3.32 ms, number of averages 2, 24 or 30 slices, 25 cardiac frames, 3 mm slice thickness, no interslice gap, flip angle 12, spatial resolution 0.94 ⁇ 0.94 mm 2 .
  • Retrospective electrocardiogram (ECG) gating was used to acquire the images.
  • Regions of interest (ROIs) were manually segmented with Osirix MD v 9.5.1 and exported using the ‘Export ROIs’ Osirix plugin.
  • the cine acquisition contains a total of 600 or 750 images from 24 slices with 25 cardiac frames per slice.
  • Right ventricle vegetations were quantified from an ECG triggered axial T2 weighted turbo spin echo (TSE) stack using the following acquisition parameters: TR 1125- 1485 ms, TE 76 ms, number of averages 4, 11-24 slices, 3 mm slice thickness, no interslice gap, spatial resolution 0.94x0.94 mm 2 .
  • ROIs were manually segmented with Osirix MD v 9.5.1. ROIs were exported using the ‘Export ROIs’ Osirix plugin. Vegetations were segmented as high intensity areas within the right ventricle while excluding the catheter whenever possible.
  • PET/MRI of piglets eight piglets underwent imaging with a clinical PET/MR system (Siemens 3T Biograph mMR).
  • the piglets received an intravenous injection of 18 F-DAB (51.8 and 25 MBq, respectively) 90 minutes before PET acquisition. Piglets were intubated and placed on the scanner bed under isoflurane anesthesia at 1.5-2 % by inhalation, and were oxygenated throughout the PET/MR imaging experiment. Vital parameters were monitored. A 6-channel body matrix product coil was used for signal reception. Following scout scans, a static thoracic PET was performed for 60 minutes while simultaneously acquiring cardiac and T2 weighted TSE anatomical MR images as detailed above. Attenuation correction of PET images was performed by using a vendor-built-in Dixon MR-based attenuation map (MR-AC) with 4 tissue compartments (soft tissue, fat, lung and air).
  • MR-AC Dixon MR-based attenuation map
  • results are reported as mean ⁇ standard error of mean (SEM). Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, Inc.). Normal distribution of variables was tested using the Kolmogorov-Smirnov-test or the D’Agostino-Pearson omnibus normality test. Data were analyzed by parametric tests if normal distribution was detected. An unpaired student t-test was applied for two-group comparisons and data presented as mean ⁇ s.e.m. with significance indicated by *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 and ****P ⁇ 0.001.
  • FDA Food and Drug Administration
  • DAB thrombin inhibitor dabigatran
  • Both fluorescent and radioactively labeled versions were prepared in two steps (Fig.1A). First, dabigatran was derivatized with an amino group.
  • the DAB-amino intermediate was then used to attach either the radioisotope 18-Fluorine ( 18 F), to yield the PET imaging agent 18 F- DAB, or the fluorochrome VivoTag 680XL (VT680XL), to synthesize the near infrared imaging agent DAB-VT680XL, which can be used for intravital microscopy and fluorescence molecular tomography.
  • Mass spectrometry after liquid chromatography confirmed the identities of DAB-VT680XL (Fig.1B) and the non- radioactive standard 19 F-DAB (Fig.1C). Note that 18 F-DAB has a specific collection window (Fig.1D) to obtain the imaging agent with high radiochemical purity (Fig.1, E and 1F).
  • dabigatran-NH2 dabigatran (50 mg, 106 ⁇ mol) was suspended in dimethylformamide (DMF, 4.0 mL) in a 20-mL vial with a magnetic stir-bar, to which N-Boc- 2,2′-(ethylenedioxy)diethylamine (105 mg, 424 ⁇ mol) and EDC (265 mg, 1.38 mmol) were then added. After stirring for 3 h, the reaction mixture was concentrated to dryness, re-dissolved in DMSO:H2O (2.0:0.1 mL) and subjected to reverse phase chromatography, resulting in 50 mg for a 67.2 % isolated yield of Dabigatran-NH-Boc.
  • DMF dimethylformamide
  • EDC EDC
  • LC-ESI-MS(+) m/z 702.5 [M+H+]+.
  • Dabigatran-NH-Boc was dissolved in H2O:MeCN (1:1, 400 ⁇ L), and then HCl (4 M) in dioxane (1 mL) was added. The homogeneous solution was stirred at room temperature for 30 min and the reaction was concentrated by rotovap to give 41 mg, a 95.6 % yield, of Dabigatran- NH2 as a colorless solid.
  • 18 F-SFB was prepared in 25.0 % isolated yield in 100 min. Dabigatran-NH2 (4 mg, 6.7 ⁇ mol) dissolved in acetonitrile (500 ⁇ L) and triethylamine (4 ⁇ L) was reacted with 18 F-SFB (447 MBq, 12 ⁇ 3 mCi) at 65 °C for 5 min, cooled and subjected to C18 reverse-phase HPLC using a Machery-Nagel Nucleodur C18 Pyramid 250 ⁇ 10 mm Vario-Prep column eluted with 75:25 water-acetonitrile (100 mM ammonium formate) at 5.5 mL/min and a 254 nm UV detector and radiodetector connected in series.
  • F-Dabigatran was synthesized in 10.7 % isolated yield (189 MBq, 5.1 ⁇ 0.2 mCi) and at 99 ⁇ 0.9 % radiochemical purity.
  • 1 8 F-Dabigatran for piglet imaging was produced using a GE FX2N automated synthesizer (GE Healthcare).
  • a QMA cartridge containing cyclotron-produced [ 18 F]fluoride ( ⁇ 30 GBq, 0.81 ⁇ 0.05 Ci) was eluted with a solution containing 9 mg 4,7,13,16,21,24-hexaoxa-1,10 diazabicyclo[8.8.8]hexacosane (Kryptofix [2.2.2]); 0.08 mL 0.15 M K2CO3 and 1.92 mL acetonitrile into a 5 mL reaction vial. Solvents were removed azeotropically at 110 °C under a slight flow of helium.
  • N- succinimidyl-4-[ 18 F]-fluorobenzoate ( 18 F-SFB) was synthesized in 30 % isolated yield (as described in previous section) and reacted with dabigatran-NH2 (4 mg, 6.7 ⁇ mol) dissolved in acetonitrile (500 ⁇ L) and triethylamine (4 ⁇ L) at 65 °C for 5 min.
  • the reaction mixture was purified by HPLC using a C-18 semi-preparative column (Luna C-18, 250 ⁇ 10 mm, 5 ⁇ m - Phenomenex) and isocratic elution with 90:10 water (75 mM ammonium formate)/ethanol at 5 mL/min and a 254 nm UV detector.
  • 18 F- dabigatran was synthesized in 8 ⁇ 1.2% decay corrected radiochemical yield (2.3 ⁇ 1.1 GBq, 0.06 ⁇ 0.03 Ci, at room temperature (RT) for 32 min) and at > 98 % radiochemical purity.
  • DAB-VT680XL visualized the typically fibrin-rich wall that surrounds bacterial vegetation (Fig.3, C to G).
  • Co-staining for CD11b + innate immune cells indicates that the DAB- VT680XL-positive vegetation barrier shields bacteria from the host immune response, as myeloid cells assembled in the vascular wall in front of this barrier but were unable to cross it into the bacterial colony (Fig.3, D and G).
  • Example 3 Noninvasively imaging S. aureus endocarditis in mice Due to light waves’ poor penetration depth in tissue, optical imaging is unsuitable for imaging deep tissues.
  • DAB-VT680XL Fluorescence molecular tomography/ X-ray computed tomography (FMT/CT) data demonstrated high DAB-VT680XL concentration in the aortic roots of mice with endocarditis (Fig.4, A and B), while some signal was also observed in more distal vasculature along the suture inserted to induce endocarditis, likely due to bacteria colonizing the foreign material.
  • FMT/CT Fluorescence molecular tomography/ X-ray computed tomography
  • mice Ex vivo scintillation counting of aortas confirmed high 18 F-DAB uptake (Fig.4H), which co-localized on autoradiography with bacteria-derived bioluminescent signal (Fig.4, I and J).
  • Fig.4H high 18 F-DAB uptake
  • Fig.4, I and J bacteria-derived bioluminescent signal
  • mice was imaged that developed renal S. aureus lesions. Twenty-four hours after injection, DAB-VT680XL accumulation as observed in infected kidneys (fig.11).
  • Example 4 - S. aureus endocarditis in piglets Ideally, molecular imaging probes that show satisfactory performance in mice are next tested in a large animal model. This step enables testing the approach using clinical imaging equipment. Perhaps even more importantly, host defense processes differ considerably between mice and large animals or humans.
  • piglets developed typical clinical signs of endocarditis, including fever (103.5-106.5 °F) and heart murmurs.
  • Bacteria presence on the porcine tricuspid valve was verified by hematoxylin and eosin staining (Fig.5A) and staphylococcal cells were identified by Gram staining (Fig.5B).
  • Fig.5A hematoxylin and eosin staining
  • Fig.5B staphylococcal cells were identified by Gram staining
  • This S. aureus endocarditis piglet model was subsequently characterized by clinical cardiac MRI on a 3 Tesla human scanner.
  • the development of two to four differently sized tricuspid endocarditis lesions were observed in the right ventricles of piglets on days 10-11 after bacterial injection (Fig.5C).
  • Piglets with left- sided endocarditis deteriorated clinically faster than piglets with right-sided endocarditis. They were imaged on day 7 after disease induction. Cardiac MRI revealed development of aortic valve lesions (Fig.5, I and J). Volumetric assessment of piglet hearts with right- and left-sided endocarditis indicated that the right ventricular ejection fraction was lower in piglets with tricuspid disease (Fig.5K). Ex vivo bioluminescence signal co-localized with lesions on autopsy, which enriched DAB-VT680XL after intravenous injection (Fig.5, L to P).
  • a proof-of-concept PET/MR imaging study was initiated, a step typically taken if a molecular imaging probe performs as desired in small animals.
  • an integrated PET/MR cardiac imaging protocol was developed on a clinical scanner to simultaneously acquire high-resolution morphological information and radiotracer distribution in piglet hearts.
  • the PET imaging probe 18 F-DAB was injected into three piglets with left- or right-sided endocarditis, respectively.
  • PET/MRI revealed focal enrichment of the PET imaging agent in the aortic (Fig.6, A and B) and tricuspid valves (Fig.6, C and D).
  • This difference in imaging signal may relate to bacterial load, supported by a positive correlation between bacterial bioluminescence signal and DAB-VT680XL binding across vegetations examined in both valves (fig.12C). These differences may have been caused by the faster clinical disease progression and the earlier imaging time point in piglets with left-sided endocarditis.
  • E xample 5 - Antibody immunotherapy disrupts vegetation anatomy and improves mouse survival Staphylocoagulase (SC) and von Willebrand factor-binding protein (vWBp) are virulence factors that S. aureus employs to activate the clotting cascade. The resulting fibrin wall protects bacterial colonies from the host’s immune system.
  • PET is the most sensitive and translatable molecular imaging modality, as demonstrated by its clinical track record.
  • the limitations of PET imaging include relatively low spatial resolution and high costs.
  • Co-developing a fluorescent agent with the same affinity ligand provides cellular resolution data for molecular targets imaged by PET.
  • DAB-VT680XL compound of the present examples proved useful in a preclinical neutralizing antibody trial in mice.
  • a lower imaging signal in endocarditis vegetations echoed therapy-induced survival benefits, suggesting this imaging biomarker may predict survival.
  • PET imaging with 18 F-DAB would track therapeutic efficiency in a trial of candidate drugs similar to the SC- and vWBp-neutralizing antibodies that we tested in mice. PET imaging with 18 F-DAB would identify patients in need of therapy or monitor therapeutic effects.
  • the PET/MRI data in piglets with endocarditis indicate that combining molecular PET with MRI is particularly informative, as MRI provides outstanding soft tissue contrast and sufficient temporal and spatial information to visualize S. aureus vegetations. Given its capacity for bedside use, facile Doppler assessment of valve function and lower cost, echocardiography will remain a mainstay for serially monitoring acute endocarditis.
  • 18 F-DAB and DAB- VT680XL of the present disclosure do not directly bind bacteria but rather report on S. aureus’ interaction with the host’s clotting system, which contributes to biofilm formation.
  • This fibrin-rich wall protects the bacterial colony against host immunity and — in combination with exopolysaccharides, extracellular DNA and other factors — hinders penetration of antibiotics.
  • Our data indicate that 18 F-DAB and DAB- VT680XL avidly bind to this critical vegetation component.
  • DAB-VT680XL in intravital microscopy visualized innate immune cells’ inability to penetrate the wall, swarm into the vegetation and kill S. aureus.
  • Treatment with antibodies against SC and vWBp decreased DAB-VT680XL uptake, and myeloid cells were able to enter the vegetations.
  • the piglet model could be useful for research on surgical management, a potentially life-saving treatment still in need of optimization and standardization.
  • aureus endocarditis vegetations activate the host’s clotting system to anchor the colony and surround it with a protective fibrin mesh.
  • Upstream interventions that curtail bacterial virulence rather than host clotting factors could ultimately be a safer alternative.
  • decreased thrombin was detected in mouse endocarditis vegetations after treatment with antibodies that neutralize SC and vWBp.
  • vegetations still evolved and mice succumbed eventually; nevertheless, antibody treatment prolonged survival while DAB-VT680XL signal decreased in the lesions and innate immune cells invaded bacterial colonies.

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des conjugués d'inhibiteur de thrombine dabigatran, ainsi que des procédés d'utilisation de ces conjugués pour détecter des maladies ou des états associés à la thrombine, par exemple pour diagnostiquer une endocardite bactérienne dans le coeur du patient.
PCT/US2021/039436 2020-06-29 2021-06-28 Procédés d'imagerie d'infections bactériennes WO2022006007A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014009966A2 (fr) * 2012-07-12 2014-01-16 Rao Davuluri Ramamohan Procédé perfectionné pour la préparation d'etexilate mésylate de dabigatran et de ses intermédiaires
WO2016044645A1 (fr) * 2014-09-17 2016-03-24 Elmaleh David R Dérivés d'anticoagulant pour l'imagerie cardio-vasculaire
US9375493B2 (en) * 2012-03-30 2016-06-28 Visen Medical, Inc. Bacterial imaging agents and methods of using same
US20160287731A1 (en) * 2013-11-14 2016-10-06 Endocyte, Inc. Compounds for Positron Emission Tomography

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9375493B2 (en) * 2012-03-30 2016-06-28 Visen Medical, Inc. Bacterial imaging agents and methods of using same
WO2014009966A2 (fr) * 2012-07-12 2014-01-16 Rao Davuluri Ramamohan Procédé perfectionné pour la préparation d'etexilate mésylate de dabigatran et de ses intermédiaires
US20160287731A1 (en) * 2013-11-14 2016-10-06 Endocyte, Inc. Compounds for Positron Emission Tomography
WO2016044645A1 (fr) * 2014-09-17 2016-03-24 Elmaleh David R Dérivés d'anticoagulant pour l'imagerie cardio-vasculaire

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