Biodistribution, Dosimetry, and Pharmacokinetics of 68Ga-CBP8: A Type I Collagen-Targeted PET Probe.

The 68Ga-Collagen Binding Probe #8, 68Ga-CBP8, is a peptide-based, type I collagen-targeted probe developed for imaging of tissue fibrosis. The aim of this study was to determine the biodistribution, dosimetry, and pharmacokinetics of 68Ga-CBP8 in healthy human subjects. Methods: Nine healthy volunteers (5 male and 4 female) underwent whole-body 68Ga-CBP8 PET/MRI using a Biograph mMR scanner. The subjects were imaged continuously for up to 2 h after injection of 68Ga-CBP8. A subset of subjects underwent an additional imaging session 2-3 h after probe injection. OLINDA/EXM software was used to calculate absorbed organ and effective dose estimates based on up to 17 regions of interest (16 for men) defined on T2-weighted MR images and copied to the PET images, assuming a uniform distribution of probe concentration in each region. Serial blood sampling up to 90 min after probe injection was performed to assess blood clearance and metabolic stability. Results: The mean injected activity (±SD) of 68Ga-CBP8 was 220 ± 100 MBq (range, 113-434 MBq). No adverse effects related to probe administration were detected. 68Ga-CBP8 demonstrated an extracellular distribution with predominantly rapid renal clearance. Doses on the urinary bladder were 0.15 versus 0.19 mGy/MBq for men versus women. The highest absorbed doses for the rest of the organs were measured in the kidneys (0.078 vs. 0.088 mGy/MBq) and the liver (0.032 vs. 0.041 mGy/MBq). The mean effective dose was 0.018 ± 0.0026 mSv/MBq using a 1-h voiding model. The 68Ga-CBP8 signal in the blood demonstrated biexponential pharmacokinetics with an initial distribution half-life of 4.9 min (95% CI, 2.4-9.4 min) and a 72-min elimination half-life (95% CI, 47-130 min). The only metabolite observed had a long blood plasma half-life, suggesting protein-bound 68Ga. Conclusion: 68Ga-CBP8 displays favorable in-human characteristics and dosimetry similar to that of other gallium-based probes. 68Ga-CBP8 could therefore be used for noninvasive collagen imaging across a range of human fibrotic diseases.

Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease.

Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ's microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.

Detection and Characterization of Thrombosis in Humans Using Fibrin-Targeted Positron Emission Tomography and Magnetic Resonance.

OBJECTIVES: The authors present a novel technique to detect and characterize LAA thrombus in humans using combined positron emission tomography (PET)/cardiac magnetic resonance (CMR) of a fibrin-binding radiotracer, [64Cu]FBP8. BACKGROUND: The detection of thrombus in the left atrial appendage (LAA) is vital in the prevention of stroke and is currently performed using transesophageal echocardiography (TEE). METHODS: The metabolism and pharmacokinetics of [64Cu]FBP8 were studied in 8 healthy volunteers. Patients with atrial fibrillation and recent TEEs of the LAA (positive n = 12, negative n = 12) were injected with [64Cu]FBP8 and imaged with PET/CMR, including mapping the longitudinal magnetic relaxation time (T1) in the LAA. RESULTS: [64Cu]FBP8 was stable to metabolism and was rapidly eliminated. The maximum standardized uptake value (SUVMax) in the LAA was significantly higher in the TEE-positive than TEE-negative subjects (median of 4.0 [interquartile range (IQR): 3.0-6.0] vs 2.3 [IQR: 2.1-2.5]; P < 0.001), with an area under the receiver-operating characteristic curve of 0.97. An SUVMax threshold of 2.6 provided a sensitivity of 100% and specificity of 84%. The minimum T1 (T1Min) in the LAA was 970 ms (IQR: 780-1,080 ms) vs 1,380 ms (IQR: 1,120-1,620 ms) (TEE positive vs TEE negative; P < 0.05), with some overlap between the groups. Logistic regression using SUVMax and T1Min allowed all TEE-positive and TEE-negative subjects to be classified with 100% accuracy. CONCLUSIONS: PET/CMR of [64Cu]FBP8 is able to detect acute as well as older platelet-poor thrombi with excellent accuracy. Furthermore, the integrated PET/CMR approach provides useful information on the biological properties of thrombus such as fibrin and methemoglobin content. (Imaging of LAA Thrombosis; NCT03830320).

68Ga-NODAGA-Indole: An Allysine-Reactive Positron Emission Tomography Probe for Molecular Imaging of Pulmonary Fibrogenesis.

Oxidized collagen, wherein lysine residues are converted to the aldehyde allysine, is a universal feature of fibrogenesis, i.e. actively progressive fibrosis. Here we report the small molecule, allysine-binding positron emission tomography probe, 68Ga-NODAGA-indole, that can noninvasively detect and quantify pulmonary fibrogenesis. We demonstrate that the uptake of 68Ga-NODAGA-indole in actively fibrotic lungs is 7-fold higher than in control groups and that uptake is linearly correlated ( R2 = 0.98) with the concentration of lung allysine.

Molecular imaging of fibrosis: recent advances and future directions.

Fibrosis, the progressive accumulation of connective tissue that occurs in response to injury, causes irreparable organ damage and may result in organ failure. The few available antifibrotic treatments modify the rate of fibrosis progression, but there are no available treatments to reverse established fibrosis. Thus, more effective therapies are urgently needed. Molecular imaging is a promising biomedical methodology that enables noninvasive visualization of cellular and subcellular processes. It provides a unique means to monitor and quantify dysregulated molecular fibrotic pathways in a noninvasive manner. Molecular imaging could be used for early detection, disease staging, and prognostication, as well as for assessing disease activity and treatment response. As fibrotic diseases are often molecularly heterogeneous, molecular imaging of a specific pathway could be used for patient stratification and cohort enrichment with the goal of improving clinical trial design and feasibility and increasing the ability to detect a definitive outcome for new therapies. Here we review currently available molecular imaging probes for detecting fibrosis and fibrogenesis, the active formation of new fibrous tissue, and their application to models of fibrosis across organ systems and fibrotic processes. We provide our opinion as to the potential roles of molecular imaging in human fibrotic diseases.

Molecular Probes for Imaging Fibrosis and Fibrogenesis.

Fibrosis, or the accumulation of extracellular matrix molecules that make up scar tissue, is a common result of chronic tissue injury. Advances in the clinical management of fibrotic diseases have been hampered by the low sensitivity and specificity of noninvasive early diagnostic options, lack of surrogate end points for use in clinical trials, and a paucity of noninvasive tools to assess fibrotic disease activity longitudinally. Hence, the development of new methods to image fibrosis and fibrogenesis is a large unmet clinical need. Herein, an overview of recent and selected molecular probes for imaging of fibrosis and fibrogenesis by magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography is provided.

Imaging the Vascular Bone Marrow Niche During Inflammatory Stress.

RATIONALE: Inflammatory stress induced by exposure to bacterial lipopolysaccharide causes hematopoietic stem cell expansion in the bone marrow niche, generating a cellular immune response. As an integral component of the hematopoietic stem cell niche, the bone marrow vasculature regulates the production and release of blood leukocytes, which protect the host against infection but also fuel inflammatory diseases. OBJECTIVE: We aimed to develop imaging tools to explore vascular changes in the bone marrow niche during acute inflammation. METHODS AND RESULTS: Using the TLR (Toll-like receptor) ligand lipopolysaccharide as a prototypical danger signal, we applied multiparametric, multimodality and multiscale imaging to characterize how the bone marrow vasculature adapts when hematopoiesis boosts leukocyte supply. In response to lipopolysaccharide, ex vivo flow cytometry and histology showed vascular changes to the bone marrow niche. Specifically, proliferating endothelial cells gave rise to new vasculature in the bone marrow during hypoxic conditions. We studied these vascular changes with complementary intravital microscopy and positron emission tomography/magnetic resonance imaging. Fluorescence and positron emission tomography integrin αVß3 imaging signal increased during lipopolysaccharide-induced vascular remodeling. Vascular leakiness, quantified by albumin-based in vivo microscopy and magnetic resonance imaging, rose when neutrophils departed and hematopoietic stem and progenitor cells proliferated more vigorously. CONCLUSIONS: Introducing a tool set to image bone marrow either with cellular resolution or noninvasively within the entire skeleton, this work sheds light on angiogenic responses that accompany emergency hematopoiesis. Understanding and monitoring bone marrow vasculature may provide a key to unlock therapeutic targets regulating systemic inflammation.

Optimization of a Collagen-Targeted PET Probe for Molecular Imaging of Pulmonary Fibrosis.

There is a large unmet need for a simple, accurate, noninvasive, quantitative, and high-resolution imaging modality to detect lung fibrosis at early stage and to monitor disease progression. Overexpression of collagen is a hallmark of organ fibrosis. Here, we describe the optimization of a collagen-targeted PET probe for staging pulmonary fibrosis. Methods: Six peptides were synthesized, conjugated to a copper chelator, and radiolabeled with 64Cu. The collagen affinity of each probe was measured in a plate-based assay. The pharmacokinetics and metabolic stability of the probes were studied in healthy rats. The capacity of these probes to detect and stage pulmonary fibrosis in vivo was assessed in a mouse model of bleomycin-induced fibrosis using PET imaging. Results: All probes exhibited affinities in the low micromolar range (1.6 µM < Kd < 14.6 µM) and had rapid blood clearance. The probes showed 2- to 8-fold-greater uptake in the lungs of bleomycin-treated mice than sham-treated mice, whereas the distribution in other organs was similar between bleomycin-treated and sham mice. The probe 64Cu-CBP7 showed the highest uptake in fibrotic lungs and the highest target-to-background ratios. The superiority of 64Cu-CBP7 was traced to a much higher metabolic stability compared with the other probes. The specificity of 64Cu-CBP7 for collagen was confirmed by comparison with a nonbinding isomer. Conclusion:64Cu-CBP7 is a promising candidate for in vivo imaging of pulmonary fibrosis.

Type I collagen-targeted PET probe for pulmonary fibrosis detection and staging in preclinical models.

Pulmonary fibrosis is scarring of the lungs that can arise from radiation injury, drug toxicity, environmental or genetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is a hallmark of organ fibrosis. We describe a peptide-based positron emission tomography (PET) probe (68Ga-CBP8) that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmonary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target/background ratios in diseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2 = 0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probe could be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associated with vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. These studies indicate that 68Ga-CBP8 is a promising candidate for noninvasive imaging of human pulmonary fibrosis.

AMD3100: A Versatile Platform for CXCR4 Targeting (68)Ga-Based Radiopharmaceuticals.

CXCR4 is a G protein-coupled receptor (GPCR), which is overexpressed in numerous diseases, particularly in multiple cancers. Therefore, this receptor represents a valuable target for imaging and therapeutic purposes. Among the different approaches, which were developed for CXCR4 imaging, a CXCR4 antagonist biscyclam system (AMD3100, also called Mozobil), currently used in the clinic for the mobilization of hematopoietic stem cells, was radiolabeled with different radiometals such as (62)Zn, (64)Cu, (67)Ga, or (99m)Tc. However, cyclam is not an ideal chelator for most of these radiometals, and could lead to the release of the radionuclide in vivo. In the current study, a new family of CXCR4 imaging agents is presented, in which AMD3100 is used as a carrier for specific delivery of an imaging reporter, i.e., a (68)Ga complex for PET imaging. AMD3100 was functionalized on the phenyl moiety with different linkers, either ethylenediamine or diamino-polyethylene glycol 3 (PEG3). The resulting AMD3100 analogues were further coupled with two different chelators, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid (NODAGA). Five potential CXCR4 targeting agents were obtained. The derived AMD3100-based ligands were labeled with (68)Ga, highlighting the influence of the spacer nature on the (68)Ga-labeling yield. The lipophilic character of the different systems was also investigated, as well as their affinity for the CXCR4 receptor. The most promising compound was further evaluated in vivo in H69 tumor xenografts by biodistribution and PET imaging studies, validating the proof of principle of our concept.

MA-NOTMP: A Triazacyclononane Trimethylphosphinate Based Bifunctional Chelator for Gallium Radiolabelling of Biomolecules.

In the past few years, gallium-68 has demonstrated significant potential as a radioisotope for positron emission tomography (PET), and the optimization of chelators for gallium coordination is a major goal in the development of radiopharmaceuticals. Methylaminotriazacyclononane trimethylphosphinate (MA-NOTMP), a new C-functionalized triazacyclononane derivative with phosphinate pendant arms, presents excellent coordination properties for (68) Ga (low ligand concentration, labelling at low pH even at room temperature). A "ready-to-be-grafted" bifunctional chelating agent (p-NCS-Bz-MA-NOTMP) was prepared to allow (68) Ga labelling of sensitive biological vectors. Conjugation to a bombesin(7-14) derivative was performed, and preliminary in vitro experiments demonstrated the potential of MA-NOTMP in the development of radiopharmaceuticals. This new chelator is therefore of major interest for labelling sensitive biomolecules, and further in vivo experiments will soon be performed.

New AMD3100 derivatives for CXCR4 chemokine receptor targeted molecular imaging studies: synthesis, anti-HIV-1 evaluation and binding affinities.

CXCR4 is a target of growing interest for the development of new therapeutic drugs and imaging agents as its role in multiple disease states has been demonstrated. AMD3100, a CXCR4 chemokine receptor antagonist that is in current clinical use as a haematopoietic stem cell mobilising drug, has been widely studied for its anti-HIV properties, potential to inhibit metastatic spread of certain cancers and, more recently, its ability to chelate radiometals for nuclear imaging. In this study, AMD3100 is functionalised on the phenyl moiety to investigate the influence of the structural modification on the anti-HIV-1 properties and receptor affinity in competition with anti-CXCR4 monoclonal antibodies and the natural ligand for CXCR4, CXCL12. The effect of complexation of nickel(II) in the cyclam cavities has been investigated. Two amino derivatives were obtained and are suitable intermediates for conjugation reactions to obtain CXCR4 molecular imaging agents. A fluorescent probe (BODIPY) and a precursor for (18)F (positron emitting isotope) radiolabelling were conjugated to validate this route to new CXCR4 imaging agents.

Remarkable inertness of copper(II) chelates of cyclen-based macrobicycles with two trans-N-acetate arms.

Two cross-bridged cyclen-based macrocycles with two trans-N-acetic acid arms, one having a dibenzofuran (DBF) moiety as the bridge, H2L1, and the other a diphenyl ether (DPE) one, H2L2, were synthesized. Both compounds behave as "proton sponges." The thermodynamic stability constants for the Cu(2+), Zn(2+), Al(3+), and Ga(3+) complexes of both compounds were determined. They exhibit an excellent thermodynamic selectivity for copper(II), ensuring that metal ions largely present in the human body do not interfere with the copper(II) chelates. All complexes are very slow to form, and [CuL2] and [CuL1] are extremely inert to demetallate, especially [CuL2]. The acid-assisted dissociation of [CuL1] led to a half-life of 4.28 h in 5 M HCl at 363.2 K, while [CuL2] needed harsher conditions of 12 M HCl at 363.2 K with a half-life of 30.8 days. To the best of our knowledge, [CuL2] exhibits the highest half-life value for a copper(II) complex of a polyazamacrocycle derivative reported in the literature until now. Single crystal X-ray diffraction determined for [Cu(H2L1)](ClO4)2 showed the copper center in a distorted octahedral environment bound to the N4O donors of the macrobicycle and one oxygen atom from a carboxylic arm, while for [CuL2] it showed the copper center in a trigonal bipyramid geometry only bound to the donors of the macrobicycle and leaving the carboxylate arms away from the coordination sphere. UV-vis-NIR and X-band EPR spectra showed that in [CuL1] the copper center adopts a distorted compressed octahedral environment, which is the only structure found in solution for this complex, while in [CuL2] a similar environment was found in the first stages of its slow formation but reached a square-pyramidal geometry upon stabilization. The acetate arms play therefore an important role during the formation of the complex, as revealed by the comparison of its complexation behavior with the corresponding parent compounds.

Proton and metal binding by cyclen-based highly rigid cryptands.

The basicity properties of the two cryptands L1 and L2, featuring, respectively, a dibenzofuran or a diphenyl ether moiety bridging the 1,7 positions of a 1,4,7,10-tetraazacyclododecane macrocycle (cyclen) have been studied by means of potentiometric, UV-vis and fluorescence emission measurements. Both ligands show a high basicity in the first protonation step, the first basicity constant of L1 being too high to be measured in aqueous solution. The crystal structure of {[HL1]L1}(+) shows that the NH(2)(+) group is involved in an intramolecular hydrogen bonding network, which justifies the observed high basicity in solution. (1)H, (13)C NMR, UV-vis and fluorescence emission measurements show that, among first row divalent metal cations, both L1 and L2 selectively bind in acetonitrile Cu(II) and Zn(II), which are encapsulated within the ligand cavities. Zn(II) coordination is accompanied by a remarkable increase of the fluorescence emission of the ligands, pointing out that the molecular architecture displayed by L1 and L2 can be used to develop new OFF/ON chemosensors for this metal cation.