Development of 68Ga-labelled DTPA galactosyl human serum albumin for liver function imaging.

PURPOSE: The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [(99m)Tc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([(99m)Tc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [(68)Ga]GSA for the potential use with positron emission tomography (PET). METHODS: Labelling of GSA with (68)Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [(68)Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [(68)Ga]GSA distribution in Lewis rats was compared with [(99m)Tc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [(68)Ga]GSA and [(99m)Tc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90). RESULTS: [(68)Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free (68)Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98%). Stability after 120 min incubation at 37 °C was high in PBS (>95% intact tracer) and low in human serum (∼27% intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [(68)Ga]GSA compared to [(99m)Tc]GSA. The [(99m)Tc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [(68)Ga]GSA curves. The mean [(68)Ga]GSA t90 was higher than the mean t90 for [(99m)Tc]GSA. The mean [(68)Ga]GSA t50 was not significantly different from the mean t50 for [(99m)Tc]GSA. CONCLUSION: This study provides a promising new (68)Ga-labelled compound based on a commercially used kit for imaging the functional hepatocellular mass.

Atomically monodispersed and fluorescent sub-nanometer gold clusters created by biomolecule-assisted etching of nanometer-sized gold particles and rods.

Atomically monodispersed gold clusters were synthesized by etching gold nanocrystals (particles and rods) with the assistance of biomolecules (amino acids, peptides, proteins, and DNA) under sonication in water. The resulting gold clusters were exclusively composed of eight atoms, as demonstrated by photoluminescence, optical absorption, electrospray ionization mass spectrometry, and transmission electron microscopy measurements. The gold clusters exhibited solvent-dependent photoluminescence properties when exposed to organic solvents, such as chloroform, tetrahydrofuran, or N,N-dimethylformamide, which was attributed to the rich surface properties of the clusters. This strategy, based on top-down etching, offers an approach to create metal clusters from nanomaterials, which show great potential applications in biological labeling/imaging and sensors that utilize photoluminescence properties as the response.

Fluorine-18 labeled galactosyl-neoglycoalbumin for imaging the hepatic asialoglycoprotein receptor.

UNLABELLED: Asialoglycoprotein receptors (ASGP-R) are well known to exist on the mammalian liver, situate on the surface of hepatocyte membrane. Quantitative imaging of asialoglycoprotein receptors could estimate the function of the liver. (99m)Tc labeled galactosyl-neoglycoalbumin (NGA) and diethylenetriaminepentaacetic acid galactosyl human serum albumin (GSA) have been developed for SPECT imaging and clinical used in Japan. In this study, we labeled the NGA with (18)F to get a novel PET tracer [(18)F]FNGA and evaluated its hepatic-targeting efficacy and pharmacokinetics. METHODS: NGA was labeled with (18)F by conjugation with N-succinimidyl-4-(18)F-fluorobenzoate ([(18)F]SFB) under a slightly basic condition. The in vivo metabolic stability of [(18)F]FNGA was determined. Ex vivo biodistribution of [(18)F]FNGA and blocking experiment was investigated in normal mice. MicroPET images were acquired in rat with and without block at 5 min and 15 min after injection of the radiotracer (3.7MBq/rat), respectively. RESULTS: Starting with (18)F(-) Kryptofix 2.2.2./K(2)CO(3) solution, the total reaction time for [(18)F]FNGA is about 150 min. Typical decay-corrected radiochemical yield is about 8-10%. After rapid purified with HiTrap desalting column, the radiochemical purity of [(18)F]FNGA was more than 99% determined by radio-HPLC. [(18)F]FNGA was metabolized to produce [(18)F]FB-Lys in urine at 30 min. Ex vivo biodistribution in mice showed that the liver accumulated 79.18+/-7.17% and 13.85+/-3.10% of the injected dose per gram at 5 and 30 min after injection, respectively. In addition, the hepatic uptake of [(18)F]FNGA was blocked by pre-injecting free NGA as blocking agent (18.55+/-2.63%ID/g at 5 min pi), indicating the specific binding to ASGP receptor. MicroPET study obtained quality images of rat at 5 and 15 min post-injection. CONCLUSION: The novel ASGP receptor tracer [(18)F]FNGA was synthesized with high radiochemical yield. The promising biological properties of [(18)F]FNGA afford potential applications for assessment of hepatocyte function in the future. It may provide quantitative information and better resolution which particularly help to the liver surgery.

Differential receptor targeting of liver cells using 99mTc-neoglycosylated human serum albumins.

Neolactosyl human serum albumin (LSA) targets asialoglycoprotein receptor and shows high liver uptake due to accumulation in hepatocytes. Although neomannosyl human serum albumin (MSA) also shows high liver uptake, it has been reported to be taken up by Kupffer cells and endothelial cells. We compared the biological properties of LSA and MSA. 99mTc-LSA and 99mTc-MSA biodistribution in mice were investigated after intravenous injection. In vivo localization of rhodaminisothiocyanate (RITC)-LSA and fluoresceineisothiocyanate (FITC)-MSA were investigated in mouse liver. Excretion routes of 99mTc-LSA and 99mTc-MSA metabolites were examined. Both 99mTc-LSA and 99mTc-MSA showed high liver uptakes. RITC-LSA was taken up by hepatocytes whereas FITC-MSA was taken up by Kupffer cells and endothelial cells. 99mTc-MSA showed higher spleen and kidney uptakes than 99mTc-LSA. 99mTc-LSA metabolites excreted in urine and feces accounted for 44.4 and 50.0% of 99mTc-LSA injected, respectively, while 99mTc-MSA metabolites accounted for 51.5 and 10.3%, respectively. In conclusion, LSA is specifically taken up by hepatcytes while MSA by Kupffer cells and endothelial cells. After taken up by the liver, LSA is metabolized by the hepatocytes and then excreted through both the hepatobiliary tract and kidney, whereas MSA is metabolized by Kupffer cells and endoghelial cells and then excreted mainly through the kidney.

Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties, and intracellular distribution.

Oligodesoxynucleotides (ODNs) or the corresponding phosphorothioates (PTOs) spontaneously form spherical nanoparticles ("proticles") with protamine in aqueous solutions. The proticles can cross cellular membranes and release the ODNs within the cells. Thus, they represent a potential drug delivery system. The major disadvantages of this system are a lack of stability in salt solutions and its inability to also release PTOs. The present study shows, using PTOs and protamine free base, that these shortcomings can be eliminated by the addition of human serum albumin (HSA) as a third component to the starting mixture. The "ternary" proticles thus obtained contain maximally a few percent of the HSA that was originally present. Nevertheless, they differ from the previously studied "binary" proticles: (1) They are stable in salt solutions for at least several hours. (2) They show a high cellular uptake into murine fibroblasts, and they readily release the PTOs after uptake. The ternary proticles therefore represent a considerable improvement over binary proticles for use as drug delivery systems.

Gallium-labeled deferoxamine-galactosyl-neoglycoalbumin: a radiopharmaceutical for regional measurement of hepatic receptor biochemistry.

Galactosyl-neoglycoalbumin (NGA) is a synthetic ligand to the hepatocyte-specific receptor, hepatic binding protein. In-vitro and in-vivo characterization of a chelation-based derivative of NGA, deferoxamine-galactosyl-neoglycoalbumin (DF-NGA), is described. A two-step glutaraldehyde method was used to covalently couple deferoxamine (DF) to NGA. Products with an average DF-to-NGA ratio of less than 2 contained less than 3% polymeric DF-NGA. All products retained the chelator after 12 mo of storage at 4 degrees C. Gallium labeling of DF-NGA-41 (41 galactose units per HSA) with an average of 1.1 DF per NGA was quantitative within 15 min after the addition of 67Ga-citrate. The labeled product was stable for at least 24 hr. Scatchard and reverse-binding assays of 67Ga-DF-NGA-41 revealed a forward binding rate constant kb similar to that of 125I-NGA-44. The %ID of 67Ga-DF-NGA-41 in rabbit liver was approximately 90% at 10 min after injection of 1.2 x 10(-9) mole DF-NGA per kilogram of body weight. This value decreased to 40% at a scaled molar dose of 1.2 x 10(-7) mol/kg. Biodistribution data of 67Ga-DF-NGA in rabbits was similar to 99mTc-NGA. High tissue specificity and facile labeling will make 68Ga-labeled neoglycoalbumin an ideal agent for regional measurements of receptor biochemistry in the investigational and clinical setting.

Technetium-99m galactosyl-neoglycoalbumin: preparation and preclinical studies.

Technetium-99m galactosyl-neoglycoalbumin ([Tc] NGA), a labeled analog ligand to the hepatocyte-specific receptor, hepatic binding protein (HBP), was prepared and tested for labeling yield, stability, biodistribution, toxicity, and dosimetry. The ligand was synthesized by the covalent coupling of a carbohydrate bifunctional reagent, 2-imino-2-ethyloxymethyl-1-thiogalactose, to human serum albumin. Testing in mice and rabbits revealed the product to be nontoxic and apyrogenic. Technetium labeling yields in excess of 95%, by the electrolytic method, did not alter the molecular weight profile of the neoglycoalbumin. The NGA-bound activity remained stable for at least 4 hr. Biodistribution studies in rabbits demonstrated the liver as the single focus of tracer uptake. Dosimetry was based on kinetic studies in three baboons. Absorbed doses to liver, small intestine, urinary bladder wall, and uterus were 0.089, 0.28, 0.56, and 0.88 rad/mCi, respectively. Total body, lens of the eye, red marrow, ovaries, and testes were less than 0.06 rad/mCi. High liver specificity imparted by receptor binding combined with high labeling yield, stability, acceptable dosimetry, and safety provide [Tc]NGA with the attributes required for routine clinical assessment of hepatocyte function.

Degradation and intrahepatic compatibility of albumin-heparin conjugate microspheres.

The in vitro degradation properties of glutaraldehyde cross-linked albumin and albumin-heparin conjugate microspheres (AMS and AHCMS respectively) were evaluated using light microscopy, turbidity measurements and heparin release determinations, showing that the microspheres are degraded by proteolytic enzymes such as trypsin, proteinase K and lysosomal enzymes. The degradation rate was inversely related to the cross-link density of the microspheres. After intrahepatic administration of AHCMS, cross-linked with 0.5% glutaraldehyde, to male Wag/Rij rats by injection into a mesenteric vein (intravenoportal: i.v.p.), the microspheres were entrapped in the hepatic vascular system. The AHCMS were entrapped within terminal portal veins predominantly at the periphery of the liver. The AHCMS were degraded by cellular enzymatic processes within 2 wk after injection, with a half life of approximately 1 d. Biocompatibility of AHCMS and adriamycin-loaded AHCMS was evaluated by histological assessment of the mitotic activity of liver parenchyma and inflammatory response, and by determination of liver damage marker enzymes during 4 wk after administration. Liver damage marker enzymes were not increased compared with controls, nor were adverse effects observed upon histological examination. There was no difference in response between empty and adriamycin-loaded AHCMS.

A simple and inexpensive preparation of [(51)Cr]-MAA particles.

The feasibility of preparing [(51)Cr]-MAA was investigated using an MAA cold kit and commercially available sodium [(51)Cr]chromate solutions. Of different conditions tested, the best required an addition of a low level of sodium dithionite to achieve a radiolabeling efficiency of 44%. The product was purified by centrifugation to give [(51)Cr]-MAA with 99 +/- 1% radiochemical purity (RCP) comprised of >94% radioactive particles >10 microm, and resulted in a rat biodistribution with >80% in the lungs. [(51)Cr]-MAA is inexpensive, easily and conveniently prepared by this procedure.

Indirect labeling of macroaggregated albumin with indium-111 via diethylenetriaminepentaacetic acid.

It is ideal to perform a simultaneous pulmonary perfusion and ventilation scan in cases of suspected pulmonary thromboembolism. Indium-111 (111In)-diethylenetriaminepentaacetic acid (DTPA)-macroaggregated albumin (MAA) was designed for this purpose. MAA was conjugated with DTPA at a molar ratio of 1:100 and incubated with 111In-chloride for 30 min at room temperature. DTPA-MAA could be labelled with 111In above a 96% labelling efficiency without MAA particle aggregates making their particles larger than desirable. The obtained 111In-DTPA-MAA was intravenously injected into normal mice and their biodistribution was studied at 15 and 180 min after injection. A gamma camera image was obtained 15 min after injection. 111In-DTPA-MAA was stable in vitro and in vivo, and gave high uptake of murine lung in the biodistribution study and clearly visualized murine lung in the scintigraph. Using 111In-DTPA-MAA as a pulmonary perfusion agent, a simultaneous pulmonary perfusion and ventilation scan with technetium-99m-ventilation agents is able to be performed using the dual-isotope technique. 111In-DTPA-MAA may be a potential pulmonary perfusion agent.

In-vivo evaluation of indium-111-diethylenetriaminepentaacetic acid-labelling for determining the sites and rates of protein catabolism in mice.

Pharmacokinetic analyses of protein pharmaceuticals are of prime importance for their clinical application. Because many proteins have pharmacological activity at low concentrations, radiolabelling of proteins is widely used to identify the sites and determine the rates of protein catabolism in-vivo due to the high sensitivity of detection of radioactivity. Recently, a metallic radionuclide, (111)In, has been used to trace the pharmacokinetics of proteins of interest after conjugation of the proteins with diethylenetriaminepentaacetic acid (DTPA). In this study, galactosyl-neoglycoalbumin (NGA) was reacted with the cyclic dianhydride of DTPA and labelled with (111)In to estimate the validity of this radiolabelling procedure for pharmacokinetic analyses. For comparison, we also evaluated direct radioiodination, because directly-radioiodinated proteins are widely used to assess the pharmacokinetics of proteins of interest. The hepatic radioactivity profile after intravenous injection of [131I]NGA or [(111)In]DTPA-NGA into mice was analysed pharmacokinetically, and the first-order rate constant representing the elimination of the respective radiometabolite from hepatic parenchymal cells was determined. The results indicated that direct radioiodination is inappropriate for pursuing the pharmacokinetics of the proteins, because of rapid elimination of the radioactivity from the sites of protein catabolism. These findings also implied that the [(111)In]DTPA label could be used to identify the catabolic sites and determine the rates of catabolism of proteins with relatively short biological half-lives, although characterization of radiolabelled species at the sites of accumulation would be required for accurate determination of the catabolic sites of proteins.

Photografting of albumin onto dimethyldichlorosilane-coated glass.

4-Azido-2-nitrophenyl albumin (ANP-albumin) was prepared by displacing the fluoro group of 4-fluoro-3-nitrophenyl azide (FNPA) by an amino group of albumin. Photolysis of phenyl azides of ANP-albumin was studied by Fourier-transform infrared (FTIR) spectroscopy. The band of phenyl azide disappeared completely after a 12-min exposure to long wave UV light (366 nm), and the photolysis was first-order. Albumin was grafted onto dimethyldichlorosilane-coated glass (DDS-glass) by photolysis of the azido groups of ANP-albumin without any premodification of the surface. The albumin-grafted DDS-glass was characterized by determining the relative amount of nitrogen resulting from the grafted albumin on the surface using electron spectroscopy for chemical analysis (ESCA). The amount of nitrogen increased when the concentration of ANP-albumin in the adsorption solution increased up to 0.1 mg/ml. As the solution concentration increased above this value, the amount of nitrogen decreased. The platelet resistance of the albumin-grafted surfaces was evaluated by measuring the number of adherent platelets and the extent of activation that was quantitated by the area of platelets spread on the surfaces. The maximum platelet-resistant effect was observed when the ANP-albumin was adsorbed for more than 50 min at the solution concentration ranging from 0.05 to 10 mg/ml.

Recent developments in receptor binding radiopharmaceuticals.

The stereoselective and saturable binding between receptor and ligand molecules plays an important role in many biological processes. Therefore ligand-receptor interactions have been increasingly studied in recent years by means of radionuclide labelled ligand molecules of high radiochemical purity and high specific activity. This contribution describes radiochemical development work of such radioligands performed at the Austrian Research Centre Seibersdorf in recent years: Tc99m-Neo-Galacto-Albumin was prepared as ligand for the Hepatic Binding Protein receptor, I-123-Low Density Lipoprotein (LDL) for LDL-receptors in the liver. Since LDL is involved in the origin of atherosclerosis it was subsequently labelled with Tc99m and In-111 as well. A pyrrolidine-methyl-benzamide derivative was separated into its stereoisomers and the active form labelled with I-123. It exhibited specific uptake in Dopamine D-2 receptor containing brain tissue. A benzazepine derivative was likewise labelled with I-123 as potential ligand for D-1 receptors.

Quantification of human hepatic binding protein (HBP) via 99mTc-galactosyl-neoglycoalbumin (NGA) liver scintigraphy.

99mTc-galactosyl-neoglycoalbumin (99mTc-NGA) was synthesized by covalent coupling of 2-imino-2-methoxyethyl-1-thio-beta-D-galactopyranoside to the primary amino groups of human serum albumin. Injection of 99mTc-NGA (150 MBq; 3.5 mg (= 50 nmol)/ml demonstrated the liver to be the exclusive site of tracer-uptake. Simulation of 99mTc-NGA-kinetics allowed quantification of binding to the hepatic binding protein (HBP). Using this model we studied 250 patients with various liver disease. In alcoholic liver cirrhosis such patients with Child B and Child C stage cirrhosis had a lower HBP-concentration in the liver compared to control individuals (0.85-1.2 mumol/l). The group with the most advanced cirrhosis (Child C stage) had a significantly lower HBP-concentration (0.20-0.45 mumol/l) than Child A patients (0.60-0.85 mumol/l; p less than 0.01) and Child B patients (0.45-0.60 mumol/l; p less than 0.05). In patients with biopsy proven liver fibrosis (0.80-1.22 mumol/l) no difference in receptor concentration to normal individuals was estimated. Patients with recently diagnosed acute viral hepatitis underwent repeated 99mTc-galactosyl-neoglycoalbumin (NGA) scanning of the liver during the course of the disease. Return of liver function tests to normal values was associated with an increased hepatic imaging size as well as increase in HBP-concentration (up to a 3-fold of initial concentration). In patients exhibiting a prolonged course of the disease changes in NGA-kinetic data were borderline and the hepatic image size unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of Holmium-166 Labelled Macroaggregates for radionuclide synovectomy.

BACKGROUND: Radionuclide synovectomy (radiation synovectomy) is an alternative method that cures patients with rheumatoid arthritis diseases without surgery. During treatment, the suspension of the (166)Ho-macroaggregates radioactive particles ((166)Ho-MA) is administrated via intra-articular injection into the target joint to destroy the inflamed synovium. MATERIAL AND METHODS: The isotope of (166)Ho (E(beta) max = 1.84 MeV, T((1/2)) = 26.8 hr) was prepared by the (165)Ho(n, gamma)(166)Ho reaction in the LWR-15 nuclear reactor (8-10 MW) using approximate neutron flux 10(14) cm-2s-1. RESULTS: The particles of Ho-Macroaggregates with suitable dimension 1-20 microm and the mean diameter of 8.5 microm were prepared. High in-vitro stability was obtained after incubation of neutron-irradiated Ho-Macroaggregates in saline solution (0.9% NaCl). The in-vivo stability on rats was verified as well. CONCLUSIONS: High in-vivo and in-vitro stability as well as supporting gamma radiation of Ho-166 make the Macroaggregates a prospective agent for radionuclide synovectomy. The method of preparation is relatively easy and allows for the production of particles of a suitable dimension with a sufficient amount of radioactivity of Ho-166 for the treatment of the rheumatoid arthritis.

Synthesis and in vitro characterization of poly(ethylene glycol)-albumin hydrogel microparticles.

High water content hydrogel microparticles based on the cross-linking of albumin with activated poly(ethylene glycol) were synthesized. The influence of different synthesis parameters on the physicochemical characteristics of the microparticles, such as the type of oil and of albumin, and the molecular weight of PEG, was evaluated. The water content of the microparticles ranged from 95 to 98%, increasing with an increase of the molecular weight of PEG. At optimal conditions, microparticles with sizes ranging from 3 to 50 µm were prepared. These microparticles showed a negatively charged surface. They were freely dispersed in PBS buffer and they were stable at 4°C for times varying from 0.5 to 10 months. Initial stirring speed and molecular weight of PEG were the 2 main factors that significantly affected microparticle size. High hydrophilicity, good stability and modulable size make this hydrogel an attractive matrix for protein or cell immobilization for biomedical applications.

Synthesis and biological evaluation of (99m)Tc-DMP-NGA as a novel hepatic asialoglycoprotein receptor imaging agent.

A novel bifunctional coupling agents-biomolecular compound DMP-NGA was prepared by coupling the SATP with galactosyl-neoglycoalbumin (NGA). The DMP-NGA was labeled with technetium-99m, and the radiochemical purity in excess of 98% after purified with HPLC. In vivo biodistribution showed that (99m)Tc-DMP-NGA had very high initial liver uptake with good retention. The liver accumulated 99.35+/-9.77%, 74.25+/-3.03%, 52.47+/-7.58% of the injected dose per gram at 5, 30 and 120min after injection, respectively. It had relative higher initial liver uptake and much lower blood uptake than that of (99m)Tc-GSA. The liver/blood ratio reached 83.4 at 30min post-injection, while the ratio of liver/kidney was 14.4. The uptakes in other organs in the abdomen were also slightly low. In addition, the hepatic uptake of (99m)Tc-DMP-NGA was blocked by preinjecting free GSA as blocking agent. The result indicates that (99m)Tc-DMP-NGA has specific binding to ASGP receptor. Images acquired with Kodak In-Vivo Imaging System FX Pro showed significant difference before and after inhibition. The promising biological properties of (99m)Tc-DMP-NGA afford potential applications in liver receptor imaging for assessment of hepatocyte function.

Carbonyldiimidazole/1-hydroxybenzotriazole activation in polymer phase synthesis of the arginine rich proalbumin hexapeptide extension.

The synthesis on different polymer phases of Arg-Gly-Val-Phe-Arg-Arg, the proalbumin extension, is reported. The peptide was prepared both on a 0.5% cross-linked polystyrene gel containing 2-oxoethyl bromide anchor functions and on a 1% cross-linked chloromethyl polystyrene. For temporary blocking of the amino groups we utilized the 2-(3,5-dimethoxyphenyl) propyl-(2)-oxycarbonyl (Ddz). The guanidino groups of the three arginine moieties were protected by the 4-toluenesulfonyl (Tos) group. As coupling procedure we used 1 equiv. carbonyldiimidazole (CDI)/2 equiv. 1-hydroxybenzotriazole (HOBt). The C-terminal activation of the Ddz-amino acids with CDI/HOBt made it possible to recover the excess Ddz-amino acids in 60-80% yield. We also investigated different procedures to cleave the 2-oxoethyl ester bond between the peptide and the polymer. This bond was completely stable against trifluoromethanesulfonic acid and was split by 1 N triethylamine in methanol/dioxane 1:1 (v/v) + 1 vol% 1 N NaOH. The optical rotation and HPLC properties of Arg-Gly-Val-Phe-Arg-Arg from this synthesis are identical to the product from a different synthesis published earlier.