Preclinical Validation of the Heparin-Reactive Peptide p5+14 as a Molecular Imaging Agent for Visceral Amyloidosis.

Amyloid is a complex pathologic matrix comprised principally of paracrystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloid diseases are rare, thus, routine diagnosis is often challenging. The glycosaminoglycans ubiquitously present in amyloid deposits are biochemically and electrochemically distinct from those found in the healthy tissues due to the high degree of sulfation. We have exploited this unique property and evaluated heparin-reactive peptides, such as p5+14, as novel agents for specifically targeting and imaging amyloid. Herein, we demonstrate that radiolabeled p5+14 effectively bound murine AA amyloid in vivo by using molecular imaging. Biotinylated peptide also reacted with the major forms of human amyloid in tissue sections as evidenced immunohistochemically. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients.

Whole-body biodistribution of 3'-deoxy-3'-[(18) f]fluorothymidine ((18) FLT) in healthy adult cats.

Positron emission tomography/computed tomography (PET/CT) utilizing 3'-deoxy-3'-[(18) F]fluorothymidine ((18) FLT), a proliferation tracer, has been found to be a useful tool for characterizing neoplastic diseases and bone marrow function in humans. As PET and PET/CT imaging become increasingly available in veterinary medicine, knowledge of radiopharmaceutical biodistribution in veterinary species is needed for lesion interpretation in the clinical setting. The purpose of this study was to describe the normal biodistribution of (18) FLT in adult domestic cats. Imaging of six healthy young adult castrated male cats was performed using a commercially available PET/CT scanner consisting of a 64-slice helical CT scanner with an integrated whole-body, high-resolution lutetium oxy-orthosilicate (LSO) PET scanner. Cats were sedated and injected intravenously with 108.60 ± 2.09 (mean ± SD) MBq of (18) FLT (greater than 99% radiochemical purity by high-performance liquid chromatography). Imaging was performed in sternal recumbency under general anesthesia. Static images utilizing multiple bed positions were acquired 80.83 ± 7.52 (mean ± SD) minutes post-injection. Regions of interest were manually drawn over major parenchymal organs and selected areas of bone marrow and increased tracer uptake. Standardized uptake values were calculated. Notable areas of uptake included hematopoietic bone marrow, intestinal tract, and the urinary and hepatobiliary systems. No appreciable uptake was observed within brain, lung, myocardium, spleen, or skeletal muscle. Findings from this study can be used as baseline data for future studies of diseases in cats.

Radioimmunodetection of amyloid deposits in patients with AL amyloidosis.

Care of patients with AL amyloidosis currently is limited by the lack of objective means to document disease extent, as well as therapeutic options that expedite removal of pathologic deposits. To address these issues, we have initiated a Phase I Exploratory IND study to determine the biodistribution of the fibril-reactive, amyloidolytic murine IgG1 mAb 11-1F4 labeled with I-124. Patients were infused with less than 1 mg (∼ 74 MBq) of GMP-grade antibody and imaged by PET/CT scan 48 and 120 hours later. Among 9 of 18 subjects, there was striking uptake of the reagent in liver, lymph nodes, bone marrow, intestine, or, unexpectedly, spleen (but not kidneys or heart). Generally, positive or negative results correlated with those obtained immunohistochemically using diagnostic tissue biopsy specimens. Based on these findings, we posit that (124)I-mAb m11-1F4 can be used to identify AL candidates for passive immunotherapy using the chimeric form of the antibody.

First in Human Evaluation and Dosimetry Calculations for Peptide 124I-p5+14-a Novel Radiotracer for the Detection of Systemic Amyloidosis Using PET/CT Imaging.

PURPOSE: Accurate diagnosis of amyloidosis remains a significant clinical challenge and unmet need for patients. The amyloid-reactive peptide p5+14 radiolabeled with iodine-124 has been developed for the detection of amyloid by PET/CT imaging. In a first-in-human evaluation, the dosimetry and tissue distribution of 124I-p5+14 peptide in patients with systemic amyloidosis. Herein, we report the dosimetry and dynamic distribution in the first three enrolled patients with light chain-associated (AL) amyloidosis. PROCEDURES: The radiotracer was assessed in a single-site, open-label phase 1 study (NCT03678259). The first three patients received a single intravenous infusion of 124I-p5+14 peptide (≤37 MBq). Serial PET/CT imaging was performed during the 48 h post-infusion. Dosimetry was determined as a primary endpoint for each patient and gender-averaged mean values were calculated. Pharmacokinetic parameters were estimated from whole blood radioactivity measurements and organ-based time activity data. Lastly, the biodistribution of radiotracer in major organs was assessed visually and compared to clinically appreciated organ involvement. RESULTS: Infusion of the 124I-p5+14 was well tolerated with rapid uptake in the heart, kidneys, liver, spleen, pancreas, and lung. The gender-averaged whole-body effective radiation dose was estimated to be 0.23 (± 0.02) mSv/MBq with elimination of the radioactivity via renal and gastrointestinal routes. The whole blood elimination t1/2 of 21.9 ± 7.6 h. Organ-based activity concentration measurements indicated that AUClast tissue:blood ratios generally correlated with the anticipated presence of amyloid. Peptide uptake was observed in 4/5 clinically suspected organs, as noted in the medical record, as well as six anatomic sites generally associated with amyloidosis in this population. CONCLUSION: Peptide 124I-p5+14 rapidly distributes to anatomic sites consistent with the presence of amyloid in patients with systemic AL. The dosimetry estimates established in this cohort are acceptable for whole-body PET/CT imaging. Pharmacokinetic parameters are heterogeneous and consistent with uptake of the tracer in an amyloid compartment. PET/CT imaging of 124I-p5+14 may facilitate non-invasive detection of amyloid in multiple organ systems.

Distribution of 2-deoxy-2-fluoro-d-glucose in the coelom of healthy bald eagles (Haliaeetus leucocephalus).

OBJECTIVE: To determine 2-deoxy-2-fluoro (fluorine 18)-d-glucose ((18)FDG) biodistribution in the coelom of bald eagles (Haliaeetus leucocephalus). ANIMALS: 8 healthy adult bald eagles. PROCEDURES: For each eagle, whole-body transmission noncontrast CT, 60-minute dynamic positron emission tomography (PET) of the celomic cavity (immediately after (18)FDG injection), whole-body static PET 60 minutes after (18)FDG injection, and whole-body contrast CT with iohexol were performed. After reconstruction, images were analyzed. Regions of interest were drawn over the ventricular myocardium, liver, spleen, proventriculus, cloaca, kidneys, and lungs on dynamic and static PET images. Standardized uptake values were calculated. RESULTS: Kidneys had the most intense (18)FDG uptake, followed by cloaca and intestinal tract; liver activity was mild and slightly more intense than that of the spleen; proventricular activity was always present, whereas little to no activity was identified in the wall of the ventriculus. Activity in the myocardium was present in all birds but varied in intensity among birds. The lungs had no visibly discernible activity. Mean ± SD standardized uptake values calculated with representative regions of interest at 60 minutes were as follows: myocardium, 1. 6 ± 0.2 (transverse plane) and 1.3 ± 0.3 (sagittal plane); liver, 1.1 ± 0.1; spleen, 0.9 ± 0.1; proventriculus, 1.0 ± 0.1; cloaca, 4.4 ± 2.7; right kidney, 17.3 ± 1.0; left kidney, 17.6 ± 0.3; and right and left lungs (each), 0.3 ± 0.02. CONCLUSIONS AND CLINICAL RELEVANCE: The study established the biodistribution of (18)FDG in adult eagles, providing a baseline for clinical investigation and future research.

Preliminary evaluation of serial (18) FDG-PET/CT to assess response to toceranib phosphate therapy in canine cancer.

Palladia(TM) (toceranib phosphate-Pfizer Animal Health) is a novel orally administered receptor tyrosine kinase inhibitor (TKI) approved for treatment of canine mast cell tumors. Receptor tyrosine kinase dysregulation leads to tumor growth, progression, and metastasis. Toceranib's targets include vascular endothelial growth factor receptor (VEGFR-2/Flk-1/KDR), platelet-derived growth factor receptor, and kit. Positron Emission Tomography/Computed Tomography (PET/CT) is used commonly to diagnose, prognosticate, and monitor response to antineoplastic therapy in human patients. In this study, serial PET/CT imaging with (18) F-fluorodeoxyglucose ((18) FDG) was used to assess response to toceranib therapy in dogs with measurable solid malignancies. Six tumor-bearing dogs underwent tumor assessment using both standard RECIST criteria and PET/CT prior to and at a median of 5 weeks postinitiation of toceranib treatment. Toceranib was prescribed initially at a target dose 3.25 mg/kg PO q48 h, with subsequent modifications based on observed toxicity. Treatment was continued in patients achieving stable disease with acceptable drug tolerance. One dog was maintained on drug despite dose modification due to toxicity; measurable clinical and image-based responses were seen after 10 weeks of therapy. All others had stable or progressive disease based on clinical restaging and PET/CT at first recheck. . Due to discordance with anatomic and metabolic imaging, further studies are needed to investigate the role of molecular imaging in assessment of drug response and identify other potential molecular targets of toceranib.

Synergistic effects of leucine and resveratrol on insulin sensitivity and fat metabolism in adipocytes and mice.

BACKGROUND: Sirtuins are important regulators of glucose and fat metabolism, and sirtuin activation has been proposed as a therapeutic target for insulin resistance and diabetes. We have shown leucine to increase mitochondrial biogenesis and fat oxidation via Sirt1 dependent pathways. Resveratrol is a widely recognized activator of Sirt; however, the biologically-effective high concentrations used in cell and animal studies are generally impractical or difficult to achieve in humans. Accordingly, we sought to determine whether leucine would exhibit synergy with low levels of resveratrol on sirtuin-dependent outcomes in adipocytes and in diet-induced obese (DIO) mice. METHODS: 3T3-L1 mouse adipocytes were treated with Leucine (0.5 mM), ß-hydroxy-ß-methyl butyrate (HMB) (5 µM) or Resveratrol (200 nM) alone or in combination. In addition, diet-induced obese mice were treated for 6-weeks with low (2 g/kg diet) or high (10 g/kg diet) dose HMB, Leucine (24 g/kg diet; 200% of normal level) or low (12.5 mg/kg diet) or high (225 mg/kg diet) dose resveratrol, alone or as combination with leucine-resveratrol or HMB-resveratrol. RESULTS: Fatty acid oxidation, AMPK, Sirt1 and Sirt3 activity in 3T3-L1 adipocytes and in muscle cells, were significantly increased by the combinations compared to the individual treatments. Similarly, 6-week feeding of low-dose resveratrol combined with either leucine or its metabolite HMB to DIO mice increased adipose Sirt1 activity, muscle glucose and palmitate uptake (measured via PET/CT), insulin sensitivity (HOMAIR), improved inflammatory stress biomarkers (CRP, IL-6, MCP-1, adiponectin) and reduced adiposity comparable to the effects of high dose resveratrol, while low-dose resveratrol exerted no independent effect. CONCLUSION: These data demonstrate that either leucine or its metabolite HMB may be combined with a low concentration of resveratrol to exert synergistic effects on Sirt1-dependent outcomes; this may result in more practical dosing of resveratrol in the management of obesity, insulin-resistance and diabetes.