Synthesis and evaluation of 6-[1-(2-[(18)F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline for positron emission tomography imaging of the metabotropic glutamate receptor type 1 in brain

The purpose of this study was to synthesize 6-[1-(2-[¹⁸F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline ([¹⁸F]FPTQ, [¹⁸F]7a) and to evaluate its potential as a positron emission tomography ligand for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [¹⁸F]7a was synthesized by [¹⁸F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline (7b) with potassium [¹⁸F]fluoride. At the end of synthesis, 1280-1830 MBq (n = 8) of [¹⁸F]7a was obtained with >98% radiochemical purity and 118-237 GBq/??mol specific activity using 3300-4000 MBq of [¹⁸F]F⁻. In vitro autoradiography showed that [¹⁸F]7a had high specific binding with mGluR1 in the rat brain. Biodistribution study using a dissection method and small-animal PET showed that [¹⁸F]7a had high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled 7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [¹⁸F]7a had in vivo specific binding with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [¹⁸F]7a may have a limiting potential for the in vivo imaging of mGluR1 by PET.     

Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function using PET imaging. In this study, the positron imaging agent [¹⁸F]FPGal was designed to specifically target hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radiosynthesis of [¹⁸F]FPGal required 50 min with total radiochemical yields of [¹⁸F]FPGal from [¹⁸F]fluoride as 10% (corrected radiochemical yield). The K_d of [¹⁸F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by 200 mM d(+)-galactose (<0.1%). In vivo biodistribution analysis showed that the liver accumulation of [¹⁸F]FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was significantly lower when assessed through dynamic data collection for 1 h. Therefore, [¹⁸F]FPGal is a feasible PET probe that provide insight into ASGPR related liver disease.     

(18)F-labelling of A-ring substituted 16alpha-fluoro-estradiols as potential radiopharmaceuticals for PET imaging

The 2-methoxy derivative of estradiol is currently in Phase II clinical trial as an anticancer agent while the 4-methyl derivative has been shown to interact with cytoplasmic and nuclear estrogen receptors in rat pituitary gland and hypothalamus. We hypothesize that the 16alpha-(18)F-analogs of these estrogens could be suitable radiotracers to evaluate action mechanisms of the parent compounds. In this study we report the synthesis of the 16alpha-(18)F and 16alpha-(19)F-analogs of the A-ring substituted estradiols in high yield via stereoselective opening of the intermediate 16beta,17beta-O-cyclic sulfones with [(18)F]F(-) or F(-) followed by deprotection.     

Drug delivery using vesicles targeted to the hepatic asialoglycoprotein receptor

We assessed the utility of liver-targeted vesicles as a drug delivery system for the treatment of liver diseases. Small, unilamellar vesicles (mean diameter, 60–80 nm) composed of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and digalactosyldiacylglycerol (mol ratios, 40:40:5:15) are rapidly cleared from the blood in rats after intravenous injection. In vivo organ distribution shows that the liver is the major site of vesicles accumulation, with roughly 60–80% of the vesicles contents delivered to the liver. Isolated, perfused rat liver experiments show that the uptake is due to the hepatic asialoglycoprotein receptor, and the uptake process occurs with minimal vesicle leakage. At low doses of the vesicles, the single pass extraction by the liver is around 50%, which means that this vesicle formulation operates close to optimal efficiency as a drug delivery system to the liver. Binding of vesicles to the liver was determined to saturate at 6.5 mg total lipid/kg body weight, with a maximum steady-state turnover rate of vesicles at 37° C of 79 μg lipid/min per kg body weight. This gives a receptor recycling time of around 80 min. We have incorporated this information into a pharmacokinetic model of vesicle distribution which quantitatively predicts the kinetics and dose dependence of vesicle uptake by the liver in vivo. This information can be used to optimize vesicle-mediated drug delivery to the liver.     

<Superscript>18</Superscript>F-labeled tyrosine derivatives: Synthesis and experimental studies on accumulation in tumors and abscesses

Tyrosine derivatives labeled with a short-lived fluorine-18 isotope (T _1/2 110 min), namely 2-[¹⁸F]fluoro-L-tyrosine (FTYR) and O-(2′-[¹⁸F]fluoroethyl)-L-tyrosine (FET), promising radiopharmaceuticals (RPs) for positron emission tomography (PET), were obtained by asymmetric syntheses. Accumulation of FTYR and FET in the rat tumor “Glioma 35 rats tumor” and in abscesses induced in Wistar rats muscles was studied and compared with that of a well-known glycolysis radiotracer 2-[¹⁸F]fluoro-2-deoxy-D-glucose (FDG). It was shown that the relative accumulation indices of amino acid RPs were considerably lower than those of FDG. At the same time, tumor/muscle ratios were high enough (2.9 for FET and 3.9 for FTYR 120 min after injection) for reliable tumor visualization. The data obtained indicated a possibility in principle to use FTYR and FET for differentiated PET diagnostics of brain tumors and inflammation lesions. Of the tyrosine derivatives studied, FET seems to be the most promising agent due to a simple and easily automated method of preparation based on direct nucleophilic substitution of the leaving tosyloxy group of an enantiomerically pure Ni-(S)-BPS-(S)-Tyr(CH₂CH₂OTs) precursor by an activated [¹⁸F]fluoride.     

Synthesis of [¹¹C]PBR06 and [¹⁸F]PBR06 as agents for positron emission tomographic (PET) imaging of the translocator protein (TSPO)

The translocator protein 18 kDa (TSPO) is an attractive target for molecular imaging of neuroinflammation and tumor progression. [¹⁸F]PBR06, a fluorine-18 labeled form of PBR06, is a promising PET TSPO radioligand originally developed at NIMH. [¹¹C]PBR06, a carbon-11 labeled form of PBR06, was designed and synthesized for the first time. The standard PBR06 was synthesized from 2,5-dimethoxybenzaldehyde in three steps with 71% overall chemical yield. The radiolabeling precursor desmethyl-PBR06 was synthesized from 2-hydroxy-5-methoxybenzaldehyde in five steps with 12% overall chemical yield. The target tracer [¹¹C]PBR06 was prepared by O-[¹¹C]methylation of desmethyl-PBR06 with [¹¹C]CH₃OTf in CH₃CN at 80 °C under basic condition and isolated by HPLC combined with SPE purification with 40–60% decay corrected radiochemical yield and 222–740 GBq/μmol specific activity at EOB. On the similar grounds, [¹⁸F]PBR06 was also designed and synthesized. The previously described Br-PBR06 precursor was synthesized from 2,5-dimethoxybenzaldehyde in two steps with 78% overall chemical yield. A new radiolabeling precursor tosyloxy-PBR06, previously undescribed tosylate congener of PBR06, was designed and synthesized from ethyl 2-hydroxyacetate, 4-methylbenzene-1-sulfonyl chloride, and N-(2,5-dimethoxybenzyl)-2-phenoxyaniline in four steps with 50% overall chemical yield. [¹⁸F]PBR06 was prepared by the nucleophilic substitution of either new tosyloxy-PBR06 precursor or known Br-PBR06 precursor in DMSO at 140 °C with K[¹⁸F]F/Kryptofix 2.2.2 for 15 min and HPLC combined with SPE purification in 20–60% decay corrected radiochemical yield, >99% radiochemical purity, 87–95% chemical purity, and 37–222 GBq/μmol specific activity at EOB. Radiosynthesis of [¹⁸F]PBR06 using new tosylated precursor gave similar radiochemical purity, and higher specific activity, radiochemical yield and chemical purity in comparison with radiosynthesis using bromine precursor.     

Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.     

Synthesis and in vitro evaluation of [18F]fluoroethyl triazole labelled [Tyr3]octreotate analogues using click chemistry

A novel class of alkyne linked [Tyr³]octreotate analogues have been labelled by a copper catalysed azide-alkyne cycloaddition reaction (CuAAC) to form a 1,4-substituted triazole using the reagent [¹⁸F]2-fluoroethyl azide. An unexpected variability in reactivity during the CuAAC reaction was observed for each alkyne analogue which has been investigated. Two lead alkyne linked [Tyr³]octreotate analogues, G-TOCA (3a) and βAG-TOCA (5a) have been identified to be highly reactive in the click reaction showing complete conversion to the [¹⁸F]2-fluoroethyl triazole linked [Tyr³]octreotate analogues FET-G-TOCA (3b) and FET-βAG-TOCA (5b) under mild conditions and with short synthesis times (5 min at 20 °C). As well as ease of synthesis, in vitro binding to the pancreatic tumour AR42J cells showed that both FET-G-TOCA and FET-βAG-TOCA have high affinity for the somatostatin receptor with IC₅₀ of 4.0 ± 1.4, and 1.6 ± 0.2 nM, respectively.     

An In Vivo Microdialysis Study of Striatal 6-[18F]Fluoro-L-m-Tyrosine Metabolism

In vivo brain microdialysis was used to monitor 6-[¹⁸F]fluoro-L-m-tyrosine (FMT) uptake and metabolism in the striatum of conscious freely moving rats for 3 hours after FMT injection (25 mg/kg, iv). Microdialysate collected 20 to 120 min post-dose, contained FMT at a concentration (0.2 to 0.3 nM) approximately ten-fold below that of its metabolite [¹⁸F]fluoro-3-hydroxyphenylacetic acid (FPAC; 3.2 to 3.3 nM). D-amphetamine (2.5 mg/kg, i.p.) injected 120 min after significantly increased microdialysate FPAC (3.27 ± 0.31 nM to 4.51 ± 0.45 nM) in control but not reserpinized rats. Taken together these data demonstrate FMT is heavily metabolized following its entry into the striatum yielding FPAC which appears to be stored, at least in part, in reserpine sensitive cytoplasmic vesicles. Presynaptic retention of FPAC may contribute to the preferential accumulation of FMT positron emission tomography (PET) signaling in dopaminergic brain areas.     

Investigation of an F-18 oxytocin receptor selective ligand via PET imaging

The compound 1-(1-(2-(2-(2-fluoroethoxy)-4-(piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquinolin-2(1H)-one (1) was synthesized and positively evaluated in vitro for high potency and selectivity with human oxytocin receptors. The positron emitting analogue, [F-18]1, was synthesized and investigated in vivo via PET imaging using rat and cynomolgus monkey models. PET imaging studies in female Sprague–Dawley rats suggested [F-18]1 reached the brain and accumulated in various regions of the brain, but washed out too rapidly for adequate quantification and localization. In vivo PET imaging studies in a male cynomolgus monkey suggested [F-18]1 had limited brain penetration while specific uptake of radioactivity significantly accumulated within the vasculature of the cerebral ventricles in areas representative of the choroid plexus.     

Direct labelling of peptides with 2-[F]fluoro-2-deoxy-d-glucose ([F]FDG)

The study describes the use of [¹⁸F]FDG as ¹⁸F building block for the direct labelling of various aminooxy-functionalised peptides via chemoselective oxime formation.     

Characterization of 1-(2-[18F] fluoro-3-pyridyl)-4-(2-isopropyl-1-oxo- isoindoline-5-yl)-5-methyl-1H-1,2,3-triazole, a PET ligand for imaging the metabotropic glutamate receptor type 1 in rat and monkey brains

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-β (Aβ). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aβ in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.     

Synthesis and biological evaluation of novel F-18 labeled pyrazolo[1,5-a]pyrimidine derivatives: potential PET imaging agents for tumor detection

Two novel pyrazolo[1,5-a]pyrimidine derivatives, 7-(2-[¹⁸F]fluoroethylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile ([¹⁸F]FEMPPC, [¹⁸F]1) and N-(2-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)ethyl)-2-[¹⁸F]fluoro-4-nitrobenzamide ([¹⁸F]FCMPPN, [¹⁸F]2), have been designed and successively labeled with ¹⁸F by the nucleophilic substitution employing tosylate and nitryl as leaving groups, respectively. The radiochemical synthesis of both compounds was completed within 60 min with final high-performance liquid chromatography purification included. The corresponding radiochemical yields (without decay correction) were approximately 35% and 30%, respectively. Meanwhile, we compared the uptake characteristics of [¹⁸F]1 and [¹⁸F]2 with those of [¹⁸F]FDG and L-[¹⁸F]FET in S180 tumor cells. Furthermore, the tumor uptake of [¹⁸F]1 and [¹⁸F]2 was assessed in mice bearing S180 tumor and compared with [¹⁸F]FDG and L-[¹⁸F]FET in the same animal model. In vitro cell uptake studies showed [¹⁸F]1 had higher uptake than [¹⁸F]FDG, [¹⁸F]2 and L-[¹⁸F]FET over the 2 h period. In ex vivo biodistribution showed tumor/brain uptake ratios of [¹⁸F]2 were 12.35, 10.44, 8.69 and 5.13 at 15 min, 30 min, 60 min and 120 min post-injection, much higher than those of L-[¹⁸F]FET (2.43, 2.54, 2.93 and 2.95) and [¹⁸F]FDG (0.59, 0.61, 1.02 and 1.33) at the same time point. What’s more, the uptake of [¹⁸F]1 in tumor was 1.88, 4.37, 5.51, 2.95 and 2.88 at 5 min, 15 min, 30 min, 60 min and 120 min post-injection, respectively. There was a remarkable increasing trend before 30 min. The same trend was present for L-[¹⁸F]FET before 30 min and [¹⁸F]FDG before 60 min. Additionally, the tumor/brain uptake ratios of [¹⁸F]1 were superior to those of [¹⁸F]FDG at all the selected time points, the tumor/muscle and tumor/blood uptake ratios of [¹⁸F]1 at 30 min were higher than those of L-[¹⁸F]FET at the same time point. MicroPET image of [¹⁸F]1 administered into S180 tumor-bearing mouse acquired at 30 min post-injection illustrated that the uptake in S180 tumor was obvious. These results suggest that compound [¹⁸F]1 could be a new probe for PET tumor imaging.     

In vivo imaging of endogenous neural stem cells in the adult brain

The discovery of endogenous neural stem cells (eNSCs) in the adult mammalian brain with their ability to self-renew and differentiate into functional neurons, astrocytes and oligodendrocytes has raised the hope for novel therapies of neurological diseases. Experimentally, those eNSCs can be mobilized in vivo, enhancing regeneration and accelerating functional recovery after, e.g., focal cerebral ischemia, thus constituting a most promising approach in stem cell research. In order to translate those current experimental approaches into a clinical setting in the future, non-invasive imaging methods are required to monitor eNSC activation in a longitudinal and intra-individual manner. As yet, imaging protocols to assess eNSC mobilization non-invasively in the live brain remain scarce, but considerable progress has been made in this field in recent years. This review summarizes and discusses the current imaging modalities suitable to monitor eNSCs in individual experimental animals over time, including optical imaging, magnetic resonance tomography and-spectroscopy, as well as positron emission tomography (PET). Special emphasis is put on the potential of each imaging method for a possible clinical translation, and on the specificity of the signal obtained. PET-imaging with the radiotracer 3’-deoxy-3’-[¹⁸F]fluoro-L-thymidine in particular constitutes a modality with excellent potential for clinical translation but low specificity; however, concomitant imaging of neuroinflammation is feasible and increases its specificity. The non-invasive imaging strategies presented here allow for the exploitation of novel treatment strategies based upon the regenerative potential of eNSCs, and will help to facilitate a translation into the clinical setting.     

18F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model

Quantification of the expression of asialoglycoprotein receptor (ASGPR), which is located on the hepatocyte membrane with high-affinity for galactose residues, can help assess ASGPR-related liver diseases. A hepatic fibrosis mouse model with lower asialoglycoprotein receptor expression was established by dimethylnitrosamine (DMN) administration. This study developed and demonstrated that 4-¹⁸F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide (¹⁸F-FBHGal), a new ¹⁸F-labeled monovalent galactose derivative, is an asialoglycoprotein receptor (ASGPR)-specific PET probe in a normal and a hepatic fibrosis mouse models. Immunoassay exhibited a linear correlation between the accumulation of GalH-FITC, a fluorescent surrogate of FBHGal, and the amount of ASGPR. A significant reduction in HepG2 cellular uptake (P <0.0001) was observed using confocal microscopy when co-incubated with 0.5 μM of asialofetuin, a well known ASGPR blocking agent. Animal studies showed the accumulation of ¹⁸F-FBHGal in fibrosis liver (14.84 ± 1.10 %ID/g) was appreciably decreased compared with that in normal liver (20.50 ± 1.51 %ID/g, P <0.01) at 30 min post-injection. The receptor indexes (liver/liver-plus-heart ratio at 30 min post-injection) of hepatic fibrosis mice derived from both microPET imaging and biodistribution study were significantly lower (P <0.01) than those of normal mice. The pharmacokinetic parameters (T_1/2α, T_1/2β, AUC and Cl) derived from microPET images revealed prolonged systemic circulation of ¹⁸F-FBHGal in hepatic fibrosis mice compared to that in normal mice. The findings in biological characterizations suggest that ¹⁸F-FBHGal is a feasible agent for PET imaging of hepatic fibrosis in mice and may provide new insights into ASGPR-related liver dysfunction.     

MicroPET imaging of noxious thermal stimuli in the conscious rat brain

Small animal positron emission tomography (microPET) has been utilized in the investigation of nociception. However, a possible drawback from previous studies is the reduced activation pattern due to the application of anesthesia. The purpose of the present study was to demonstrate a potential means of avoiding anesthesia during stimulation, as well as minimizing the confounding anesthetic effect. Sodium pentobarbital and ketamine were first evaluated to determine their effect on microPET images in the current study. [¹⁸F]-Fluorodeoxyglucose (¹⁸F-FDG) was an appropriate radiotracer to reveal activated regions in rat brains. Pentobarbital anesthesia significantly reduced ¹⁸F-FDG uptake in neural tissues, blurrier to lower contrast; therefore, ketamine was used to anesthetize animals during microPET. After the rats were anesthetized and secured in a laboratory-made stereotaxic frame, a simple, noninvasive stereotaxic technique was used to position their heads in the microPET scanner and to roughly conform the images in the stereotaxic atlas. For functional imaging, conscious rats were restrained in cages with minimal ambient noise; short repetitive thermal stimuli were applied to each rat's tail subsequently. The rats were adequately anesthetized with ketamine following 30 min of scanning without stimulation. An activation index (AI) was calculated from microPET data to quantify the local metabolic activity changes according to the normalized ¹⁸F-FDG dosage. The average AI indicated a side-to-side difference for all innocuous stimulations in the thalamus. However, such side-to-side difference was only observed for noxious heat and cold stimulations in primary somatosensory cortex (SI), secondary somatosensory cortex (SII), and agranular insular cortex (AIC). The present study demonstrated the feasibility of the microPET technique to image metabolic functions of the conscious rat brain, offering better rationale and protocol designs for future pain studies.     

Development of a Model For Estimating the Size and Dynamics of the Pet Dog Population

Abstract

A conceptual model was developed to describe the dynamics of the pet dog population. The model synthesizes existing data collected for a variety of purposes to estimate the size of the various components of the pet dog population in Washington and Iowa during 1991. The total population mortality rate was estimated as 12.4% per year. Animal shelters in Washington and Iowa handled 7.6% and euthanized 4.0% of the dog population in those two states. When these estimates were extrapolated to the entire U.S. dog population, the model predicted that the total annual turnover in owned dogs was 14.7%, or 7.71 million dogs, that 4 million dogs were handled by animal shelters, and that 2.1 million were euthanized. It was also estimated that 79% of all female dogs were spayed, that household breeding could be attributed to less than one-fifth (18.7%) of the female dogs in the reproductive pool, and that the number of owners contributing to total dog population turnover through failure to retain their dog (103,453) was approximately three times the number of owners who allowed their female dogs to be bred (32,513).     

Trivalent, Gal/GalNAc-containing ligands designed for the asialoglycoprotein receptor

A series of novel, fluorescent ligands designed to bind with high affinity and specificity to the asialoglycoprotein receptor (ASGP-R) has been synthesized and tested on human liver cells. The compounds bear three non-reducing, β-linked Gal or GalNAc moieties linked to flexible spacers for an optimal spatial interaction with the binding site of the ASGP-R. The final constructs were selectively endocytosed by HepG2 cells derived from parenchymal liver cells—the major human liver cell type—in a process that was visualized with the aid of fluorescence microscopy. Furthermore, the internalization was analyzed with flow cytometry, which showed the process to be receptor-mediated and selective. The compounds described in this work could serve as valuable tools for studying hepatic endocytosis, and are suited as carriers for site-specific drug delivery to the liver.     

Monitoring the correction of glycogen storage disease type 1a in a mouse model using [(18)F]FDG and a dedicated animal scanner

Monitoring gene therapy of glycogen storage disease type 1a in a mouse model was achieved using [(18)F]FDG and a dedicated animal scanner. The G6Pase knockout (KO) mice were compared to the same mice after infusion with a recombinant adenovirus containing the murine G6Pase gene (Ad-mG6Pase). Serial images of the same mouse before and after therapy were obtained and compared with wild-type (WT) mice of the same strain to determine the uptake and retention of [(18)F]FDG in the liver. Image data were acquired from heart, blood pool and liver for twenty minutes after injection of [(18)F]FDG. The retention of [(18)F]FDG was lower for the WT mice compared to the KO mice. The mice treated with adenovirus-mediated gene therapy had retention similar to that found in age-matched WT mice. These studies show that FDG can be used to monitor the G6Pase concentration in liver of WT mice as compared to G6Pase KO mice. In these mice, gene therapy returned the liver function to that found in age matched WT controls as measured by the FDG kinetics in the liver compared to that found in age matched wild type controls.     

Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody

The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.