99mTc-CXCR4-L for Imaging of the Chemokine-4 Receptor Associated with Brain Tumor Invasiveness: Biokinetics, Radiation Dosimetry, and Proof of Concept in Humans.

Overexpression of the chemokine-4 receptor (CXCR4) in brain tumors is associated with high cancer cell invasiveness. Recently, we reported the preclinical evaluation of 99mTc-CXCR4-L (cyclo-D-Tyr-D-[NMe]Orn[EDDA-99mTc-6-hydrazinylnicotinyl]-Arg-NaI-Gly) as a SPECT radioligand capable of specifically detecting the CXCR4 protein. This research aimed to estimate the biokinetic behavior and radiation dosimetry of 99mTc-CXCR4-L in healthy subjects, as well as to correlate the radiotracer uptake by brain tumors in patients, with the histological grade of differentiation and CXCR4 expression evaluated by immunohistochemistry. 99mTc-CXCR4-L was obtained from freeze-dried kits prepared under GMP conditions (radiochemical purities >97%). Whole-body scans from six healthy volunteers were acquired at 0.3, 1, 2, 4, 6, and 24 h after 99mTc-CXCR4-L administration (0.37 GBq). Time-activity curves of different source organs were obtained from the image sequence to adjust the biokinetic models. The OLINDA/EXM code was employed to calculate the equivalent and effective radiation doses. Nine patients with evidence of brain tumor injury (6 primaries and 3 recurrent), determined by MRI, underwent cerebral SPECT at 3 h after administration of 99mTc-CXCR4-L (0.74 GBq). Data were expressed as a T/B (tumor uptake/background) ratio. Biopsy examinations included histological grading and anti-CXCR4 immunohistochemistry. Results showed a fast blood activity clearance (T 1/2 α = 0.81 min and T 1/2 ß = 12.19 min) with renal and hepatobiliary elimination. The average equivalent doses were 6.10E - 04, 1.41E - 04, and 3.13E - 05 mSv/MBq for the intestine, liver, and kidney, respectively. The effective dose was 3.92E - 03 mSv/MBq. SPECT was positive in 7/9 patients diagnosed as grade II oligodendroglioma (two patients), grade IV glioblastoma (two patients), grade IV gliosarcoma (one patient), metastasis, and diffuse astrocytoma with T/B ratios of 1.3, 2.3, 13, 7, 19, 5.5, and 3.9, respectively, all of them with positive immunohistochemistry. A direct relationship between the grade of differentiation and the expression of CXCR4 was found. The two negative SPECT studies showed negative immunohistochemistry with a diagnosis of reactive gliosis. This "proof-of-concept" research warrants further clinical studies to establish the usefulness of 99mTc-CXCR4-L in the diagnosis and prognosis of brain tumors.

Paclitaxel-loaded folate-coated long circulating and pH-sensitive liposomes as a potential drug delivery system: A biodistribution study.

A range of antitumor agents for cancer treatment is available; however, they show low specificity, which often limit their use. Recently, we have reported the preparation of folate-coated long-circulating and pH-sensitive liposomes (SpHL-folate-PTX) loaded with paclitaxel (PTX), an effective drug for the treatment of solid tumors, including breast cancer. The purpose of this study was to prepare and characterize SpHL-PTX and SpHL-folate-PTX radiolabeled with technetium-99m (99mTc). Biodistribution studies and scintigraphic images were performed after intravenous administration of 99mTc-PTX, 99mTc-SpHL-PTX and 99mTc-SpHL-folate-PTX into healthy and tumor-bearing mice. High radiochemical purity (>98%) and in vitro stability (>90%) were achieved for both liposome formulations. The pharmacokinetic properties of 99mTc-SpHL-DTPA-PTX and 99mTc-SpHL-folate-DTPA-PTX decreased in a monophasic manner showing half-life of 400.1 and 541.8min, respectively. Scintigraphic images and biodistribution studies showed a significant uptake in liver, spleen and kidneys, demonstrating these routes as way for excretion. At 8h post-injection, the liposomal tumor uptake was higher than 99mTc-PTX. Interesting, 4h after administration, the liposome folate coated showed higher tumor-to-muscle ratio than 99mTc-SpHL-DTPA-PTX and 99mTc-PTX. In conclusion, the liposomal systems, showed high tumor uptake by scintigraphic images, especially the 99mTc-SpHL-folate-DTPA-PTX that showed a sustained and higher tumor-to-muscle ratio than non-functionalized liposome, which indicate its feasibility as a PTX delivery system to folate positive tumors.

Cinnamomum zeylanicum extract on the radiolabelling of blood constituents and the morphometry of red blood cells: in vitro assay.

Effects of Cinnamomum zeylanicum (cinnamon) on the labelling of blood constituents with technetium-99m(99mTc) and on the morphology of red blood cells were studied. Blood samples from Wistar rats were incubated with cinnamon extract for 1 hour or with 0.9% NaCl, as control. Labelling of blood constituents with 99mTc was performed. Plasma (P) and blood cells (BC), soluble (SF-P and SF-BC) and insoluble (IF-P and IF-BC) fractions were separated. The radioactivity in each fraction was counted and the percentage of radioactivity incorporated (%ATI) was calculated. Blood smears were prepared, fixed, stained and the qualitative and quantitative morphological analysis of the red blood cells was evaluated. The data showed that the cinnamon extract decreased significantly (p<0.05) the %ATI on BC, IF-P and IF-BC. No modifications were verified on shape of red blood cells. Cinnamon extracts could alter the labelling of blood constituents with 99mTc, and although our results were obtained with animals, precaution is suggested in interpretations of nuclear medicine examinations involving the labelling of blood constituents in patients who are using cinnamon.

The effect of Glucantime on the labeling of blood constituents with technetium-99m.

PURPOSE: The labeling of red blood cells (C) with 99mTc is employed in clinical nuclear medicine for a variety of diagnostic procedures. Drugs can alter this labeling method and modify the disposition of the radiopharmaceuticals. In this paper, the influence of glucantime on the labeling of blood constituents with 9mTc was reported. METHODS: Blood was withdrawn from rats and incubated with glucantime. Stannous chloride and 99mTc were added. After centrifugation, plasma (P) and (C) were isolated. Samples of P and C were precipitated with TCA 5%, centrifuged and insoluble (IF) and soluble fractions (SF) separated. The percentages of total activity injected (%ATI) in C, IF-P and IF-C were calculated (p < 0.05). RESULTS: The %ATI on C decreased from control to following concentrations of glucantime (6.25%; 12.5%; 25%; 50%; 100%), respectively: 94.06 +/- 1.29 (control) to 77.15 +/- 2.79; to 76.68 +/- 1.88; to 75.15 +/- 2.79; to 72.64 +/- 4.40 and to 63.05 +/- 3.84. On IF-C the %ATI decreased from control to all the concentrations of glucantime (3.125%;6.25%; 12.5%; 25%; 50%; 100%), respectively: 93.34 +/- 1.18 (control) to 78.81 +/- 2.76; to 74.76 +/- 4.82; to 74.02 +/- 5.32; to 64.35 +/- 4.82; to 62.81 +/- 1.97 and to 54.55 +/- 3.58. CONCLUSIONS: This effect was probably due to products present in this drug that may complex with ions (Sn(+2) and 99mTcO4) or have a direct or indirect effect on intracellular stannous ion concentration.

The effect of glucantime™ on the labeling of blood constituents with technetium-99m

OBJETIVO: A marcação de hemácias sangüíneas (C) com 99mTc é muito utilizada nos procedimentos diagnósticos na medicina nuclear. Drogas podem alterar este método de marcação e modificar a biodisponibilidade de radiofármacos. Neste trabalho, foi avaliada a influência de glucantime na marcação de elementos sangüíneos com 99mTc. MÉTODOS: Sangue foi retirado de ratos e incubado com glucantime. Adicionou-se cloreto estanoso e 99mTc. Após centrifugação, plasma (P) e (C) foram isolados. Amostras de P e C foram precipitadas com TCA 5 por cento, centrifugadas e separadas em frações solúveis (FS) e insolúveis (FI). Os percentuais de atividade total injetada (por cento ATI) em C, FI-P e FI-C foram calculados (p<0,05). RESULTADOS: O %ATI em C diminuiu, em relação ao controle, nas seguintes concentrações de glucantime (6,25 por cento;12,5 por cento; 25 por cento; 50 por cento; 100por cento), respectivamente: 94,06±1,29 (controle) para 77,15±2,79; para 76,68±1,88; para 75,15±2,79; para 72,64±4,40 e para 63,05±3,84. Em FI-C, o %ATI diminuiu, em relação ao controle, em todas as concentrações de glucantime (3,125 por cento; 6,25 por cento; 12, 5 por cento; 25 por cento; 50 por cento; 100 por cento), respectivamente: 93,34±1,18 (controle) para 78,81±2,76; para 74,76±4,82; para 74,02±5,32; para 64,35±4,82; para 62,81±1,97 e para 54,55±3,58. CONCLUSÕES: Este efeito provavelmente foi devido a produtos presentes nesta droga que podem se complexar com íons (Sn+2 e 99mTcO-4) ou ter um efeito direto ou indireto na concentração intracelular do íon estanoso.

Protection of plasmid DNA by a Ginkgo biloba extract from the effects of stannous chloride and the action on the labeling of blood elements with technetium-99m.

Ginkgo biloba extract (EGb) is a phytotherapeutic agent used for the treatment of ischemic and neurological disorders. Because the action of this important extract is not fully known, assays using different biological systems need to be performed. Red blood cells (RBC) are labeled with technetium-99m (Tc-99m) and used in nuclear medicine. The labeling depends on a reducing agent, usually stannous chloride (SnCl2). We assessed the effect of different concentrations of EGb on the labeling of blood constituents with Tc-99m, as sodium pertechnetate (3.7 MBq), and on the mobility of a plasmid DNA treated with SnCl2 (1.2 microg/ml) at room temperature. Blood was incubated with EGb before the addition of SnCl2 and Tc-99m. Plasma (P) and RBC were separated and precipitated with trichloroacetic acid, and soluble (SF-P and SF-RBC) and insoluble (IF-P and IF-RBC) fractions were isolated. The plasmid was incubated with Egb, SnCl2 or EGb plus SnCl2 and agarose gel electrophoresis was performed. The gel was stained with ethidium bromide and the DNA bands were visualized by fluorescence in an ultraviolet transilluminator system. EGb decreased the labeling of RBC, IF-P and IF-RBC. The supercoiled form of the plasmid was modified by treatment with SnCl2 and protected by 40 mg/ml EGb. The effect of EGb on the tested systems may be due to its chelating action with the stannous ions and/or pertechnetate or to the capability to generate reactive oxygen species that could oxidize the stannous ion.

Lys and Arg in UBI: a specific site for a stable Tc-99m complex?

The aim of this study was to help establish if ubiquicidin peptide 29-41 fragment (UBI) contains a specific site for 99mTc labeling by a new direct method under alkaline conditions. Since this peptide does not have cysteine residues, it is possible that neighboring arginine and lysine in the peptide amino acid sequence (Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) could be a specific coordination site to form a stable 99mTc-UBI complex. Following direct labeling, the in vitro stability of 99mTc-UBI was compared to UBI radiolabeled by one indirect method using HYNIC/tricine and HYNIC/tricine/EDDA. Radiochemical purity of 99mTc-UBI averaged 97% compared to 88% for 99mTc-HYNIC-UBI/tricine and 98% for 99mTc-HYNIC-UBI/tricine/EDDA. Both 99mTc-HYNIC-UBI (tricine or EDDA) and 99mTc-UBI showed stability in human serum and solutions of cysteine. 99mTc-UBI radiochemical purity 24 h after dilution in 0.9% NaCl was greater than 90% at pH 9 and greater than 95% at pH 6.5. Under one set of experimental conditions, in vitro binding to bacteria of 99mTc-UBI was 35% and identical to that of 99mTc-HYNIC-UBI/tricine and 99mTc-HYNIC-UBI/tricine/EDDA at 32% and 31% respectively. The biodistribution of 99mTc-UBI in mice showed a rapid renal clearance. To help identify the site(s) of 99mTc binding following direct labeling, molecular mechanics and quantum-mechanical calculations were performed which showed that the amine groups of Arg(7) and Lys are the most probable site. The calculations show that these groups can form a square pyramid with two water molecules for the Tc cation (dxysp(3)). It will be necessary to isolate and characterize the 99Tc(V)(O)-UBI.(H2O)n complex to confirm these results.

Evaluation of the phytic acid effect on the labeling of blood elements with technetium-99m and on the survival of a strain of Escherichia coli treated with stannous fluoride.

The labeling of red blood cells with technetium-99m (99mTc) depends on a reducing agent and stannous ions, as chloride or fluoride, are widely utilized. This labeling may also be altered by drugs. Moreover, some authors have reported that the survival of Escherichia coli (E. coli) cultures decreases in presence of stannous ions. Phytic acid is present in the daily diet and we evaluated its influence on: (i) the labeling of blood elements with 99mTc and (ii) on the survival of an E. coli strain treated with stannous fluoride. Heparinized whole blood was withdrawn from Wistar rats and it was incubated with stannous chloride and with 99mTc, as sodium pertechnetate, centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P and BC were also precipitated with trichloroacetic acid, centrifuged and soluble (SF) and insoluble fractions (IF) isolated. E. coli culture was treated with stannous fluoride in presence of phytic acid. As phytic acid altered the fixation of 99mTc on BC, on IF-P and on IF-BC and, moreover, it abolished the lethal effect of stannous fluoride on the E. coli culture, we can suggest that, probably, phytic acid would have chelating properties to the stannous ions.

Assessment of the effect of Fucus vesiculosus extract on the labeling of blood constituents with technetium-99m and the histological modifications on the shape of the red blood cells.

Natural products are widely used as food or food additives or medicines for humans. We are trying to develop a model to assess the possible toxic properties of natural products, such as Fucus vesiculosus, utilized in popular medicine. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used in various procedures in nuclear medicine. This labeling procedure depends on a reducing agent, and stannous chloride is used. There is evidence that this labeling may be altered by drugs. We have investigated the possibility that F. vesiculosus extract is capable of altering the labeling of blood elements with 99mTc. Blood was incubated with F. vesiculosus extract and stannous chloride solution and Tc-99m added. Blood was centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P or BC were also precipitated, centrifuged and insoluble (IF) and soluble (SF) were separated. The percentages of radioativity (%ATI) in BC, IF-P and IF-BC were calculated. Histological preparations of the RBC treated with F. vesiculosus revealed that this extract is capable of promoting important modifications on the shape of the RBC. The%ATI decreased on BC from 93.6+/-2.3 to 29.0+/-2.7, on IF-P from 77.6+/-1.2 to 7.5+/-1.0 and on IF-BC from 80.0+/-3.4 to 12.6+/-4.8. Once the RBC labeling procedure with 99mTc depends on the presence of stannous (+2) ions, the substances present in the F. vesiculosus extract should increase the valence of these ions to stannic (+4). This would decrease the%ATI on blood elements and indicate the presence of oxidant agents in the F. vesiculosus extract.

Effect of a chayotte (Sechium edule) extract on the labeling of red blood cells and plasma proteins with technetium-99m: in vitro and in vivo studies.

Sechium edule (chayotte) is used as food or as medication in popular medicine. The labeling of blood elements with technetium-99m (99mTc) has been altered by drugs (synthetic and natural). Some authors have reported biological effects concerning the chayotte. We have evaluated the influence of chayotte extracts (macerated and infusion) on the labeling of blood elements with 99mTc. In vitro study, blood was incubated with the extracts, (6.25, 12.5, 25, 50 and 100% v/v). In in vivo study, the animals were treated with the extracts (100% v/v), as drinking water (15 and 60 days) and samples of blood were withdrawn. The blood samples were incubated with stannous chloride and with 99mTc. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. There was a (p < 0.05) decrease in the radioactivity in BC, IF-BC and IF-P with the infusion (100%) and a slight decrease in the uptake of 99mTc by BC and a strong decrease in the fixation in IF-P with the macerated when the extracts were administrated in vivo (15 days). In 60 days, there was a decrease in BC (98.77 to 53.53%), in IF-BC (90.36 to 21.20%) and in IF-P (77.20 to 11.01%). In vitro study no alterations on the labeling of blood elements were found, however, we have found alterations on the fixation of 99mTc in the in vivo study, probably, due to the metabolization of chayotte capable to induce the generation of active metabolites.

Assessment of the effect of antiseizure drugs on the labeling process of red blood cells and plasma proteins with technetium-99m.

It is estimated that about 2.5 million people only in the United States are affected by epilepsy. Labelled red blood cells (RBC) and plasma proteins (PP) are used for several evaluations in nuclear medicine and drugs affecting those labelings have previously been described. The aim of this study was to evaluate whether the most popular antiseizure drugs interfere with the 99mTc labeling process of RBC and PP. Heparinized blood withdrawn from Wistar rats was incubated with phenobarbital (0.2, 2, 20, 200, 2,000 microg/ml), phenytoin (0.15, 1.5, 15, 150, 1,500 microg/ml), carbamazepine (0.7, 7, 70 microg/ml), clonazepam (0.5, 5, 50, 500 microg/ml) or valproic acid (0.5, 5, 50, 500 microg/ml) for I hr. Stannous chloride (SnCl2), in two different concentrations (0.012 or 1.2 microg/ml) and 99mTc were added. Plasma and cellular fractions were isolated by centrifugation, soluble and insoluble fractions were separated by trichloroacetic acid precipitation. The percentage of radioactivity was calculated for each fraction. Statistical analysis was performed with ANOVA and Dunnet tests. The analysis of the results has shown that phenobarbital (2,000 microg/ml) and clonazepam (50 microg/ml) significantly have reduced the RBC labeling efficiency when it was used the optimal SnCl2 concentration (1.2 microg/ml) and clonazepam (5, 50 microg/ml) has significantly decreased the PP labeling efficiency with 99mTc. Phenytoin (1,500 microg/ml) has decreased the RBC labeling efficiency when the experiments were carried out with a small SnCl2 concentration (0.012 microg/ml). We can suggest that with this in vitro assay, at the therapeutic level of phenytoin, phenobarbital, carbamazepine and valproic acid will not interfere on the 99mTc labeling process of RBC. Interference is displayed at higher phenobarbital concentrations (2,000 microg/ml). However, humans do not tolerate this concentration. On the other hand, a decreased RBC and PP labeling efficiency with 99mTc may be expected for clonazepam at therapeutic levels.

The effect of drugs on the labeling of blood elements with technetium-99m.

The influence of drugs on the labeling of red blood cells and plasma proteins with 99mTc has been reported. Any drug, which alters the labeling of the tracer, could be expected to modify the disposition of the radiopharmaceuticals. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used for several evaluations in nuclear medicine. We have evaluated the effect of Thuya occidentalis, Peumus boldus and Nicotiana tabacum (tobacco) extracts on the labeling of RBC and plasma and cellular proteins with 99mTc. Blood was incubated with the drugs. Stannous chloride (SnCl2) solutions and 99mTc were added. Plasma (P) and blood cells (BC) were separated. The percentage of radioactivity (%ATI) bound to P and BC was determined. The %ATI on the plasma and cellular proteins was also evaluated by precipitation of P and BC samples with trichloroacetic acid (TCA) and isolation of soluble (SF) and insoluble (IF) fractions. The analysis of the results shows that there is a decrease in %ATI (from 97.64 to 75.89 percent) in BC with Thuya occidentalis extract. The labeling of RBC and plasma proteins can be decreased in presence of tobacco. This can be due either a direct or indirect effect (reactive oxygen species) of tobacco. The analysis of radioactivity in samples of P and BC isolated from samples of whole blood treated with Peumus boldus showed a rapid uptake of the radioactivity by blood cells in the presence of the Peumus boldus, whereas there was a slight decrease in the amount of 99mTc radioactivity in the TCA-insoluble fraction of plasma. This study shows that extracts of some medicinal plants can affect the radiolabeling of red blood cells with 99mTc using an in vitro technique.

Effect of Peumus boldus on the labeling of red blood cells and plasma proteins with technetium-99m.

Peumus boldus is used in popular medicine in Brazil. The influence of Peumus boldus on the labeling of red blood cells and plasma proteins with 99mTc was studied. Stannous chloride and 99mTc pertechnetate were incubated with blood and a tincture of Peumus boldus. Aliquots of plasma and blood cells were isolated from the mixture and treated with trichloroacetic acid (TCA). After separation, analysis of the soluble and insoluble fractions showed a rapid uptake of the radioactivity by blood cells in the presence of the drug, whereas there was a slight decrease in the amount of 99mTc radioactivity in the TCA-insoluble fraction of plasma.

Assessment of the diagnostic value of 99mTc-radiolabelled specific antibodies in experimental hydatidosis.

We assessed the potential of 99mTc labelled specific polyclonal antibodies (99mTc-PoAb) for the diagnosis of hydatid disease by immunoscintigraphy. Experimentally infected mice and rabbits were used for this purpose. A specific rabbit antibody recognizing total somatic antigen from hydatid membranes (HCMA) was obtained. PoAb biological activity before labelling was checked according to Barbieri et al. 99mTc-PoAb labelling was performed according to Thakur et al.; the radiochemical purity was higher than 90%. The following studies of 99mTc-PoAb were made: post-labelling biological activity; in vitro stability; blood and renal kinetics in normal mice up to 24 hours after intravenous (i.v.) and intraperitoneal (i.p.) administration; biodistribution in normal and infected mice after i.p. or i.v. injection, and in rabbits after i.v. administration. Biodistribution studies in normal mice, after both administration routes, showed considerable hepatic uptake of activity. An important uptake in cysts after i.p. administration in mice, indicating successful targeting, was also confirmed by autoradiography images. Intravenously administered 99mTc PoAb was not significantly targeted to peritoneal cysts in either animal species, due to inherent limitations to these animal models. Results obtained with i.p. administration suggest that specific hydatid imaging may be possible. Both the mice and rabbit models revealed hepatic uptake which, combined with the short isotope half-life, prevent the drawing of any final conclusions regarding the usefulness of 99mTc-labelling in hydatid disease.

Effect of different anticoagulants on the labelling of red blood cells and plasma proteins with 99Tcm.

There are controversies about the effect of different anticoagulants on the labelling of blood elements with 99Tcm. Our results show that the type of anticoagulant employed to withdraw the whole blood can modify the 99Tcm labelling of red blood cells (RBC) and plasma proteins (PP). The anticoagulants ACD (citric acid, sodium citrate and dextrose solution), heparin and sodium oxalate present similar results for the 99Tcm labelling of RBC with the exception of 0.13 microM stannous chloride. In this assay oxalate provides the best RBC labelling. In addition, with ethylenediaminetetraacetic acid (EDTA) the labelling of RBC is almost always lower than with the other anticoagulants, probably due to its high chelating capacity. The anticoagulants ACD, oxalate and heparin show the same results as expected with 99Tcm labelling of PP. The lowest labelling at 13.00 microM stannous chloride in the presence of oxalate is probably due to its low chelating capacity. The results also reinforce the idea that the erythrocyte membrane exerts an important role in the regulation of stannous ion transport into RBCs.

Assessment of glomerular filtration rate and effective renal plasma flow in cystic fibrosis.

A study was conducted to examine renal function in 10 healthy control subjects and eight patients with cystic fibrosis in stable condition. Sequential bolus injections of 99mTc-DTPA and 125I-OIH were administered to assess glomerular filtration rate and effective renal plasma flow, respectively. Blood was subsequently collected for 3 hours, and urine for 24 hours. Renal clearances of both radioisotope markers were virtually identical in patients and controls. Inasmuch as neither glomerular filtration rate nor effective renal plasma flow was enhanced in patients with cystic fibrosis, increased clearance of drugs in these patients is unlikely to be the result of enhanced glomerular filtration or tubular secretion.