Steatosis Alters the Activity of Hepatocyte Membrane Transporters in Obese Rats

Fat accumulation (steatosis) in ballooned hepatocytes alters the expression of membrane transporters in Zucker fatty (fa/fa) rats. The aim of the study was to quantify the functions of these transporters and their impact on hepatocyte concentrations using a clinical hepatobiliary contrast agent (Gadobenate dimeglumine, BOPTA) for liver imaging. In isolated and perfused rat livers, we quantified BOPTA accumulation and decay profiles in fa/+ (normal) and fa/fa hepatocytes by placing a gamma counter over livers. Profiles of BOPTA accumulation and decay in hepatocytes were analysed with nonlinear regressions to characterise BOPTA influx and efflux across hepatocyte transporters. At the end of the accumulation period, BOPTA hepatocyte concentrations and influx clearances were not significantly different in fa/+ and fa/fa livers. In contrast, bile clearance was significantly lower in fatty hepatocytes while efflux clearance back to sinusoids compensated the low efflux into canaliculi. The time when BOPTA cellular efflux impacts the accumulation profile of hepatocyte concentrations was slightly delayed (2 min) by steatosis, anticipating a delayed emptying of hepatocytes. The experimental model is useful for quantifying the functions of hepatocyte transporters in liver diseases.

The influence of temperature on biodistribution of N,N,N’,N’- ethylenediaminetetrakis(methylene phosphonic) acid labeled with gallium-68

Bone metastases are serious complication in the progression of various types of cancer. It determines the requirement of modern nuclear diagnostic tools, including positron emission tomography (PET). In this study the biodistribution of EDTMP labeled with gallium-68 (68Ga-EDTMP) prepared at different temperature (20, 50, and 95 °C) was investigated. All experimental studies were performed in healthy intact Wistar rats by measuring the radioactivity in organs and tissues with gamma counter. All 68Ga-EDTMP formulations accumulated predominantly in bones. Only in tibia the uptake of 68Ga-EDTMP prepared at 95 °C was higher (p < 0.05) than 68Ga-EDTMP prepared at 20 °C, but in other bones there weren’t any statistical differences in uptake of 68Ga-EDTMP formulations. The amounts of 68Ga-EDTMP formulations in soft organs and tissues were lower when compared with bones. In conclusion, a temperature of reaction mixture had an influence on the biodistribution of 68Ga-EDTMP in bones.

Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE−/− mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.

Protection From Contamination by 211At, an Enigmatic but Promising Alpha Emitter

Purpose 211At, a promising alpha emitter, can easily volatilize and contaminate the environment. To safely manage this unique alpha emitter, we investigated the permeability of four types of plastic films and gloves against 211At and identified suitable materials to avoid contamination by 211At. Methods Four types of plastic films, polyethylene, polyvinylidene chloride, polyvinyl chloride, and a laminated film, and two types of rubber gloves, latex and nitrile, were examined. Small pieces of filter paper were covered with these materials, and a drop containing 100 kBq of 211At was placed on them. The radioactivity of pieces of filter paper under the materials was evaluated by measuring counts using a gamma counter and obtaining autoradiograms 3.5 h later. These experiments were also performed using 225Ac, 125I, 111In, 201Tl, and 99mTc. Results 211At solution easily penetrated polyethylene, polyvinyl chloride, and latex rubber. Similar results were obtained for 125I, while other radionuclides did not penetrate films or gloves. These results suggest that halogenic radionuclides under anionic conditions are likely to penetrate plastic films and rubber gloves. Conclusion Our evaluation revealed that, when 211At solution is used, the protection by polyvinylidene chloride, a laminated film, or nitrile rubber would be more effective than that by polyethylene, polyvinyl chloride, or latex rubber.

3D printing 18F radioactive phantoms for PET imaging

Purpose Phantoms are routinely used in molecular imaging to assess scanner performance. However, traditional phantoms with fillable shapes do not replicate human anatomy. 3D-printed phantoms have overcome this by creating phantoms which replicate human anatomy which can be filled with radioactive material. The problem with these is that small objects suffer to a greater extent than larger objects from the effects of inactive walls, and therefore, phantoms without these are desirable. The purpose of this study was to explore the feasibility of creating resin-based 3D-printed phantoms using 18F. Methods Radioactive resin was created using an emulsion of printer resin and 18F-FDG. A series of test objects were printed including twenty identical cylinders, ten spheres with increasing diameters (2 to 20 mm), and a double helix. Radioactive concentration uniformity, printing accuracy and the amount of leaching were assessed. Results Creating radioactive resin was simple and effective. The radioactive concentration was uniform among identical objects; the CoV of the signal was 0.7% using a gamma counter. The printed cylinders and spheres were found to be within 4% of the model dimensions. A double helix was successfully printed as a test for the printer and appeared as expected on the PET scanner. The amount of radioactivity leached into the water was measurable (0.72%) but not visible above background on the imaging. Conclusions Creating an 18F radioactive resin emulsion is a simple and effective way to create accurate and complex phantoms without inactive walls. This technique could be used to print clinically realistic phantoms. However, they are single use and cannot be made hollow without an exit hole. Also, there is a small amount of leaching of the radioactivity to take into consideration.

Ceramide analog [18F]F-HPA-12 detects sphingolipid disbalance in the brain of Alzheimer’s disease transgenic mice by functioning as a metabolic probe

The metabolism of ceramides is deregulated in the brain of Alzheimer’s disease (AD) patients and is associated with apolipoprotein (APO) APOE4 and amyloid-β pathology. However, how the ceramide metabolism changes over time in AD, in vivo, remains unknown. Distribution and metabolism of [18F]F-HPA-12, a radio-fluorinated version of the ceramide analog N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide, was investigated in the brain of AD transgenic mouse models (FAD) on an APOE4 or APOE3 genetic background, by positron emission tomography and by gamma counter. We found that FAD mice displayed a higher uptake of [18F]F-HPA-12 in the brain, independently from the APOE4 or APOE3 genetic background. FAD mice could be distinguished from littermate control animals with a sensitivity of 85.7% and a specificity of 87.5%, by gamma counter measurements. Metabolic analysis of [18F]F-HPA-12 in the brain suggested that the tracer is degraded less efficiently in the FAD mice. Furthermore, the radioactive signal registered in the hippocampus correlated with an increase of Cer d18:1/20:2 levels measured in the same brain region by mass spectrometry. Our data gives additional proof that ceramide metabolism is different in FAD mice compared to controls. Ceramide analogs like HPA-12 may function as metabolic probes to study ceramide disbalance in the brain.

Highly Sensitive Identification of Lymphatic and Hematogenous Metastasis Routes of Novel Radiolabeled Exosomes Using Non-invasive PET Imaging

Clinically, there has been significant interest in the use of exosomes for diagnostic applications as promising biomarkers and therapeutic applications as therapeutic vehicles. However, knowledge of in vivo physiological biodistribution of exosomes was difficult to assess until now. Physiological distribution of exosomes in the body must be elucidated for clinical application. In this study, we aimed to develop reliable and novel methods to monitor biodistribution of exosomes using in vivo PET and optical imaging.MethodsExosomes were isolated from cultured medium of 4T1, mouse breast cancer cells. Exosomes were labeled with Cy7 and 64Cu (or 68Ga). In mice, radio/fluorescent dye-labeled exosomes were injected through the lymphatic routes (footpad injection) and hematogenous metastatic routes (tail vein injection). Fluorescence and PET images were obtained and quantified. Radio-activity of ex vivo organs was measured by gamma counter.ResultsPET signals from exosomes in the lymphatic metastatic route were observed in the draining lymph nodes, which are not distinguishable with optical imaging. Immunohistochemistry revealed greater uptake of exosomes in brachial and axillary lymph nodes than inguinal lymph node. After administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in PET images in the lungs, liver, and spleen, showing results similar to ex vivo gamma counter data.ConclusionExosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and visualized by PET imaging. These results suggest that this cell type-independent, quick, and easy exosome labeling method using PET isotopes could provide valuable information for further application of exosomes in the clinic.

IN VIVO BIODISTRIBUTION OF 99mTc-MDP FOR EARLY OSTEOPOROSIS MONITORING IN OVARIECTOMIZED BALB/C MICE

Technetium-99m Methylene Diphosphonate (99mTc-MDP) has been utilized in a variety of clinical situations to identify bone areas due to the strong affinity of hydroxyapatite crystals in the mineral phase of the bone with the diphosphonate compounds. Osteoporosis is a disease characterized by decreased bone mass and increased fracture risk and represents a significant population health issue. It has been observed that 99mTc-MDP can be used for bone scintigraphy especially in case of bone cancer, but biodistribution study of 99mTc-MDP on ovariectomized mice for early monitoring of osteoporosis model remains unclear. Therefore, we aimed to investigate the biodistribution of 99mTc-MDP both in normal and ovariectomized mice. The experiment was performed on BALB/c mice weighing approximately 30 g. Mice were divided into a normal and ovariectomized group. After the first, second and third hours, mice were euthanized using the accepted protocol and the tissue of interest was collected. All tissue and blood were weighed using an analytical scale and counted for radioactivity using Automatic Gamma Counter with NaI(Tl) detector. Administration of 99mTc-MDP showed in normal mice compared with an animal model of osteoporosis, there are significant differences at 1 hour post-injection from (20.32±1.38) %ID/g decreased to (7.42±2.61) %ID/g, 2 hours from (13.75±0.01) %ID/g to (5.25±0.25) %ID/g and 3 hours from (12.18±1.44)%ID/g to (4.86±1.34) %ID/g uptake in the bones with (p<0.05). This study can be a consideration for the clinical application of 99mTc-MDP for early detection of osteoporosis conditions by looking at bone uptake and become a concern in the application for bone scintigraphy if the patient is indicated osteoporosis because it will affect visualization of the organ.