Amphiphilic beta-cyclodextrin (betaCDa) nanospheres (mean diameter 90-110 nm) prepared by the solvent displacement method were developed as a colloidal drug delivery system. In order to survey the fate of these nanoparticles, the amphiphilic beta-cyclodextrin was first iodinated by a two-step procedure involving iodination of the primary face followed by an acylation of the secondary face. After radiolabeling of this derivative with (125)I, nanospheres made of betaCDa/betaCDa (125)I were formulated. After a single intravenous injection of labeled nanoparticles in mice, the organ distribution was analyzed from 10 min to 6 days. A rapid clearance of (125)I-labeled betaCDa nanospheres from the blood circulation to the mononuclear phagocyte system was visualized by non-invasive planar imaging study. Radioactivity measurements in organs showed that the nanospheres mainly concentrated in the liver and the spleen where 28 and 24% of the radioactivity per gram of organ was, respectively, found 10 min after injection. At the opposite, the blood activity was low at that time and become negligible thereafter. Finally, the fact that no particular sign of toxicity is observed in injected animals should be emphasized since it is the first report on intravenous administration of betaCDa nanoparticles.

In addition to improving drug solubilization, cyclodextrins (CDs) also affect the biological behavior of the included compound. We evaluated the effects of two natural CDs beta-CD and gamma-CD, and six beta-CD derivatives, Dimeb, Trimeb, SBb, 2-HP, 6AD, and 6 MTU on the biological behavior of (99m)TcN-NOET, a technetium-99m-labeled, lipophilic compound readily detectable through radioactivity assessment. Determination of CDs' affinities for (99m)TcN-NOET indicated that the cavity size of gamma-CD was not suitable for (99m)TcN-NOET inclusion, and that beta-CD derivatization mostly resulted in decreased CDs affinities for (99m)TcN-NOET to various extents compared with the natural beta-CD. In vitro and ex vivo experiments performed on newborn rat cardiomyocytes and isolated perfused rat hearts, respectively, showed 1.7- and 2.3-fold maximal differences in (99m)TcN-NOET cellular and tissue activities. Regression analyzes indicated no significant correlation between these observed biological differences and the affinities of the eight CDs tested for (99m)TcN-NOET or for cellular membranes. In conclusion, CD derivatization often resulted in impaired affinity of the derivatives for the lipophilic compound (99m)TcN-NOET. Moreover, the in vitro and ex vivo biological behavior of (99m)TcN-NOET was greatly affected depending on the CD used for inclusion of the tracer.

UNLABELLED Bis(N-ethoxy,N-ethyldithiocarbamato)nitrido technetium (V) ((99m)Tc) ((99m)TcN-NOET) is a new myocardial perfusion imaging agent currently undergoing phase III clinical trials in the United States and in Europe. (99m)TcN-NOET cellular uptake has been shown to be inhibited by the calcium channel inhibitor verapamil in cultured newborn rat cardiomyocytes. However, the effect of verapamil on in situ (99m)TcN-NOET myocardial uptake remains unknown. Therefore, the aim of this study was to evaluate whether the inhibitory effect of verapamil on the cellular uptake of (99m)TcN-NOET shown in vitro could be reproduced in situ in a canine model of normal and ischemic myocardium. METHODS (99m)TcN-NOET uptake in normal and ischemic myocardium (70% flow reduction in the left anterior descending coronary artery) was measured in the absence or presence of verapamil (0.015 mg/kg/min x 10 min) in anesthetized, open-chest dogs (n = 17). Control animals were infused with adenosine (0.2 mg/kg/min) to match the verapamil-induced increase in flow. RESULTS By verapamil treatment, a clinically relevant plasma concentration of the calcium channel inhibitor was attained (mean +/- SEM, 290 +/- 152 ng/mL). In normal myocardium (n = 8), regional blood flow at the time of (99m)TcN-NOET injection was not statistically different in verapamil- and adenosine-treated dogs (1.69 +/- 0.03 vs. 1.61 +/- 0.04 mL/min/g, respectively). (99m)TcN-NOET uptake was slightly higher in the presence of verapamil (0.39 +/- 0.01 vs. 0.38 +/- 0.01 counts per minute [cpm]/[Bq/kg]/g for adenosine; P = 0.04). However, no significant difference in (99m)TcN-NOET myocardial uptake was observed after normalization of the tracer uptake to regional myocardial blood flow. In ischemic myocardium (n = 9), regional blood flow was lower in verapamil-treated than in adenosine-treated animals (0.22 +/- 0.02 vs. 0.29 +/- 0.03 mL/min/g; P < 0.05). (99m)TcN-NOET uptake in the ischemic area was not inhibited by verapamil (0.09 +/- 0.01 vs. 0.10 +/- 0.01 cpm/[Bq/kg]/g; P = not significant). CONCLUSION Verapamil does not inhibit (99m)TcN-NOET uptake in situ in normal and ischemic canine myocardium. These results suggest that verapamil should not affect (99m)TcN-NOET myocardial uptake in patients referred for myocardial perfusion imaging.

Background Impairment of insulin-stimulated glucose transport is a characteristic of type 2 diabetes. A radioactive glucose analogue has been synthesized: [I-125]-6-deoxy-6-iodo-D-glucose. Its biological behaviour in vitro is similar to that of 3-O-methyl-D-glucose, the reference tracer of glucose transport. The aim of the present study was to determine the ability of [I-125]-6-deoxy-6-iodo-D-glucose to evaluate variations in glucose transport in vivo.Methods Biodistributions of [I-125]-6-deoxy-6-iodo-D-glucose were performed with or without exogenous insulin (iv injection of 1.5 IU/kg) in db/+ non-diabetic control mice and in db/db type 2 diabetic mice, exhibiting a severe insulin resistance characterized by a lack of increase in glucose uptake in response to insulin.Results In db/+ mice, insulin increased [I-125]-6-deoxy-6-iodo-D-glucose transport by 30% in most insulin-sensitive tissues (heart, diaphragm and skeletal muscle, p < 0.05) and had no effect in other organs. In db/db mice, [I-125]-6-deoxy-6-iodo-D-glucose transport in these organs was not modified by insulin.Conclusion [I-125]-6-deoxy-6-iodo-D-glucose is able to trace in vivo an increase in glucose transport with insulin in non-diabetic mice and a defect of glucose transport in type 2 diabetic mice. It is the first time that an iodinated analogue of glucose has shown such promising results after in vivo injection. The use of this tracer to assess glucose transport in vivo in humans via nuclear imaging warrants further investigation. Copyright (C) 2003 John Wiley Sons, Ltd.

Aims: Radiolabeled antisense oligonucleotide to target the mRNA of the hmdr1 gene for diagnostic purposes is a new concept for evaluating the chemoresistance of tumors in vivo. Methods and Results: An 18 mer complementary to the zone which contains the translation initiation codon of the hmdr1 gene was modified using one phosphoramidate group and one. dimethoxytrityle group at the 5' and 3'ends. It permitted probe radiolabeling by I-125 Chemical modifications made to the antisense probe ensured the stability in biological media tested by incubation with human serum at 37degreesC from 5 minutes to 24 hours. These modifications did not interfere with recognition of the target. Retention of the antisense probe followed the expression level of the target transcript in in vitro and in vivo studies. In vitro, after a 2-hour incubation in the presence of K562 - sensitive (S) and- resistant (R) cell lines, uptake was respectively 4.27 +/- 0.96% ID/mg protein and 7.78 +/- 0.46% ID/mg protein (p<0.001). In vivo, the ratios between radioactivity found in the tumor and that found in the striated muscle and in the blood were, respectively, 20 and 3 for IGR OV1 resistant tumor and 1 and 0.3 for the sensitive one. Conclusion: In our study, resistant cell lines and tumor showed greater retention of the specific probe than the sensistive ones. This constitutes a further advance towards non invasive imaging of resistant genes involved in chemoresistance. These results are encouraging: the current trend in innovative cancer therapy is moving towards targeting the genes of interest.

BACKGROUND:Bis[N-ethoxy,N-ethyl(dithiocarbamato)]nitrido Tc (V) (TcN-NOET) is a new technetium complex proposed as a tracer of myocardial perfusion. However, its cellular uptake mechanisms are unknown, although membrane localization on rat heart preparations and preferential binding to polymorphonuclear neutrophils (PMNs) have been reported. Because of the central role of calcium in PMN actions, a relationship was hypothesized between this ion flux and TcN-NOET cellular uptake.METHODS AND RESULTS:The mechanisms of cellular uptake of TcN-NOET were investigated in newborn rat cardiomyocytes by study of the effect of calcium channel modulators on tracer binding. Nifedipine had no effect on tracer uptake at 1 minute. However, verapamil 0.1 micromol/L and diltiazem 0.5 micromol/L induced a 40% decrease in uptake. Conversely, Bay K 8644 0.25 micromol/L increased TcN-NOET uptake by 73%. Alterations in other membrane ion transports failed to modify tracer uptake, indicating the specificity of the relationship between TcN-NOET uptake and calcium channels. Kinetic studies indicated that cellular net accumulation of the tracer was slow (t1/2=28.5 minutes) and retention was prolonged (84% of initial activity retained after 120 minutes of washout). The energy dependence of TcN-NOET uptake was investigated after 60 minutes of metabolic inhibition by iodoacetic acid plus rotenone. The ATP decrease was not associated with reduction in tracer uptake at 1 minute (114.9+/-21.9% of control, P=NS).CONCLUSIONS:The decrease in uptake observed with verapamil and diltiazem, the increase with Bay K 8644, and the lack of effect with nifedipine suggest that TcN-NOET binds to L-type calcium channels in the open configuration, without entering cardiomyocytes. The kinetics of TcN-NOET accumulation and retention are slow, and the mechanism for cellular uptake is not energy-dependent. From a clinical point of view, the effect of concurrent treatment by calcium inhibitors on myocardial binding of TcN-NOET should be taken into account.

Two anomeric analogues of glucose labelled with 123 iodine in position 6, proposed as tracers of glucose transport in vivo, have been synthesized: alpha- and beta-methyl-6-deoxy-6-iodo-D-glucopyranoside (alpha MDIG and beta MDIG). The aim of this study was to determine whether these molecules interact with the glucose transporter and whether they could be used as tracers of glucose transport in vivo. The biodistribution of alpha MDIG and beta MDIG was studied in the mouse in vivo. To determine if these two anomers enter the cell via the glucose transporter, their uptake was measured in isolated perfused rat hearts, in human erythrocytes in suspension, and in cardiomyocytes of neonatal rat in culture. Both alpha MDIG and beta MDIG had similar repartitions in the mouse: myocardial uptake averaged 7% of the injected dose/g of organ at 2 min postinjection and alpha MDIG competed with D-glucose to enter the cells. Insulin produced a 123% increase of its uptake in isolated perfused rat hearts and a 100% increase in cardiomyocytes of neonatal rat in culture. alpha MDIG uptake was lowered in the presence of glucose transport inhibitors in each experimental model. An interaction between beta MDIG and glucose transporters was observed only in human erythrocytes in suspension. Only alpha MDIG interacts with the glucose transporter, and thus could be used to estimate glucose transport in vivo.

A glucose analogue labelled with iodine-123 in position 6 has been synthesized: [123I]-6-deoxy-6-iodo-D-glucose (6DIG). The aim of this study was to examine its biological behaviour in order to assess whether it could be used to evaluate glucose transport with SPECT. To establish whether 6DIG enters the cells using the glucose transporter, four biological models have been used: human erythrocytes in suspension, neonatal rat cardiomyocytes in culture, isolated perfused rat hearts, and biodistribution in mice. 6DIG competed with D-glucose to enter the cells and its entry was increased by insulin and inhibited in the presence of cytochalasin B. The biological behaviour of 6DIG was similar to that of 3-O-methyl-D-glucose. 6DIG is a tracer of glucose transport which is very promising for clinical studies.

La synthèse de tétramines cycliques, de tétramines cycliques mono-et tétra-N-alkylées est développée ainsi que la complexation due technétium par ces ligands à partir de 99mTcO−4. Plusieurs réducteurs du pertechnétate sont étudiés ainsi que le rendement de complexation, la charge et la lipophilie des complexes. Leur étude biologique chez la souris (biodistribution) et chez le chien (scintigraphie) a permis de mettre en évidence une excellente captation hépatobiliaire du trans-99mTcO2(1-dodécyl-1,4,8,11-tétraazacylotétradécane).

Analogues of glucose labeled with 123 iodine in positions 1, 2 or 3 have been synthesized. The aim of this study was to examine their biological behavior in four experimental models in order to assess whether they could be used to evaluate the uptake of glucose with single photon emission computed tomography (SPECT). The results obtained have shown that none of these molecules enters the cell using the glucose transporter. Therefore, they cannot be used as tracers of glucose uptake.

123-Iodine labelled 6-iodo-4-oxa-hexanoic acid, an analogue of 6-iodo-hexanoic acid, has been prepared and its radiochemical stability and in vivo distribution in mice evaluated. The β-iodoethoxyl group has been found to be suitable moiety for iodine radiolabelling.

For an iodinated analogue of glucose to be useful for evaluating glucose uptake using single-photon emission computed tomography (SPECT), it must enter the cell via the same transporter as glucose and accumulate within the cell without being degraded. The biological behavior of the iodinated tracer must therefore be similar to that of 2-deoxy-d-[1-14C]-glucose (2-DG). In the present study, four experimental models (biodistribution in mouse, isolated rat heart, human erythrocytes in suspension and cultured neonatal rat cardiomyocytes) have been chosen and protocols have been set up which allow for the examination of small quantities of iodinated analogues of glucose. The uptakes of 2-DG and of l-[1-14C]-glucose have been measured in these models to establish reference values which will be compared with uptake values for iodinated analogues of glucose.

UNLABELLED:[Bis (N-etoxy, N ethyl dithiocarbamato) nitrido] 99mTc (V) (TcN-NOET) is a new neutral lipophilic myocardial imaging agent proposed for clinical use for detecting coronary artery disease. We studied the relation between myocardial retention of TcN-NOET and myocardial blood flow (MBF) in a canine model.METHODS:A wide range of MBF was induced by partial regional coronary occlusion and dipyridamole infusion (protocols 1,2 and 3). Myocardial activity of TcN-NOET was determined by in vitro tissue counting at 15 or 90 min postinjection. Tracer activity was correlated with radiolabeled microspheres using linear regression analysis.RESULTS:There was a linear correlation between myocardial TcN-NOET activity and microspheres in protocol 1 (r = 094, 15 min postinjection, protocol 2 (r = 0.94, 15 min postinjection after dipyridamole) and protocol 3 (r = 0.91, 90 min postinjection after dipyridamole). When arterial occlusion was discontinued (protocol 4), there was no longer a close linear correlation (r = 0.26). The first-pass myocardial extraction action of TcN-NOET was 75.5% +/- 4% under basal conditions and 85% +/- 2% under hyperemic conditions (p < 0.01).CONCLUSION:Up to 90 min after injection, the relationship between TcN-NOET myocardial retention and blood flow is excellent over a wide range of flows. After reflow, TcN-NOET redistributes almost completely within 90 min.

La complexation du coeur 99mTcO3+ par le 2,10-diméthyl-4,8-dithiaundécane-2,10-dithiol 1 a été réalisée en utilisant un sel d'étain comme réducteur et a conduit à la formation d'une espèce I stable et neutre. Avec le 5-butyl-3,7-dithianonane-1,9-dithiol 2, la même réaction conduit à la formation d'un complexe II instable difficile à isoler. Les études biologiques sur la souris Swiss ont montré que seul I était intéressant (fixation myocardique supérieure à celle du RP 30). Dans le cas des complexes à coeur TcN, on obtient chaque fois deux espèces neutres, soit à partir de [99mTcNCl4]−, soit à partir de 99mTcNCl2[P(CH2CH2CN)3]2; il est possible, selon les conditions opératoires, de favoriser la formation de l'une ou l'autre espèce et de les isoler par purification. Les résultats biologiques se sont avérés assez décevants.

Les conditions d'obtention exclusive de [99mTcNCl4]− à partir de 99mTcO4−, NaN3 et HCl ont été déterminées. Les conditions d'analyse chromatographique sur papier de cet ion ont été précisées (éluant, pH, type de papier). L'espèce obtenue a servi à préparer, par échange de ligandes, une série de complexes de type N2S2 à partir de diaminodithiols substitués ou non sur l'azote. L'échange a également été obtenu avec 99mTcNCl2[P(CH2CH2CN)3]2 comme précurseur. Suivant la structure du diaminodithiol de départ on obtient un ou deux complexes neutres. La séparation éventuelle des deux espèces (dans ce cas seule la plus lipophile est récupérée) et l'élimination de l'excès de ligande ont été effectuées par chromatographie sur papier. Les complexes ont donné lieu à une étude biologique sur la souris Swiss. La captation myocardique est peu satisfaisante. Par contre, dans certains cas et en particulier avec l'espèce obtenue à partir du 5,8-diaza-3,10-diéthyldodécane-3,10-dithiol 6 la captation cérébrale est au moins èquivalence à celle obtenue avec le HMPAO.

The association of verapamil with halothane causes ischaemic-like myocardial dysfunction. Using an isolated rat heart model perfused with a radiolabelled fatty acid (123I-labelled iodohexadecenoic acid) as a sensitive marker of ischaemia this study investigated whether or not this dysfunction is of ischaemic origin. Hearts were perfused with a control solution or with solutions containing either 1% of halothane or 150 ng ml-1 of verapamil or the association of 0.75% halothane + 120 ng ml-1 verapamil. The ischaemic group was perfused at a reduced perfusion rate (-50%). Intracellular fate of IHA was assessed, and its esterification ratio computed. Ischaemia and the drugs induced a similar depression of haemodynamics. The esterification ratio in the ischaemic group was significantly higher (0.723 +/- 0.04) than in controls (0.0526 +/- 0.03) and than in the treated groups: halothane (0.533 +/- 0.06), verapamil (0.411 +/- 0.027) or the association halothane+verapamil (0.408 +/- 0.05), suggesting a non-ischaemic origin for the dysfunction caused by halothane-verapamil.