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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Uptake of Gallium-67 in Colonic and Rectal Tumours

The uptake of ⁶⁷Ga in biopsy specimens of normal and abnormal tissue was measured in 20 cases of colonic and rectal disease and the ratio between the ⁶⁷Ga uptake in these diseases and in the normal colon or rectum was determined. Uptake of the isotope was high in most of the 14 primary malignant tumours, being highest in poorly differentiated tumours. This uptake was found to be concentrated at the active tumour edge. Uptake of the isotope was generally low in non-malignant lesions.     

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.     

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.     

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.     

Development and characterization of a 68Ga-labeled A20FMDV2 peptide probe for the PET imaging of αvβ6 integrin-positive pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal cancers. Since the majority of patients are diagnosed at an advanced stage, development of a detection method for PDAC at an earlier stage of disease progression is strongly desirable. Integrin α_Vβ6 is a promising target for early PDAC detection because its expression increases during precancerous changes. The present study aimed to develop an imaging probe for positron emission tomography (PET) which targets α_Vβ6 integrin-positive PDAC. We selected A20FMDV2 peptide, which binds specifically to αvβ6 integrin, as a probe scaffold, and ⁶⁸Ga as a radioisotope. A20FMDV2 peptide has not been previously labeled with ⁶⁸Ga. A cysteine residue was introduced to the N-terminus of the probe at a site-specific conjugation of maleimide-NOTA (mal-NOTA) chelate. Different numbers of glycine residues were also introduced between cysteine and the A20FMDV2 sequence as a spacer in order to reduce the steric hindrance of the mal-NOTA on the binding probe to αVβ6 integrin. In vitro, the competitive binding assay revealed that probes containing a 6-glycine linker ([^natGa]CG6 and [^natGa]Ac-CG6) showed high affinity to αVβ6 integrin. Both probes could be labeled by ^67/68Ga with high radiochemical yield (>50%) and purity (>98%). On biodistribution analysis, [⁶⁷Ga]Ac-CG6 showed higher tumor accumulation, faster blood clearance, and lower accumulation in the surrounding organs of pancreas than did [⁶⁷Ga]CG6. The α_Vβ6 integrin-positive xenografts were clearly visualized by PET imaging with [⁶⁸Ga]Ac-CG6. The intratumoral distribution of [⁶⁸Ga]Ac-CG6 coincided with the α_Vβ6 integrin-positive regions detected by immunohistochemistry. Thus, [⁶⁸Ga]Ac-CG6 is a useful peptide probe for the imaging of α_Vβ6 integrin in PDAC.     

68Ga‐labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators

The dual interaction with integrins and neuropilin‐1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with ⁶⁸GaCl₃. Negatively charged ⁶⁸Ga‐DOTAGA‐iRGD and neutral ⁶⁸Ga‐NODAGA‐iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral ⁶⁸Ga‐NODAGA‐iRGD, but ⁶⁸Ga‐DOTAGA‐iRGD exhibited better tumor‐to‐blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.     

An Improved Method for in Vivo Tracing and Imaging of Liposomes Using a Gallium 67-Deferoxamine Complex

Abstract

We have developed a new method for tracing and imaging liposomes in vivo based on the encapsulation of a gallium 67-deferoxamine ⁶⁷Ga-DF) complex in the liposomal water phase. This method combines several advantages over other published methods: extremely high affinity of ⁶⁷Ga for DF, thus avoiding the problem of metal translocation to various plasma proteins; rapid renal clearance rate of⁶⁷Ga-DF complex, thus minimizing the background of radioactivity in non-liposome-associated form; and use of⁶⁷Ga, a readily available, short half-life gamma-emitter convenient for dosimetry and imaging, which can be efficiently loaded into preformed liposomes.     

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.     

An antibody-desferrioxamine conjugate labelled with 67Ga

The optimum conditions for producing a ⁶⁷Ga labelled antibody-desferrioxamine conjugate were determined. The mouse monoclonal antibody LICR-LON-M8 was coupled to the metal chelating compound desferrioxamine (DFO) using glutaraldehyde (GLUT). A DFO: GLUT: M8 molar ratio of 500: 150: 1 gave an immunoreactive antibody with low amounts (< 5%) of high molecular weight polymer. The labelling efficiency with ⁶⁷Ga was >90%, with a specific activity of 2–4 MBq/mg antibody. This ⁶⁷Ga labelled antibody is suitable for evaluation as a diagnostic imaging agent.     

Comparative studies of radiocitrates in oncological models—I. 99mTc citrate and 67Ga citrate uptake by EMT-6 tumors in mice

⁶⁷Ga citrate and ^99mTc citrate (Solcocitran) were injected sequentially, with an interval of 48 h, into Balb/c mice bearing transplanted EMT-6 tumors. Tissue distributions of ⁶⁷Ga and ^99mTe were measured simultaneously at intervals of 1, 3, 5 and 8 h after injection of the ^99mTc citrate (49, 51, 53 and 56 h after ⁶⁷Ga citrate). Maximal tumor:blood ratios for ^99mGa and ^99mTc were 13.8 ± 3.2 and 4.0 ± 1.0 respectively, both occurring at the final period. The maximum tumor index (T.I. = T:B x % dose/g) for ⁶⁷Ga was 71 ± 23% 56 h after injection, and for Tc was 13 ± 12% 1 h after injection. Liver, kidney and spleen had equal or higher concentrations of radioactivity than tumor for either radiotracer. The somewhat higher tumor:blood ratio for ⁶⁷Ga citrate was offset by the time required for this optimum to be reached. Alternatively, the best ^99mTc citrate tumor:blood ratios were attained within 8 h, with less liver and gut radioactivity. These data fall within the range of results from other clinical and animal model studies of ⁶⁷Ga citrate and Tc citrate. In view of the radiation dose, the inconvenience of the 48–72 h wait, and the cost of ⁶⁷Ga, and because neither radiopharmaceutical is tumor specific, ^99mTc citrate may have a place in early oncological screening. The results are discussed as part of a comprehensive review of the ^99mTc citrate literature.     

Autoradiographic model experiments with 67Ga and 99mTc

Summary In order to evaluate the feasibility to localize correctly ⁶⁷Ga citrate and ^99mTc pertechnetate in tissues, the resolution of these radioactive compounds were measured in a model system using four different autoradiographic techniques, wet as well as dry.Wet autoradiographic techniques gave an almost complete loss of ⁶⁷Ga. In deparaffinized sections of fixed and paraffin-embedded tissue the remaining ⁶⁷Ga, which was probably bound to proteins, gave a resolution of 2.6 . ^99mTc was either completely lost in wet techniques or the procedure could not be performed at all because of the very short half life of ^99mTc.The resolution of ⁶⁷Ga in a dry autoradiographic technique (according to Stumpf) was 6.9 and the resolution of ^99mTc 22.6 .The technique in which frozen sections are thawed on dry film and consequently dryed, gave slightly better resolutions than the dry technique (according to Stumpf) with ⁶⁷Ga as well as ^99mTc.It is concluded that for the histological localization of ⁶⁷Ga and ^99mTc a dry technique is required. However, the use of a wet technique can be considered, provided a loss of the radioisotope is acceptable and the procedure is controlled by a dry technique.     

Biodistribution and tumor uptake of [67Ga]chlorogallium-tetraoctadecyloxy phthalocyanine and its sulfonation products in tumor bearing C3H mice

To evaluate the biodistribution and tumor uptake of chlorogallium tetraoctadecyloxy phthalocyanine, a potential new drug for the photodynamic therapy of cancer, we prepared the radioactive ⁶⁷Ga-labeled complex and its water soluble sulfonated derivative. The non-sulfonated dye was obtained by condensation of octadecyloxyphthalonitrile in the presence of a mixture of ⁶⁷Ga and ⁶⁹Ga chloride. The sulfonated derivative was obtained by treatment of the condensation product with oleum. As singlet molecular oxygen has been implicated in the photodynamic action of phthalocyanines (Pcs), the quantum yield of singlet oxygen (φ_Δ) was evaluated for chlorogallium tetraoctadecyloxy Pc, and also its zinc, aluminum and metal free analogues. After intraperitoneal administration of the non-sulfonated dye into RIF tumor bearing C3H mice, a very high ⁶⁷Ga-uptake was observed in the spleen, while tumor radioactivity remained low during the 3 day study. The in vivo stability of the ⁶⁷Ga-phthalocyanine complex was confirmed by comparing the distribution pattern with that of ⁶⁷Ga-citrate, which proved to be significantly different. Intravenous injection of the sulfonated dye resulted in an overall lowering of ⁶⁷Ga-uptake by most tissues, particularly in the spleen, while tumor radioactivities were slightly higher. These data suggest that amphiphilic photosensitizers, containing both polar sulfonate groups and long aliphatic substituents, exhibit the best distribution pattern for both imaging and photodynamic therapy of tumors.