We have previously shown that transplantation of autologously derived, respiration-competent mitochondria by direct injection into the heart following transient ischemia and reperfusion enhances cell ...viability and contractile function. To increase the therapeutic potential of this approach, we investigated whether exogenous mitochondria can be effectively delivered through the coronary vasculature to protect the ischemic myocardium and studied the fate of these transplanted organelles in the heart. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and then reperfused for 10 minutes. Mitochondria were labeled with 18F-rhodamine 6G and iron oxide nanoparticles. The labeled mitochondria were either directly injected into the ischemic region or delivered by vascular perfusion through the coronary arteries at the onset of reperfusion. These hearts were used for positron emission tomography, microcomputed tomography, and magnetic resonance imaging with subsequent microscopic analyses of tissue sections to confirm the uptake and distribution of exogenous mitochondria. Injected mitochondria were localized near the site of delivery; while, vascular perfusion of mitochondria resulted in rapid and extensive dispersal throughout the heart. Both injected and perfused mitochondria were observed in interstitial spaces and were associated with blood vessels and cardiomyocytes. To determine the efficacy of vascular perfusion of mitochondria, an additional group of rabbit hearts were subjected to 30 minutes of regional ischemia and reperfused for 120 minutes. Immediately following regional ischemia, the hearts received unlabeled, autologous mitochondria delivered through the coronary arteries. Autologous mitochondria perfused through the coronary vasculature significantly decreased infarct size and significantly enhanced post-ischemic myocardial function. In conclusion, the delivery of mitochondria through the coronary arteries resulted in their rapid integration and widespread distribution throughout the heart and provided cardioprotection from ischemia-reperfusion injury.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The dopamine D2 agonist MCL-524 is selective for the D2 receptor in the high-affinity state (D2high), and, therefore, the PET analogue, 18FMCL-524, may facilitate the elucidation of the role of ...D2high in disorders such as schizophrenia. However, the previously reported synthesis of 18FMCL-524 proved difficult to replicate and was lacking experimental details. We therefore developed a new synthesis of 18FMCL-524 using a “non-anhydrous, minimally basic” (NAMB) approach. In this method, 18FF− is eluted from a small (10–12 mg) trap-and-release column with tetraethylammonium tosylate (2.37 mg) in 7:3 MeCN:H2O (0.1 mL), rather than the basic carbonate or bicarbonate solution that is most often used for 18FF− recovery. The tosylated precursor (1 mg) in 0.9 mL anhydrous acetonitrile was added directly to the eluate, without azeotropic drying, and the solution was heated (150 °C/15 min). The catechol was then deprotected with the Lewis acid In(OTf)3 (10 equiv.; 150 °C/20 min). In contrast to deprotection with protic acids, Lewis-acid-based deprotection facilitated the efficient removal of byproducts by HPLC and eliminated the need for SPE extraction prior to HPLC purification. Using the NAMB approach, 18FMCL-524 was obtained in 5–9% RCY (decay-corrected, n = 3), confirming the utility of this improved method for the multistep synthesis of 18FMCL-524 and suggesting that it may applicable to the synthesis of other 18F-labeled radiotracers.
In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. ...In this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, Ki = 33 nM for CA IX) has been conjugated with bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG2) tetradendate ligands and the conjugates radiolabelled with 99mTc, to obtain anionic and neutral 99mTc-labeled sulfonamide derivatives, respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory activities against hCA IX (Ki = 59–66 nM). In addition, a second generation bis AEBS was conjugated with MAG2 and labeled with 99mTc, and the obtained diastereomers were also evaluated in targeting CA IX. Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG2 tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28% intact). This preliminary data suggest that negatively charged 99mTc-labeled sulfonamide derivatives with modest lipophilicity and longer circulation time could be promising markers to target CA IX.
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•The sulfonamide derivative AEBS was conjugated with a BAT and MAG2 ligand.•The new sulfonamide conjugates were successfully radiolabelled with 99mTc.•The corresponding rhenium analogs showed a good inhibitory constant against CA IX.•Biodistribution in mice bearing HT-29 tumor xenografts was studied.•All new tracers showed a limited retention in tumors (≤0.5% ID at 0.5–4 h p.i).
Objectives: There is considerable interest in the development of an 18F-labeled myocardial perfusion agent, and we are developing 18F-labeled rhodamine dyes to address this significant clinical need. ...After the preclinical evaluation of several rhodamine derivatives, we found that the 18F-labeled diethylene glycol ester of rhodamine 6G (Rho6G, Fig. 1) shows high uptake in the myocardium and low uptake/rapid clearance from non-target tissues. We previously reported the automated synthesis of Rho6G (J Nucl Med 2014; 55:1252); and now, in preparation for first-in-human studies, we report the results of the microdose toxicology study. Methods: Male and female rats (Sprague Dawley SD) were assigned to two groups, and were administered (non-radioactive) 19F-rhodamine 6G in vehicle (10% v/v ethanol in 0.9% sterile saline) or vehicle alone via intravenous injection in a tail vein at a volume of 0.8 mg/kg, which corresponds to approximately 650 times the expected maximum human dose. The animals were observed for 2 days (Day 3 interim sacrifice) or 14 days (Day 15 terminal sacrifice) to assess possible adverse effects (i.e., mortality, changes in body weight and/or food consumption, injection site reactions, ophthalmic examinations, and clinical and anatomic pathology). Results: On Day 3 of the dosing phase, one female rat died of accidental causes following blood sample collection. All other animals survived to their scheduled sacrifice. No 19F-rhodamine 6G-related clinical observations or ophthalmic findings were noted. No definitive 19F-rhodamine 6G-related alterations in body weight, body weight change, or food consumption were noted, and any changes were inconclusive, sporadic, and/or incidental and nonadverse. Transient erythema was noted at the injection site but this was most likely due to the deep red color of 19F-rhodamine 6G solution and was nonadverse. 19F-rhodamine 6G administration had no effects on hematology, coagulation, or clinical chemistry results. At the interim sacrifice, 19F-rhodamine 6G-related increased heart and pituitary weight occurred in females given 0.8 mg/kg; however, no microscopic correlates were observed for these changes. 19F-rhodamine 6G-related microscopic findings at the intravenous injection site in interim sacrifice animals given 0.8 mg/kg included increased incidences of mixed cell infiltrates and edema. No test article-related organ weight parameters or microscopic findings were noted at the terminal sacrifice. No macroscopic findings were observed at the interim or terminal sacrifice. Conclusion: Intravenous injection of 19F-rhodamine 6G as a single dose at ~650 times the expected human dose was well tolerated in rats: No clinical observations or ophthalmic findings, and no definitive alterations in body weight, body weight change, or food consumption were noted, and no effects on clinical pathology were noted. Nonadverse, transient erythema was noted at the injection site. At the interim sacrifice, 19F-rhodamine 6G-related increased heart and pituitary weight was noted, and mixed cell infiltrates and edema were present at the injection site, none of which were adverse. At the terminal sacrifice, no organ weight changes or microscopic findings were present. No macroscopic findings were noted. Based on these findings, a single intravenous dose of 19F-rhodamine 6G at 0.8 mg/kg was not deemed adverse.
Aberrant redox signaling underlies the pathophysiology of many chronic metabolic diseases, including type 2 diabetes (T2D). Methodologies aimed at rebalancing systemic redox homeostasis have had ...limited success. A noninvasive, sustained approach would enable the long-term control of redox signaling for the treatment of T2D. We report that static magnetic and electric fields (sBE) noninvasively modulate the systemic GSH-to-GSSG redox couple to promote a healthier systemic redox environment that is reducing. Strikingly, when applied to mouse models of T2D, sBE rapidly ameliorates insulin resistance and glucose intolerance in as few as 3 days with no observed adverse effects. Scavenging paramagnetic byproducts of oxygen metabolism with SOD2 in hepatic mitochondria fully abolishes these insulin sensitizing effects, demonstrating that mitochondrial superoxide mediates induction of these therapeutic changes. Our findings introduce a remarkable redox-modulating phenomenon that exploits endogenous electromagneto-receptive mechanisms for the noninvasive treatment of T2D, and potentially other redox-related diseases.
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•Combined static magnetic and electric fields (sBE) enhance insulin sensitivity•sBE is not associated with adverse effects•sBE triggers a systemic redox response to modulate insulin sensitivity•Scavenging mitochondrial superoxide in the liver abolishes the effects of sBE
Abnormal redox homeostasis contributes to the pathogenesis of type 2 diabetes. However, targeting redox systems remains a challenge. In this issue, Carter, Huang et al. demonstrate that static magnetic and electric fields can be used to modulate redox systems for the noninvasive treatment of type 2 diabetes.
Background
To date, few PET tracers for
in vivo
labeling of red blood cells (RBCs) are available. In this study, we report the radiosynthesis and
in vitro
and
in vivo
evaluation of
11
C and
18
F ...sulfonamide derivatives targeting carbonic anhydrase II (CA II), a metallo-enzyme expressed in RBCs, as potential blood pool tracers. A proof-of-concept
in vivo
imaging study was performed to demonstrate the feasibility to assess cardiac function and volumes using electrocardiogram (ECG)-gated positron emission tomography (PET) acquisition in comparison with cine magnetic resonance imaging (cMRI) in rats and a pig model of myocardial infarction.
Methods
The inhibition constants (
K
i
) of CA II were determined
in vitro
for the different compounds by assaying CA-catalyzed CO
2
hydration activity. Binding to human RBCs was estimated after
in vitro
incubation of the compounds with whole blood. Biodistribution studies were performed to evaluate tracer kinetics in NMRI mice. ECG-gated PET acquisition was performed in Wistar rats at rest and during pharmacological stress by infusing dobutamine at 10 μg/kg/min and in a pig model of myocardial infarction. Left ventricular ejection fraction (LVEF) and volumes were compared with values from cMRI.
Results
The
K
i
of the investigated compounds for human CA II was found to be in the range of 8 to 422 nM. The fraction of radioactivity associated with RBCs was found to be ≥90% at 10- and 60-min incubation of tracers with heparinized human blood at room temperature for all tracers studied. Biodistribution studies in mice indicated that 30% to 67% of the injected dose was retained in the blood pool at 60 min post injection. A rapid and sustained tracer uptake in the heart region with an average standardized uptake value of 2.5 was observed from micro-PET images. The LVEF values obtained after pharmacological stress in rats closely matched between the cMRI and micro-PET values, whereas at rest, a larger variation between LVEF values obtained by both techniques was observed. In the pig model, a good agreement was observed between PET and MRI for quantification of left ventricular volumes and ejection fraction.
Conclusions
The
11
C and
18
F sulfonamide derivatives can be used for efficient
in vivo
radiolabeling of RBCs, and proof-of-concept
in vivo
imaging studies have shown the feasibility and potential of these novel tracers to assess cardiac function.
Objectives: Gallium-68 labeled DOTATOC is a promising clinical PET radiotracer for detection of neuroendocrine tumors that express somatostatin receptors. For regulatory and clinical trial protocol ...compliance, it is important the radiopharmaceutical produced with different radionuclide sources and synthesizers should meet the quality control and release criteria as per USP or 21 CFR part 212. Here, we report for the first time in U.S., the utility of 68Ga sourced from Galli Eo® generator and production of clinical grade 68Ga-DOTATOC using two different synthesizers. Methods: 68Ga-DOTATOC was synthesized using Galli Eo® 68Ge/68Ga generator (IRE-ELiT) coupled with a GRP 3V (Scintomics) or Modular-Lab PharmTracer (Eckert & Ziegler) synthesis module. The GMP grade precursor and appropriate DOTATOC reagent kits or cassettes are obtained commercially. Ascorbic acid solution is added to the reactor prior to synthesis in order to reduce radiolysis byproducts. The 68Ga-DOTATOC is purified by in-line solid phase extraction, followed by reformulation and filtration through a 0.22-μm sterilizing filter. The phosphate buffered saline from the Scintomics kit is replaced with normal saline so that it matches the final product formulation of the Eckert & Ziegler synthesis module. Quality control parameters including radiochemical purity, radionuclide identity, pH, endotoxin content and sterility were performed by standard techniques. Results: 68Ga-DOTATOC is obtained in < 30 minutes from generator elution to final product. In both synthesizers the radiochemical yields were 83 ± 5%, radiochemical purity 99 ± 1% and the final product consists of <10% ethanol in saline. The average molar activity based on starting DOTATOC mass was found to be 18 GBq/micromole. Conclusions: We successfully implemented a simple and reliable radiosynthesis of 68Ga-DOTATOC using a novel generator with two different synthesizers. Despite variances with synthesizer work flow and their respective DOTATOC reagent kit/cassette, the final product was produced in < 30 min with identical end product specifications and met all quality control specifications.
Introduction: Mucociliary Clearance (MCC) is an innate respiratory host defense mechanism that removes inhaled and aspirated particles and pathogens from the airways. Defects in MCC contribute to ...many airway diseases such as primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and cystic fibrosis. MCC has been visualized using inhaled aerosols containing insoluble Tc-99m labeled colloids with either planar scintigraphy or SPECT. However, the low temporal and spatial resolution of single photon imaging in this application makes it difficult to investigate the mechanisms of MCC defects in airways disease or to determine the efficacy of therapeutics. An imaging modality with higher temporal and spatial resolution, such as PET, will allow for quantitative measurement of MCC and opportunities to evaluate the efficacy of newer therapeutics. Objectives: The objective of the study was to develop a method to visualize and quantify MCC in pulmonary pathophysiology using PET imaging. Pre-clinical imaging was performed using a porcine model. Methods: 18F-fluoride labeled alumina nanoparticles were obtained by adding aqueous 18F-fluoride to γ-alumina nanoparticles (<50 nm). The suspension was incubated at room temperature with continuous shaking (20 min) followed by centrifugation (10 min). The unbound 18F-fluoride was separated from the pellet, and the pellet was then washed with water, re-suspended, shaken (5 min) and centrifuged (5 min). The washing step was repeated two times, and the 18F-fluoride alumina nanoparticles were formulated in saline. We used newborn piglets for this study. In one experiment, we targeted small distal airways by generating aerosols of 18F-fluoride alumina nanoparticles in saline with mass median aerodynamic diameter (MMAD) of ~0.31 µm. We used 14 mCi with effective delivery of ~ 1 mCi into the airways. In another experiment, we targeted large proximal airways by generating aerosols of 18F-fluoride alumina nanoparticles in saline with MMAD of ~10 µm. We delivered 1 mCi directly into the airways. An endotracheal tube was needed for the delivery of the aerosolized 18F-fluoride alumina nanoparticles. Immediately after delivery of the aerosolized nanoparticles, the endotracheal tube was removed. The piglet breathed spontaneously throughout the study and maintained its own airway humidification. We obtained PET scans in list mode for a period of 60 minutes, along with a CT scan for attenuation correction. Decay- and attenuation-corrected data was binned into 30-second frames and the binned static images were used to assess MCC over time. Results: 18F-fluoride alumina nanoparticles were obtained with radiochemical yields 95 ± 3% in < 70 minutes. Repeated washing of 18F-labeled alumina nanoparticles with water and incubation in saline failed to release any significant 18F-fluoride over 2-hour period. We achieved a diffuse and homogeneous delivery of the radiotracer regardless of whether a nebulizer or microsprayer was used. As predicted, the fine particles generated with the nebulizer deposited in distal airways. In contrast, the larger particles generated with a microsprayer deposited in proximal airways. With time, the radiotracer cleared out of the airways. Most of the clearance happened in the first few minutes of the study. Conclusions: A simple and highly efficient synthesis of 18F-labeled alumina nanoparticles was developed and the 18F-labeled alumina nanoparticles show good in vitro stability. The 18F-labeled alumina nanoparticles were successfully delivered to pig airways via nebulizer or micro-sprayer. Varying the size of the droplets affected the deposition of the radioactivity within the airways. MCC clearance was observed using PET, with most of the clearance occurring within the first few minutes of radiotracer administration.
Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane ...conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans
, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-Cre
::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-Cre
::CFTR
). By comparing these models with cystic fibrosis ferrets
, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-Cre
::CFTR
ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl
and HCO
. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.
Objectives: Abnormality of dopamine D2 and D3 receptors is implicated in several neuropsychiatric disorders, including Parkinson's disease and schizophrenia. Our earlier studies (Bioorg Med Chem. ...2004, 12, 3553) showed that several non-radiolabeled 11-hydroxynoraporphines and their esters show high behavioral potency for D2 receptors in rodents, presumably due to their lipophilicity and prodrug nature. Here we report our more recent efforts to extend these results to the development and evaluation of 18F-labeled D2 imaging agents based on the acetate (18F-MCL-562) and valerate (18F-MCL-572) esters of R-(-)-N-n-propyl-2-(3-fluoropropanoxy-11-hydroxynoraporphine) (18F-MCL-536) (J. Label. Compd. Radiopharm, 2014, 57, 725-729). Methods: The desired radiotracers, 18F-MCL-562 and 18F-MCL-572 (Fig. 1), were synthesized from the same bis-tosylated precursor MCL-563, (R)-N-n-propyl-11-toluenesulfonyloxy-2-(3-toluenesulfonyloxypropanoxy)noraporphine. Following initial radiolabeling using standard radiofluorination procedures (i.e., K2.2.2, K2CO3, CH3CN, 110 °C), the compound was deprotected (3 M NaOH, 15 min, 70 °C), the resulting 3-18Ffluoropropanoxy 11-OH compound (18F-MCL-536) was purified by semi-preparative HPLC, and the ester was generated using either acetic anhydride (18F-MCL-562) or valeric anhydride (18F-MCL-572) (room temperature, 5 min). The final product is isolated using a C18 cartridge and formulated in 10% ethanol/saline. LogD7.4 was measured by the shake-flask method. Non-invasive imaging was performed in rats using dedicated small-animal PET/CT system. In addition, the stability of the 11-hydroxyaporphine valerate ester was measured in human and rat serum. Results: The radiotracers 18F-MCL-562 and 18F-MCL-572 were obtained with 5% decay-corrected radiochemical yield and >99% radiochemical purity (n=2) in a total synthesis time of 2 h. The specific activities were 5 GBq/°mol and 2.5 GBq/μmol, respectively. LogD7.4 was found to be 1.54+0.09. The PET images indicated both the tracers had no specific uptake in the rat brain. The in vitro stability study showed the valerate ester was <4% intact in rodent serum at 5 min and 54% intact in human serum at 5 min. Conclusion: The radiotracers 18F-MCL-562 and 18F-MCL-572 were synthesized in modest radiochemical yield but high purity. Unfortunately, neither tracer showed high or specific uptake in the rat brain, presumably due to the rapid metabolism in the rat serum. However, based on in vitro human serum stability, the valerate ester of aporphine could be the promising candidate for more extensive evaluation and imaging studies are underway in non-human primates.
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