Chronic ß3 adrenergic agonist treatment improves brain microvascular endothelial function and cognition in aged mice.

Microvascular endothelial dysfunction, characterized by impaired neurovascular coupling, reduced glucose uptake, blood-brain barrier disruption, and microvascular rarefaction, plays a critical role in the pathogenesis of age-related vascular cognitive impairment (VCI). Emerging evidence points to non-cell autonomous mechanisms mediated by adverse circulating milieu (an increased ratio of pro-geronic to anti-geronic circulating factors) in the pathogenesis of endothelial dysfunction leading to impaired cerebral blood flow and cognitive decline in the aging population. In particular, age-related adipose dysfunction contributes, at least in part, to an unfavorable systemic milieu characterized by chronic hyperglycemia, hyperinsulinemia, dyslipidemia, and altered adipokine profile, which together contribute to microvascular endothelial dysfunction. Hence, in the present study, we aimed to test whether thermogenic stimulation, an intervention known to improve adipose and systemic metabolism by increasing cellular energy expenditure, could mitigate brain endothelial dysfunction and improve cognition in the aging population. Eighteen-month-old old C57BL/6J mice were treated with saline or CL (ß3-adrenergic agonist) for 6 weeks followed by functional analysis to assess endothelial function and cognition. CL treatment improved neurovascular coupling responses and rescued brain glucose uptake in aged animals. In addition, CL treatment also attenuated blood-brain barrier leakage and associated neuroinflammation in the cortex of aged animals. More importantly, these beneficial changes in microvascular function translated to improved cognitive performance in radial arm water maze and Y-maze tests. Our results suggest that ß3-adrenergic agonist treatment improves multiple aspects of brain microvascular endothelial function and can be potentially repurposed for treating age-associated cognitive decline.

Corrigendum: IgG2 rules: N-acetyl-ß-D-glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes.

[This corrects the article DOI: 10.3389/fcvm.2022.919700.].

Evaluation of anti-inflammatory diphenyldihaloketone EF24 in transient ischemic stroke model.

OBJECTIVES: Revascularization is necessary in patients with ischemic stroke, however it does not address inflammation that contribute to reperfusion injury and the early growth of ischemic core. We investigated EF24, an anti-inflammatory agent, in a stroke model. METHODS: Ischemic stroke was induced in mice by occluding middle cerebral artery for 1 h followed by reperfusion. EF24 was given either 10 min post-reperfusion (EF24Post) or 10 min before occlusion (prophylactic, EF24Pro). Survival, ipsilateral uptake of radioactive infarct marker 18F-fluoroglucaric acid (FGA), inflammatory cytokines, and tetrazolium chloride (TTC) staining were assessed. RESULTS: Survival was increased in both EF24-treated groups compared to the stroke+vehicle group. Ipsilateral 18F-FGA uptake increased 2.6-fold in stroke+vehicle group compared to sham group (p < 0.05); the uptake in EF24-treated groups and sham group was not significantly different. TTC-staining also showed reduction in infarct size by EF24 treatment. Plasma IL-6, TNF-α, and corticosterone did not show significant changes among groups. However, ipsilateral tissue in stroke+vehicle mice showed increased IL-6 (>90-fold) and TNF-α (3-fold); the tissue IL-6 and TNF-α were significantly reduced in stroke+EF24Pro and stroke+EF24Post groups. 18F-FGA uptake significantly correlated with tissue IL-6 levels. CONCLUSIONS: EF24 controls infarct growth and suppresses tissue inflammation in ischemic stroke, which can be monitored by 18F-FGA uptake.

Positron Emission Tomography (PET) with 18F-FGA for Diagnosis of Myocardial Infarction in a Coronary Artery Ligation Model.

Location and extent of necrosis are valuable information in the management of myocardial infarction (MI). Methods. We investigated 2-deoxy-2-18F-fluoro glucaric acid (FGA), a novel infarct-avid agent, for positron emission tomography (PET) of MI. We synthesized FGA from commercially available 18F-fluoro-2-deoxy-2-D-glucose (FDG). MI was induced in mice by permanently occluding the left anterior descending coronary artery. Biodistribution of FGA was assessed 1 h after FGA injection (11 MBq). PET/CT was conducted 1 h, 6 h, 1 d, 3 d, and 4 d after MI. Subcellular compartment of FGA accumulation in necrosis was studied by tracing the uptake of biotin-labeled glucaric acid with streptavidin-HRP in H2O2-treated H9c2 cardiomyoblasts. Streptavidin-reactive protein bands were identified by LC-MS/MS. Results. We obtained a quantitative yield of FGA from FDG within 7 min (radiochemical purity > 99%). Cardiac uptake of FGA was significantly higher in MI mice than that in control mice. Imaging after 1 h of FGA injection delineated MI for 3 days after MI induction, with negligible background signal from surrounding tissues. Myocardial injury was verified by tetrazolium staining and plasma troponin (47.63 pg/mL control versus 311.77 pg/mL MI). In necrotic H9c2 myoblasts, biotinylated glucaric acid accumulated in nuclear fraction. LC-MS/MS primarily identified fibronectin in necrotic cells as a putative high fidelity target of glucaric acid. Conclusion. FGA/PET detects infarct early after onset of MI and FGA accumulation in infarct persists for 3 days. Its retention in necrotic cells appears to be a result of interaction with fibronectin that is known to accumulate in injured cardiac tissue.

IgG2 rules: N-acetyl-ß-D-glucosamine-specific IgG2 and Th17/Th1 cooperation may promote the pathogenesis of acute rheumatic heart disease and be a biomarker of the autoimmune sequelae of Streptococcus pyogenes.

Antecedent group A streptococcal pharyngitis is a well-established cause of acute rheumatic fever (ARF) where rheumatic valvular heart disease (RHD) and Sydenham chorea (SC) are major manifestations. In ARF, crossreactive antibodies and T cells respond to streptococcal antigens, group A carbohydrate, N-acetyl-ß-D-glucosamine (GlcNAc), and M protein, respectively, and through molecular mimicry target heart and brain tissues. In this translational human study, we further address our hypothesis regarding specific pathogenic humoral and cellular immune mechanisms leading to streptococcal sequelae in a small pilot study. The aims of the study were to (1) better understand specific mechanisms of pathogenesis in ARF, (2) identify a potential early biomarker of ARF, (3) determine immunoglobulin G (IgG) subclasses directed against GlcNAc, the immunodominant epitope of the group A carbohydrate, by reaction of ARF serum IgG with GlcNAc, M protein, and human neuronal cells (SK-N-SH), and (4) determine IgG subclasses deposited on heart tissues from RHD. In 10 pediatric patients with RHD and 6 pediatric patients with SC, the serum IgG2 subclass reacted significantly with GlcNAc, and distinguished ARF from 7 pediatric patients with uncomplicated pharyngitis. Three pediatric patients who demonstrated only polymigrating arthritis, a major manifestation of ARF and part of the Jones criteria for diagnosis, lacked the elevated IgG2 subclass GlcNAc-specific reactivity. In SC, the GlcNAc-specific IgG2 subclass in cerebrospinal fluid (CSF) selectively targeted human neuronal cells as well as GlcNAc in the ELISA. In rheumatic carditis, the IgG2 subclass preferentially and strongly deposited in valve tissues (n = 4) despite elevated concentrations of IgG1 and IgG3 in RHD sera as detected by ELISA to group A streptococcal M protein. Although our human study of ARF includes a very small limited sample set, our novel research findings suggest a strong IgG2 autoantibody response against GlcNAc in RHD and SC, which targeted heart valves and neuronal cells. Cardiac IgG2 deposition was identified with an associated IL-17A/IFN-γ cooperative signature in RHD tissue which displayed both IgG2 deposition and cellular infiltrates demonstrating these cytokines simultaneously. GlcNAc-specific IgG2 may be an important autoantibody in initial stages of the pathogenesis of group A streptococcal sequelae, and future studies will determine if it can serve as a biomarker for risk of RHD and SC or early diagnosis of ARF.

Design of 99mTc-labeled zinc-chelating imaging probe for SPECT imaging of the pancreas.

Early and sensitive diagnosis of pancreatic diseases is a contemporary clinical challenge. Zinc level in pancreatic tissue and its secretion in pancreatic juice has long been considered a surrogate marker of pancreatic function. The objective of this study was to design a Zn-chelating imaging probe (ZCIP) which could be labeled with 99mTc radionuclide for imaging of pancreas using single photon emission tomography (SPECT). We synthesized ZCIP as a bifunctional chelate consisting of diethylene triamine pentaacetic acid for 99mTc-chelation at one end and bispicolylethylamine for Zn-complexation at the other end. ZCIP was labeled with 99mTc by standard Sn2+-based reduction method. The 99mTc-labeled ZCIP was studied in normal mice (0.3 mCi) for SPECT imaging. We found that ZCIP consistently labeled with 99mTc radionuclide with over 95% efficiency. Addition of ZCIP altered the spectrum of standard dithizone-Zn complex, indicating its ability to chelate Zn. SPECT data demonstrated the ability of 99mTc-ZCIP to image pancreas with high sensitivity in a non-invasive manner; liver and spleen were the other major organs of 99mTc-ZCIP uptake. Based on these results, we conclude that 99mTc-ZCIP presents as a novel radiotracer for pancreas imaging for diagnosis of diseases such as pancreatitis.

Drug-induced cardiomyopathy: Characterization of a rat model by [18F]FDG/PET and [99mTc]MIBI/SPECT.

BACKGROUND: Drug-induced cardiomyopathy is a significant medical problem. Clinical diagnosis of myocardial injury is based on initial electrocardiogram, levels of circulating biomarkers, and perfusion imaging with single photon emission computed tomography (SPECT). Positron emission tomography (PET) is an alternative imaging modality that provides better resolution and sensitivity than SPECT, improves diagnostic accuracy, and allows therapeutic monitoring. The objective of this study was to assess the detection of drug-induced cardiomyopathy by PET using 2-deoxy-2-[18F]fluoro-D-glucose (FDG) and compare it with the conventional SPECT technique with [99mTc]-Sestamibi (MIBI). METHODS: Cardiomyopathy was induced in Sprague Dawley rats using high-dose isoproterenol. Nuclear [18F]FDG/PET and [99mTc]MIBI/SPECT were performed before and after isoproterenol administration. [18F]FDG (0.1 mCi, 200-400 µL) and [99mTc]MIBI (2 mCi, 200-600 µL) were administered via the tail vein and imaging was performed 1 hour postinjection. Isoproterenol-induced injury was confirmed by the plasma level of cardiac troponin and triphenyltetrazolium chloride (TTC) staining. RESULTS: Isoproterenol administration resulted in an increase in circulating cardiac troponin I and showed histologic damage in the myocardium. Visually, preisoproterenol and postisoproterenol images showed alterations in cardiac accumulation of [18F]FDG, but not of [99mTc]MIBI. Image analysis revealed that myocardial uptake of [18F]FDG reduced by 60% after isoproterenol treatment, whereas that of [99mTc]MIBI decreased by 45%. CONCLUSION: We conclude that [18F]FDG is a more sensitive radiotracer than [99mTc]MIBI for imaging of drug-induced cardiomyopathy. We theorize that isoproterenol-induced cardiomyopathy impacts cellular metabolism more than perfusion, which results in more substantial changes in [18F]FDG uptake than in [99mTc]MIBI accumulation in cardiac tissue.

Production of [13N]ammonia from [13C]methanol on a 7.5 MeV cyclotron using 13C(p, n)13N reaction: Detection of myocardial infarction in a mouse model.

[13N]Ammonia is commonly produced using 16O(p, α)13N reaction but one of the limiting factor of this reaction is the relatively small nuclear cross-section at proton energies of <10 MeV. An alternative production method using 13C(p, n)13N reaction, which has a higher nuclear cross-section at low proton energies, is more suitable for a preclinical PET imaging facility equipped with a <10 MeV cyclotron. Here, we report a novel method to produce [13N]ammonia from [13C]methanol for preclinical use on a 7.5 MeV cyclotron. A tantalum solution target (80 µl) consisting of a havar window supplied by the cyclotron manufacturer for the production of [18F]fluoride was used without any modifications. The final bombardment parameters were optimized as follow: [13C]methanol concentration in target solution - 10%, bombardment time - 8 min, and beam current - 2.2 µA. These parameters provided doses of [13N]ammonia which were sufficient to conduct preclinical PET imaging studies in a mouse model of myocardial infarction. Under optimized conditions, the operational lifetime of the target was approximately 150 µAmin. Radionuclide identity of the product as 13N was confirmed by measuring the decay half-life and its radionuclide purity was confirmed by γ-ray spectroscopic analysis. Gas chromatography revealed that the final [13N]ammonia dose was not distinguishable from water, showing no traces of methanol. As expected, PET/CT imaging in healthy CD-1 mice indicated the accumulation of [13N]ammonia in myocardial tissue; mice with myocardial infarction created by left ascending coronary ligation showed clear perfusion deficit in affected tissue. This work demonstrates the proof-of-concept of using 13C(p, n)13N reaction to produce [13N]ammonia from [13C]methanol with a <10 MeV cyclotron, and its diagnostic application in imaging cardiac perfusion.

Imaging of isoproterenol-induced myocardial injury with 18F labeled fluoroglucaric acid in a rat model.

Positron emission tomography (PET) of myocardial infarction (MI) by infarct avid imaging has the potential to reduce the time to diagnosis and improve diagnostic accuracy. The objective of this work was to synthesize 18F-labeled glucaric acid (FGA) for PET imaging of isoproterenol-induced cardiomyopathy in a rat model. METHODS: We synthesized 18F-FGA by controlled oxidation of 18F-fluorodeoxy glucose (FDG), mediated by 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) in presence of NaBr and NaOCl in highly-buffered reaction conditions. After ascertaining preferential uptake of 18F-FGA in necrotic as compared to normal H9c2 myoblasts, the biodistribution and circulation kinetics of 18F-FGA was assessed in mice. Moreover, the potential of 18F-FGA to image myocardial damage was investigated in a rat model of isoproterenol-induced cardiomyopathy. Isoproterenol-induced myocardial injury was verified at necropsy by tissue staining and plasma cardiac troponin levels. RESULTS: Synthesis of radiochemically pure 18F-FGA was accomplished by a 5 min, one step oxidation of 18F-FDG. Reaction yield was quantitative and no side-products were detected. Biodistribution studies showed rapid elimination from the body (ke = 0.83 h-1); the major organ of 18F-FGA accumulation was kidney. In the rat model, isoproterenol-treatment resulted in significant increase in cardiac troponin. PET images showed that the hearts of isoproterenol-treated rats accumulated significant amounts of 18F-FGA, whereas healthy hearts showed negligible uptake of 18F-FGA. Target-to-nontarget contrast for 18F-FGA accumulation became significantly more pronounced in 4 h images as compared to images acquired 1 h post-injection. CONCLUSION: 18F-FGA can be easily and quantitatively synthesized from ubiquitously available 18F-FDG as a precursor. The resultant 18F-FGA has a potential to serve as an infarct-avid agent for PET imaging of MI. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: 18F-FGA/PET will complement existing perfusion imaging protocols in therapeutic decision making, determination of revascularization candidacy and success, differentiation of ischemia from necrosis in MI, discrimination of myocarditis from infarction, and surveillance of heart transplant rejection.

Nanovesicular liposome-encapsulated hemoglobin (LEH) prevents multi-organ injuries in a rat model of hemorrhagic shock.

The goals of resuscitation in hemorrhagic shock are to correct oxygen deficit and to maintain perfusion pressure to the vital organs. We created liposome-encapsulated hemoglobin (LEH) as a nanoparticulate oxygen carrier (216±2nm) containing 7.2g/dl hemoglobin, and examined its ability to prevent the systemic manifestations of hemorrhagic shock (45% blood loss) in a rat model. We collected plasma after 6h of shock and LEH resuscitation, and determined the circulating biomarkers of systemic inflammation and functions of liver, gut, heart, and kidney. As is typical of the shock pathology, a significant increase in the plasma levels of cardiac troponin, liver function enzymes, soluble CD163 (macrophage activation), and creatinine, and the liver/gut myeloperoxidase activity was observed in the hemorrhaged rats. The plasma levels of TNF-α, IL-6, IL-1α, CINC-1, and IL-22 also increased after hemorrhagic shock. LEH administration prevented the hemorrhagic shock-induced accumulation of the markers of injury to the critical organs and pro-inflammatory cytokines. LEH also decreased the plasma levels of stress hormone corticosterone in hemorrhaged rats. Although saline also reduced the circulating corticosterone and a few other tissue injury markers, it was not as effective as LEH in restraining the plasma levels of creatinine, alanine transaminase, CD163, TNF-α, IL-6, and IL-1α. These results indicate that resuscitation with nanoparticulate LEH creates a pro-survival phenotype in hemorrhaged rats, and because of its oxygen-carrying capacity, LEH performs significantly better than saline in hemorrhagic shock.

Synthesis and in vivo evaluation of ortho-[(124)I]iodohippurate for PET renography in healthy rats.

Ortho-[(131)I]iodohippurate [(131)I]OIH (marketed as Hippuran I 131), a gold standard for radionuclide renography, and [(123)I]OIH were in clinical use for decades. Here we radiolabeled OIH with (124)I (a positron emitter) to combine the desirable biological properties of OIH and to enable the use of positron emission tomography (PET) for renography. [(124)I]OIH was synthesized with a slight modification to a previously reported method for the kit preparation of [(123)I]OIH based on the Cu(II) catalyzed isotope-exchange reaction. Our method utilized heating at 120°C under sealed condition in a heating block instead of autoclaving. [(124)I]OIH was obtained with a radiochemical purity of >99.3%, radiochemical yield of 94.5%, and specific activity of ~17 MBq/mg. Biodistribution studies in healthy Sprague Dawley rats revealed results comparable to that of [(131)I]OIH as reported in the literature. The PET-derived [(124)I]OIH renograms revealed an average time-to-peak of 2.8±0.4min and the average time-to-half-maximal activity of 11.4±1.5min, which are also comparable to that of [(131)I]OIH as reported in the literature. Results from this study indicate that the synthesis of [(124)I]OIH without using an autoclave and [(124)I]OIH PET renography are feasible.

Facile synthesis of para-[(18)F]fluorohippurate via iodonium ylide-mediated radiofluorination for PET renography.

para-[(18)F]fluorohippurate ([(18)F]PFH) is a renal tubular agent suitable for conducting positron emission tomography (PET) renography. [(18)F]PFH is currently synthesized by a four-step two-pot procedure utilizing a classical prosthetic group, N-succinimidyl-4-[(18)F]fluorobenzoate, followed by glycine conjugation. Considering the short half-life of fluorine-18 (110min), it is important to reduce the number of synthetic steps and overall production time for successful translation of any fluorine-18 radiopharmaceutical in to clinical practice. Here, we report a new two-step one-pot procedure using a novel spirocyclic iodonium ylide precursor for producing a dose of [(18)F]PFH suitable for human use in 45min including HPLC purification with an overall decay-corrected radiochemical yield of 46.4±2.9% (n=3) and radiochemical purity of >99%.

Evaluation of [¹8F]PFH PET renography to predict future disease progression in a rat model of autosomal dominant polycystic kidney disease.

INTRODUCTION: Prognostic markers for progression of polycystic kidney disease (PKD) are limited. We evaluated the potential of early para-[(18)F]fluorohippurate ([(18)F]PFH) positron emission tomography (PET) renography to predict future progression of PKD in Han:SPRD rats with slowly progressive autosomal dominant PKD. MATERIALS AND METHODS: Male and female heterozygous (Cy/+) and normal littermate (+/+) Han:SPRD rats underwent [(18)F]PFH PET renography and blood sampling to measure serum creatinine (S-Cr) and serum urea nitrogen (SUN) concentrations at 6 and 26 wk of age. T2 and T20 values, which represent the percent of the injected dose of [(18)F]PFH in kidneys at 2 and 20 min after injection, were determined from imaging data. T20/T2 ratio was assessed as a prognostic marker. Rats were euthanized after renography at 26 wk of age, and kidney weight/body weight ratios (KW/BW%) were determined as a measure of PKD progression. RESULTS: Male and female Cy/+ rats are known to manifest PKD of different severity, male Cy/+ rats display much more severe PKD than female rats. S-Cr and SUN concentrations did not differ between +/+ and Cy/+ rats and between female and male Cy/+ rats at 6 wk of age, but they were higher at 26 wk of age and male rats displayed higher values than female rats, which indicates inability of S-Cr and SUN to measure disease severity at an early stage. T20/T2 ratios were higher for Cy/+ than +/+ rats at 6 wk of age. Importantly, male Cy/+ rats displayed higher T20/T2 ratios than female Cy/+ rats. T20/T2 ratios obtained at 6 wk of age correlated well with S-Cr, SUN, and KW/BW% values obtained at 26 wk of age. CONCLUSIONS: This study indicates that T20/T2 ratio derived from [(18)F]PFH PET renography at an early age could be useful as a novel prognostic marker to predict future disease severity in a rat model of ADPKD.

Hemorrhagic shock-induced cerebral bioenergetic imbalance is corrected by pharmacologic treatment with EF24 in a rat model.

Maintenance of cerebral viability and function is an important goal of critical care in victims of injury due to ischemia and hypovolemia. As part of the multiple organ dysfunction syndrome, the brain function after trauma is influenced by the systemic inflammatory response. We investigated the effect of EF24, an anti-inflammatory bis-chalcone, on cerebral bioenergetics in a rat model of 45% hemorrhagic shock. The rats were treated with EF24 (0.4 mg/kg) or EF24 with an artificial oxygen carrier liposome-encapsulated hemoglobin (LEH). The volume of LEH administered was equal to the shed blood. The brain was collected after 6 h of shock for biochemical assays. EF24 treatment showed significant recovery of ATP, phosphocreatine, and NAD/NADH ratio. It also increased citrate synthase activity and cytochrome c oxidase subunit IV expression which were reduced in shock brain. Furthermore, it reduced the shock-induced accumulation of pyruvate and pyruvate dehydrogenase kinase-1 expression, suggesting that EF24 treatment improves cerebral energetics by restoring perturbed pyruvate metabolism in the mitochondria. These effects of EF24 were associated with reduced poly(ADP-ribose) polymerase cleavage and a significant improvement in the levels of nerve growth factor and brain-derived neurotrophic factor in shock brain. Co-administration of LEH with EF24 was only marginally more effective as compared to the treatment with EF24 alone. These results show that EF24 treatment sets up a pro-survival phenotype in shock by resurrecting cerebral bioenergetics. Since EF24 was effective in the absence of accompanying fluid resuscitation, it has potential utility as a pre-hospital pharmacotherapy in shock due to accidental blood loss.

The brain metabolic activity after resuscitation with liposome-encapsulated hemoglobin in a rat model of hypovolemic shock.

We examined the effect of resuscitation with liposome-encapsulated hemoglobin (LEH) on cerebral bioenergetics in a rat model of 45% hypovolemia. The rats were resuscitated with isovolemic LEH or saline after 15 minutes of shock and followed up to 6 hours. Untreated hypovolemic rats received no fluid. The cerebral uptake of F-18-fluorodeoxyglucose (FDG) was measured by PET, and at 6 hours, the brain was collected for various assays. Hypovolemia decreased cellular adenosine triphosphate (ATP), phosphocreatine, nicotinamide adenine dinucleotide (NAD)/NADH ratio, citrate synthase activity, glucose-6-phosphate, and nerve growth factor (NGF), even when FDG uptake remained unchanged. The FDG uptake was reduced by saline, but not by LEH infusion. The reduced FDG uptake in saline group was associated with a decrease in hexokinase I expression. The LEH infusion effectively restored ATP content, NAD/NADH ratio, and NGF expression, and reduced the hypovolemia-induced accumulation of pyruvate and ubiquitinated proteins; in comparison, saline was significantly less effective. The LEH infusion was associated with low pH and high anion gap, indicating anionic gap acidosis. The results suggest that hypovolemic shock perturbs glucose metabolism at the level of pyruvate utilization, resulting in deranged cerebral energy stores. The correction of volume and oxygen deficits by LEH recovers the cerebral metabolism and creates a prosurvival phenotype.

Synthesis and in vivo evaluation of gallium-68-labeled glycine and hippurate conjugates for positron emission tomography renography.

The objective of this study was to evaluate four new (68) Ga-labeled 1,4,7,10-cyclododeca-1,4,7,10-tetraacetic acid (DOTA)/1,4,7-triazacyclononane-1,4,7-triacetic acid derived (NODAGA)-glycine/hippurate conjugates and select a lead candidate for potential application in positron emission tomography (PET) renography. The non-metallated conjugates were synthesized by a solid phase peptide synthesis method. The (68) Ga labeling was achieved by reacting an excess of the non-metallated conjugate with (68) GaCl4 (-) at pH -4.5 and 10-min incubation either at room temperature for NODAGA or 90 °C for DOTA. Radiochemical purity of all (68) Ga conjugates was found to be >98%. (68) Ga-NODAGA-glycine displayed the lowest serum protein binding (0.4%) in vitro among the four (68) Ga conjugates. Biodistribution of (68) Ga conjugates in healthy Sprague Dawley rats at 1-h post-injection revealed an efficient clearance from circulation primarily through the renal-urinary pathway with <0.2% of injected dose per gram remaining in the blood. The kidney/blood and kidney/muscle ratios of (68) Ga-NODAGA-glycine were significantly higher than other (68) Ga conjugates. On the basis of these results, (68) Ga-NODAGA-glycine was selected as the lead candidate. (68) Ga-NODAGA-glycine PET renograms obtained in healthy rats suggest (68) Ga-NODAGA-glycine as a PET alternate of (99m) Tc-Diethylenetriaminepentaacetic acid (DTPA).

Liposomes modified with superhydrophilic polymer linked to a nonphospholipid anchor exhibit reduced complement activation and enhanced circulation.

We report the synthesis of an acyl-anchored superhydrophilic polymer (SHP) for external surface modification of liposome surface. N¹-(2-aminoethyl)-N4-hexadecyl-2-tetradecylsuccinamide conjugated with SHP (HDAS-SHP) was synthesized and used for modifying the liposome surface. Unlike polyethylene glycol (PEG)-phospholipids, which are commonly used for manufacturing stealth liposomes, HDAS-SHP is devoid of both PEG and phosphoryl groups and possesses a zwitterionic polymeric chain. Circulation persistence of the 99(m)Tc-labeled HDAS-SHP liposomes was documented by gamma camera imaging. After 24 h postinjection, approximately 30% of injected HDAS-SHP liposomes were present in blood as compared with only 4.5% of the plain liposomes. HDAS-SHP liposomes inhibited complement activation. They were found to be amenable to pH-gradient-based active loading of Adriamycin in a stable manner. At 37°C, HDAS-SHP liposomes provided better encapsulation efficiencies than the liposomes modified with DSPE-PEG2000. These results provide a strong basis for HDAS-SHP as a viable alternative to PEG-phospholipids for imparting stealth characteristics to drug delivery vehicles such as liposomes. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:114-123, 2015.

Evaluation of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney aminopeptidase N expression.

Aminopeptidase N (APN; CD13; EC 3.4.11.2) is a zinc-dependent membrane-bound exopeptidase that catalyzes the removal of N-terminal amino acids from peptides. APN is known to be highly expressed on renal cortical proximal tubules. APN expression levels are markedly decreased under the influence of nephrotoxins and in the tumor regions of renal cancers. Thus, molecular imaging of kidney APN expression could provide pathophysiological information about kidneys noninvasively. Probestin is a potent APN inhibitor and binds to APN. Abdominal SPECT imaging was conducted at 1 h postinjection of (99m)Tc-probestin in a group of 12 UPII-SV40T transgenic and wild-type mice. UPII-SV40T mice spontaneously develop urothelial carcinoma in situ and invasive transitional cell carcinoma (TCC) that invade kidneys. Histopathology and immunohistochemistry analysis were used to confirm the presence of tumor and to evaluate APN expression in kidney. Radioactivity in normal tissue regions of renal cortex was clearly visible in SPECT images, whereas tumor regions of renal cortex displayed significantly lower or no radioactivity uptake. Histopathological analysis of kidney sections showed normal morphology for both renal pelvic and cortical regions in wild-type mice and abnormal morphology in some transgenic mice. Proliferating cell nuclear antigen staining confirmed the presence of tumor in those abnormal regions. Immunohistochemical analysis of kidney sections using anti-CD13 antibody showed significantly lower APN expression in tumor regions compared to normal regions. Results obtained in this study demonstrate the potential use of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney APN expression.

Evaluation of 99mTc-probestin for imaging APN expressing tumors by SPECT.

Aminopeptidase N (APN) is known to play important roles in tumor angiogenesis, tumor cell invasion, and metastasis. Thus, APN is an attractive biomarker for imaging tumor angiogenesis. Here we report results obtained from biodistribution and single photon emission computed tomography (SPECT) imaging studies of a technetium-99m labeled probestin (a potent APN inhibitor) conjugate containing a tripeptide, Asp-DAP-Cys (DAP=2,3-diaminopropionic acid), chelator and a 8-amino-3,6-dioxaoctanoic acid (PEG2) linker conducted in nude mice xenografted with HT-1080 human fibrosarcoma tumors (APN-positive tumors). These results collectively demonstrate that (99m)Tc-probestin uptake by tumors and other APN expressing tissues in vivo is specific and validate the use of probestin as a vector for targeting APN in vivo.

Post-modification of preformed liposomes with novel non-phospholipid poly(ethylene glycol)-conjugated hexadecylcarbamoylmethyl hexadecanoic acid for enhanced circulation persistence in vivo.

We report synthesis and characterization of a novel PEG2000-conjugated hexadecylcarbamoylmethyl hexadecanoate (HDAS-PEG) as a PEG-phospholipid substitute for enhancing circulation persistence of liposomes. HDAS-PEG showed critical micelle concentration of 4.25 µM. We used post-insertion technique to introduce HDAS-PEG in outer lipid layer of the preformed liposomes. The presence of surface HDAS-PEG was confirmed by altered electrophoretic mobility, confocal microscopy and PEG estimation by ELISA. The post-inserted HDAS-PEG desorbed at approximately half the rate at which post-inserted DSPE-PEG desorbed from the liposome surface. HDAS-PEG significantly reduced liposome-induced complement activation (C4d, Bb and SC5b); HDAS-PEG was more effective than more commonly used DSPE-PEG in this capacity. For studying circulation persistence, the liposomes were labeled with (99m)Tc radionuclide and administered in rats. (99m)Tc-HDAS-PEG-liposomes showed prolonged persistence in blood as compared to that shown by (99m)Tc-plain liposomes. After 24 h of administration, <1% of (99m)Tc-plain liposomes remained in blood, whereas approximately 28% of injected (99m)Tc-HDAS-PEG-liposomes were present in blood. In comparison, only 4.8% of (99m)Tc-DSPE-PEG-liposomes were measured in blood after 24 h. As expected, the clearance route of the liposomes was through liver and spleen. These results demonstrate the potential of a novel non-phosphoryl HDAS-PEG for surface modification of preformed liposomes with a goal of prolonging their circulation persistence and more effective inhibition of complement activation.