Influence of various factors on binding of 67Ga to polymorphonuclear leukocytes

We have studied in vitro, factors that influence the uptake of ⁶⁷Ga-citrate by human polymorphonuclear leukocytes. Citrate at 20 mM concentration decreased the uptake to 1% of control values. Uptake increased as a function of increased μCi of ⁶⁷Ga/10⁷ cells added and incubation time from 0 to 120 min. Uptake decreased somewhat as the incubation pH was lowered from 7.4 to 6.0. Our results suggest that, in vivo, these cells would accumulate ⁶⁷Ga as the inflammatory lesion progresses while the acidic milieu would modestly reduce uptake.     

Involvement of transferrin in the uptake of 67Ga in inflammatory and normal tissues

The results from gel chromatography and electrophoresis showed that ⁶⁷Ga is exclusively bound with transferrin both in vitro and in vivo, but high concentrations of sodium citrate strongly inhibited the binding of ⁶⁷Ga to transferrin. The influence of sodium citrate on the uptake of ⁶⁷Ga into inflammatory and normal soft tissues was also investigated. Sodium citrate decreased the uptake of ⁶⁷Ga into the liver and spleen, but had no influence on the uptake of ⁶⁷Ga into inflammatory tissue. These results suggest that the uptake of ⁶⁷Ga into normal soft tissues occurs in a transferrin-bound form but into inflammatory tissue in an unbound form.     

Gallium-67 Scans in the Diagnosis of Pyelonephritis

Gallium-67 (⁶⁷Ga) citrate was administered intravenously (50 microcuries per kg of body weight) to patients in whom acute and chronic urinary tract infections were suspected. Scanning was done, using both the Anger-type scintillation camera and the rectilinear scanner, 24 to 78 hours after injection of the isotope.The preliminary results imply that ⁶⁷Ga renal uptake is present in patients with pyelonephritis whether overt or silent, as well as in patients with uretero-sigmoidostomies. However, ⁶⁷Ga renal uptake is not present in patients with radiographic evidence of chronic pyelonephritis without active infection and in patients without renal disease.     

Uptake of aluminum and gallium into tissues of the rat

Transport of aluminum and gallium from blood into rat tissues following continuous iv infusion of metals in different chemical forms has been investigated. Tissue uptake of aluminum and gallium was similar and highly dependent on the chemical species of the metals. Aluminum and gallium accumulated in liver and spleen when infused in the chloride form. Raised citrate markedly enhanced aluminum and gallium uptake into renal cortex and bone; in contrast with gallium-transferrin, citrate increased uptake of⁶⁷Ga into renal cortex and bone by 8- and 14-fold respectively. Uptake of⁶⁷Ga with citrate into renal cortex was around 3 times smaller than that of aluminum. The antitransferrin receptor antibody OX-26 enhanced⁶⁷Ga uptake from gallium citrate into all rat tissues.⁶⁷Ga from purified gallium-transferrin was also taken into all tissues in the presence of OX-26, the effect being greatest in renal cortex and bone. No influence of antibody on aluminum transport into rat tissues was, however, observed when aluminum was infused in the citrate form. Therefore, transport of aluminum and gallium into tissues is not similar under all conditions. Transport of each metal occurs into all tissues in the presence of antitransferrin receptor antibody. The potential for such transport is much greater in the case of gallium. Transport of aluminum and gallium citrate complexes appears important especially in the renal cortex and bone.     

Studies on 67Ga uptake by mouse granuloma tissues

In connection with the uptake of ⁶⁷Ga into the inflammatory tissues, such as granuloma tissues produced with turpentine oil, the influence of Fe³⁺ on the uptake of ⁶⁷Ga into mouse granuloma and normal tissues and on the uptake of ¹²⁵I-labeled transferrin and ⁵⁹Fe were investigated. Fe³⁺ decreased the uptake of ⁶⁷Ga into the liver and spleen, but had no influence on the uptake of ⁶⁷Ga into the granuloma tissues. The uptake patterns of ¹²⁵I-labeled transferrin and ⁵⁹Fe in the granuloma tissues were not consistent with that of ⁶⁷Ga at all. These results show that the uptake of ⁶⁷Ga into the granuloma tissues occurs in a free, transferrin-unbound form, but into the liver and spleen in a transferrin-bound form.     

Iron-uptake in the Euryarchaeon Halobacterium salinarum

Iron-uptake is well studied in a plethora of pro- and eukaryotic organisms with the exception of Archaea, which thrive mainly in extreme environments. In this study, the mechanism of iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum strain JW 5 was analyzed. Under low-iron growth conditions no siderophores were detectable in culture supernatants. However, various xenosiderophores support growth of H. salinarum. In [⁵⁵Fe]–[¹⁴C] double-label experiments, H. salinarum displays uptake of iron but not of the chelator citrate. Uptake of iron was inhibited by cyanide and at higher concentrations by Ga. Furthermore, a K_M for iron uptake in cells of 2.36 μM and a V_max of approximately 67 pmol Fe/min/mg protein was determined. [⁵⁵Fe]-uptake kinetics were measured in the absence and presence of Ga. Uptake of iron was inhibited merely at very high Ga concentrations. The results indicate an energy dependent iron uptake process in H. salinarum and suggest reduction of the metal at the membrane level.     

Résultats atypiques et faux-négatifs de la scintigraphie au 67Ga et de la TEP-TDM au 18F-FDG chez une patiente avec sarcoïdose et cancer du sein bilateral

We describe the case of a patient with biopsy proven sinonasal sarcoidosis and a bilateral breast cancer that was unknown at the time of ⁶⁷Ga scintigraphy and ¹⁸F-FDG PET-CT. ⁶⁷Ga scintigraphy showed low sensibility in the assessement of sarcoidosis localizations. Conversely, multiple foci of intense ¹⁸F-FDG uptake were assessed suggesting the presence of active granulomatous disease in sinonasal region, rhinopharynx, skin and several peripheral lymphadenopathy, which were not previously detected by conventional evaluation. On the other hand, an atypical focal accumulation of ⁶⁷Ga uptake was showed in right breast parenchyma, proving to be a grade III infiltrating canalar carcinoma coexistent with pleomorphic type, without axillary metastastic lymphadenopathy. Surprisingly, no ¹⁸F-FDG uptake abnormalities were detected in the right breast in correspondence of ⁶⁷Ga pathological uptake. The whole of these results is discussed according to histological nature of the lesions and the data of the literature.     

Brain Transfer Coefficients for 67Ga: Comparison to 55Fe and Effect of Calcium Deficiency

The transfer coefficients (Kin) for the uptake of gallium-67 (67Ga) into brain and CSF were determined in unanesthetized male Fischer-344 rats fed either a normal or a low-Ca diet. Kin for 67Ga was also compared with transfer coefficients for the uptake of iron-55 (55Fe) and 125I-albumin in control animals. The value of CSF 67Ga Kin was 3 x 10(-7) ml.g-1.s-1 and was 50% larger in low-Ca animals. Brain regional Kin values for 67Ga were 3-9 x 10(-7) ml.g-1.s-1 with no differences in Kin between normal and low-Ca rats. CSF Kin values for 55Fe were 40% and those for albumin were 15% of Kin for 67Ga. For brain, Kin values for 55Fe were 15-40% smaller than for 67Ga, but for albumin the Kin values were 85% less than for 67Ga. 67Ga was found to be 99% bound to plasma proteins, whereas 55Fe was 99.9% bound. The results indicate that metals that are primarily bound to transferrin enter the CSF and brain very slowly. Uptake of both metals was faster than albumin, which may indicate that metal bound to small chelates contributes significantly to brain uptake. In addition, Ca deficiency does not enhance entry of Ga into the brain.     

Relationship between binding activity of 67Ga and low sulfated acid glycosaminoglycans

The sulfate content of acid glycosaminoglycan (AGAG) extracted from granuloma which had been produced by turpentine oil was inversely proportional to the amount of ⁶⁷Ga accumulation in the granuloma. Additionally, the lowest sulfation occurred in granuloma at a peak of inflammation when the uptake of ⁶⁷Ga had reached a maximum. On the basis of electrophoretic pattern, sulfate content, and specific optical rotation, it was concluded that acid glycosaminoglycans obtained from granuloma are mainly composed of chondroitin sulfate-A, -B, and desulfated heparin, while heparan sulfate was a minor component. From in vitro assays, desulfated acid glycosaminoglycans, especially desulfated-heparin and desulfated-heparan sulfate, were found to have a high affinity to ⁶⁷Ga. These results suggest that low- or de-sulfation of AGAG is related to the accumulation of ⁶⁷Ga in inflammatory lesions such as granuloma. Moreover, these results suggest that ⁶⁷Ga does not bind to glycosaminoglycans via sulfuric acid residues.     

Tumor uptake of 5-fluorouracil,methotrexate, and Adriamycin vs gallium

Since the imaging pattern of ⁶⁷Ga-citrate is well known, it can be the benchmark for evaluation of potential tumor imaging agents. Tritium labeled 5-fluorouracil, Adriamycin, (doxorubicin/Adria) and Methotrexate/Lederle were compared at 2 h to ⁶⁷Ga-citrate at 2 and 48 h by percent uptake per gram of various tissues and tumor in rats implanted with Walker 256 carcinosarcoma. ⁶⁷Ga at 48 h showed approximately three times more uptake in the tumor than 5 fluorouracil and eight times more than the others. 5 Fluorouracil showed approximately three times greater uptake than Methotrexate or doxorubicin and 1.4 times the 2 h uptake of ⁶⁷Ga. 5 Fluorouracil shows potential as an imaging agent especially where the waiting time between injection and imaging must be short.     

Effects of iron-binding proteins on in vitro uptake of 67Ga-citrate by tumor cells

A comparative study of carrier-free ⁶⁷Ga-citrate uptake by Ehrlich ascites tumor cells in the presence of lactoferrin, transferrin and ferritin has demonstrated that lactoferrin considerably increases the uptake of ⁶⁷Ga, and that this increase seems to be determined by its iron-load. The other iron-binding proteins assayed have a null or negative effect. Their behavior in the presence of sodium citrate supports the concept of lactoferrin-binding by the cells as responsible for the uptake. The different behavior of ⁶⁷Ga-citrate iron-binding protein complexes appears to support this hypothesis.     

Distribution of 103Ru—chloride in tumor-bearing animals and the mechanism for accumulation in tumor and liver

Tumor uptake rates of ¹⁰³Ru—chloride were smaller than those for ⁶⁷Ga—citrate. In three tumors and liver, ¹⁰³Ru in the mitochondrial fraction containing lysosome increased with time after the administration of ¹⁰³Ru—chloride. The concentration of ¹⁰³Ru was more dominant in connective tissue (especially inflammatory tissue) than in viable tumor tissue or in necrotic tissue. Quite large amounts of ¹⁰³Ru in the tumor and liver were bound to the acid mucopolysaccharide whose molecular masses exceeded 40,000. Behavior of this nuclide was essentially similar to that of ⁶⁷Ga.     

Role of transferrin in uptake of non-physiological metals into cells

At physiological concentrations of citrate the uptake of 59Fe, 67Ga, and 239Pu into human type B lymphocytes of splenic origin is the same in viable and in non-viable cells. Addition of transferrin has no effect on the uptake into non-viable cells but in viable cells it increases the uptake of Fe and Ga but decreases that of Pu. Uptake decreases as transferrin concentration increases although this is less marked with Ga.     

Role of transferrin in metal uptake by human lymphoblasts in vitro

The uptake and binding of 59Fe, 67Ga and 239Pu complexed with citrate of transferrin (Tf) and of 125I-labelled Fe-Tf by human lymphoblasts (WI-L2 cells) have been studied. Uptake kinetics of 59Fe-Tf and [125I]-Tf point to internalization by receptor mediated endocytosis. 67Ga binding and uptake is always less. This may be explained by a lower affinity of Ga-complexes for the cell surface. Factors which influence Fe uptake have a similar effect on Ga. 239Pu uptake and binding, however, are different, especially in that Tf does not stimulate 239Pu uptake and may actually decrease it.     

Fate of a liposome-associated agent injected into normal and tumour-bearing rodents. Attempts to improve localization in tumour tissues.

Liposomes containing ¹¹¹In-labelled bleomycin were injected intravenously into normal and tumour-bearing rodents and the fate of radioactivity followed. ¹¹¹In levels in tissues retained their maximum values for up to 48h after treatment thereby enabling accurate estimations of tissue participation which with a variety of tumours (Meth ‘A’, 6C3HED, Lewis lung carcinoma and Novikoff hepatoma) in mice and rats was secondary to that of the liver and spleen. Reductions in the size of liposomes decreased liver and spleen participation and increased tumour and kidney involvement. Uptake by lungs, skeletal muscle and brain was also augmented albeit to a lesser extent. Incorporation of anti-Meth ‘A’ cells IgG immunoglobulin into the liposomal carrier led to a modest increase in the uptake of co-entrapped ¹¹¹In by the Meth ‘A’ tumour implanted subcutaneously. Although at the same time, liposomal IgG reduced uptake by the kidney, it effected a drastic increase in hepatic and splenic involvement. This could be prevented by the concurrent administration of excess “empty” liposomes which, however, did not interfere with uptake by tumour tissue.     

Triaza-based amphiphilic chelators: Synthetic route, in vitro characterization and in vivo studies of their Ga(III) and Al(III) chelates

Radiogallium chelates are important for diagnostic imaging in nuclear medicine (PET (positron emission tomography) and γ-scintigraphy). Micelles are adequate colloidal vehicles for the delivery of therapeutic and diagnostic agents to organs and tissues. In this paper we describe the synthesis and in vitro and in vivo studies of a series of micelles-forming Ga(III) chelates targeted for the liver. The amphiphilic ligands are based on NOTA (NOTA = 1,4,7-triazacyclonoane-N,N′N″-triacetic acid) and bear a α-alkyl chain in one of the pendant acetate arms (the size of the chain changes from four to fourteen carbon atoms). A multinuclear NMR study (¹H, ¹³C, ²⁷Al and ⁷¹Ga) gave some insights into the structure and dynamics of the metal chelates in solution, consistent with their rigidity and octahedral or pseudo-octahedral geometry. The critical micellar concentration of the chelates was determined using a fluorescence method and ²⁷Al NMR spectroscopy (Al(III) was used as a surrogate of Ga(III)), both showing similar results and suggesting that the chelates of NOTAC6 form pre-micellar aggregates. The logP (octanol-water) determination showed enhancement of the lipophilic character of the Ga(III) chelates with the increase of the number of carbons in the α-alkyl chain. Biodistribution and γ-scintigraphic studies of the ⁶⁷Ga(III) labeled chelates were performed on Wistar rats, showing higher liver uptake for [⁶⁷Ga](NOTAC8) in comparison to [⁶⁷Ga](NOTAC6), consistent with a longer α-alkyl chain and a higher lipophilicity. After 24 h both chelates were completely cleared off from the tissues and organs with no deposition in the bones and liver/spleen. [⁶⁷Ga](NOTAC8) showed high kinetic stability in blood serum.     

l-DOPA (l-3,4-dihydroxyphenylalanine) uptake by human red blood cells

The uptake of l-DOPA (l-3,4-dihydroxyphenylalanine) was studied in normal human red blood cells in vitro using l-[3-¹⁴C]DOPA. Uptake was slow, tending towards a distribution ratio close to unity with a half-time to equilibrium of one hour. Uptake was not Na⁺-dependent. Concentration dependence studies showed both saturable and non-saturable components of uptake, and inhibition studies using l-leucine and l-tryptophan suggest that the L and T systems of red cell amino acid uptake are involved. A powerful inhibitor of both systems, 3,4-dihydroxy-2-methylpropriophenone (U-0521), is described. It is concluded that uptake is by carrier-mediated facilitated diffusion via the L and T systems for which l-DOPA has low affinity.     

Design of Ga–DOTA-based bifunctional radiopharmaceuticals: Two functional moieties can be conjugated to radiogallium–DOTA without reducing the complex stability

From the X-ray crystal structures of Ga–DOTA chelates, we were able to deduce that two free carboxylate groups of the radiogallium–DOTA complex may be utilized for coupling to functional moieties that recognize molecular targets for in vivo imaging without reducing the radiogallium-complex stability. Thus, we designed 2,2′-[4,10-bis(2-{[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]amino}-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl]diacetic acid (DOTA-MN2) (7), employing a metronidazole moiety as the recognition site of hypoxic lesions, based on the drug design concept of bifunctional radiopharmaceuticals. Coupling of DOTA-bis(tert-butyl)ester 5 with 1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride, followed by deprotection, afforded the required 7 (DOTA-MN2). ⁶⁷Ga-labeling was carried out by reaction of DOTA-MN2 with ⁶⁷Ga-citrate. When ⁶⁷Ga–DOTA-MN2 was incubated in phosphate-buffered saline or mouse plasma, no measurable decomposition occurred over a 24-h period. In biodistribution experiments in NFSa tumor-bearing mice, ⁶⁷Ga–DOTA-MN2 displayed not only a significant tumor uptake, but also rapid blood clearance and low accumulations in nontarget tissues, resulting in high target-to-nontarget ratios of radioactivity. These results indicate the potential benefits of the drug design of ⁶⁷Ga–DOTA-MN2. The present findings provide helpful information for the development of radiogallium-labeled radiopharmaceuticals for SPECT and PET studies.     

KINETICS OF SILICIC ACID UPTAKE AND RATES OF SILICA DISSOLUTION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA1,2

Tracer techniques using the stable isotope ³⁰Si were used to measure rates of silicic acid uptake and silica dissolution in silicon replete and silicon depleted populations of 2 clones of the marine diatom Thalassiosira pseudonana Hasle & Heimdal. Uptake kinetics were describable using the Michaelis-Menten equation for enzyme kinetics, and no threshold concentration for uptake was evident. The maximum specific uptake rate of the estuarine clone 3H (0.062–0.092 · h^?1) was higher than that of the Sargasso Sea clone 13-1 (0.028–0.031 · h^?1), but half-saturation constants for uptake by the 2 clones were not measurably different (0.8–2.3 μM for 3H; 1.4–1.5 μM for 13-1). There was little or no light dependence of uptake in populations grown under optimal light conditions prior to the experiment. Exponentially growing populations released silicic acid to the medium by dissolution of cellular silica at rates ranging from 6.5 to 15% of the maximum uptake rate.     

Chelator-induced enhancement of 67Ga tumour imaging: Comparison of desferrioxamine with N,N′-ethylene-bis[2-hydroxy-5-carboxyphenylglycine]

Intravenous injection of the gallium chelating agents, N,N′-ethylene bis[2-hydroxy-5-carboxyphenylglycine], (COOH-EHPG), or desferrioxamine (DFO) after gallium-67 (⁶⁷Ga) injection, improved tumour/non-tumour tissue uptake ratios in mice bearing the EMT-6 sarcoma. Six hours post injection of gallium and 2 h post chelator, COOH-EHPG enhanced the tumour/blood ratios by an order of magnitude compared to untreated controls, whereas DFO increased the ratios three-fold. Twenty two hours post gallium and 2 h post chelator, the increases in ratios compared to controls were seven-fold for COOH-EHPG and two-fold for DFO. The study thus demonstrated the superior ability of COOH-EHPG for the enhancement of ⁶⁷Ga tumour/blood ratios compared with DFO.