Bépridil a new effector of oxidative phosphorylations.

Bépridil action on oxidative phosphorylations depends on the nature of the oxidized substrate. Thus it seemed to act as an uncoupler on the FAD-linked substrates as we have shown with succinate. But this property could not be applied to the NAD-linked substrates. Thus, in the presence of Bépridil, ADP would have opposite effects on the electron flow across the first site of oxidative phosphorylations; from one side the direct electron flow was decreased while from the other side, the reverse one was increased. Confirming this difference in its action, Bépridil did not affect the oxidative phosphorylation property of the cytochrome oxidase as could be deduced from the invariability of TMPD + ascorbate oxidation whether Bépridil was added or not. Moreover the effect of Bépridil on the P/O ratio was a dramatic demonstration of its selectivity. As a matter of fact this ratio was increased to a value near 5 for the NAD-linked substrate while it was decreased to near 0.4 with the FAD-linked substrate.

Myocardial imaging in dogs treated with grisorixin: relationship between thallium-201 uptake and coronary blood flow.

Thallium-201 myocardial imaging was performed in dogs after pretreatment with grisorixin, which appeared to increase the myocardial uptake of Tl-201. This effect of grisorixin was found to be dose dependent, with an optimal dose of 60 microgram/kg. The myocardial-to-background ratio, which was 1.92 in the control dogs, rose to 4.45. The increase in the absolute myocardial uptake was demonstrated in guinea pigs that received Tl-201 after similar pretreatment with grisorixin. In the animals treated with 500 microgram/kg, the uptake of Tl-201 by the heart was 35% over the control value. With 60 microgram/kg grisorixin, the coronary blood flow increased from 40 to 176 ml/min 5 min after the injection. This dose, optimal for imaging, induced the maximum vasodilator effect with only a very slight concomitant increase in the left-ventricular pressure and myocardial contractility. Above 60 microgram/kg, grisorixin appeared to be a potent inotropic agent, whereas below this dose it showed only coronary vasodilator properties. Some evidence for an ionophore effect of this compound was found in dogs pretreated with 60 microgram/kg. In these the radionuclide was injected when the coronary vasodilatation had become insignificant, but a significant improvement of the M/B ratio was still evident.

Assessment of drugs by functional imaging of thallium-201 distribution in the dog.

The effect of drugs on thallium-201 biodistribution in the body has been studied in dogs using a new functional imaging method. The calculation is based upon the comparison of the activities of the two scintigraphic images obtained after two successive injections of thallium-201 separated by an interval of at least 10 min, during which some drug of interest can be administered. This imaging technique was applied in control dogs (n = 6) and in animals treated with dipyridamole (n = 7) or grisorixin (n = 7). As expected, these two vasoactive drugs increased mainly the myocardial uptake, whereas smaller variations were noted in the other organs studied: liver, kidney, lungs, and skeletal muscles. Thus this method should allow a rapid and reliable noninvasive assessment of cardiovascular drugs with thallium-201.

Hexakis (2-methoxy isobutylisonitrile) technetium-99m and thallium-201 chloride: uptake and release in cultured myocardial cells.

Hexakis (2-methoxy isobutylisonitrile) technetium-99m [(99mTc]MIBI), a new tracer of myocardial blood flow, was compared with 201TI CI in cultures of myocardial cells of newborn rats. The kinetics of uptake and release of both tracers were assessed in basal conditions and in the presence of 5 mM cyanide, an inhibitor of the respiratory chain, 0.1 mM iodoacetate, an inhibitor of glycolysis, 10 microM ouabain, an inhibitor of the Na-K ATPase, or with various pH values. The amplitude and frequency of contractions of the cells were also monitored in the same conditions. Results show that the washin and washout kinetics of [99mTc]MIBI are slower than 201TI(T1/2) of the washout curves were, respectively, of 28 min and 6 min). The kinetics of release of both tracers were not influenced by any of the inhibitors. There was a strong effect of the pH on the 201TI uptake only. Moreover 201TI uptake was decreased by 34% in the presence of cyanide plus iodoacetate. Otherwise the uptakes of 201TI and [99mTc]MIBI were not decreased by any of the drugs. The cellular contractility was significantly diminished by cyanide and it was abolished by cyanide plus iodoacetate. It is concluded that (a) impaired contractility can be associated with normal 201TI and [99mTc]MIBI kinetics in myocardial cells in culture, (b) that 201TI uptake may depend on the level of ATP devoted to the maintenance of membrane integrity, (c) that [99mTc]MIBI shows slower kinetics but is less sensitive to metabolic inhibitors than 201TI.

Myocardial imaging in dogs with thallium-201 and the ionophore grisorixin.

The effect of grisorixin, a monocarboxylic ionophore, upon the myocardium-to-background ratio in thallium-201 scintigrams has been studied in 20 dogs. Three sequences of injection and two doses of the ionophore have been used. A significant improvement was obtained when grisorixin was injected 10 min before thallium-201 and at a submaximal dose; the myocardium-to-background ratio was at least twice that of the controls during the first 20 min following the tracer injection.

Comparison between technetium-99m-sestamibi and hydrogen-3-daunomycin myocardial cellular retention in vitro.

UNLABELLED: This study was undertaken to verify whether 99mTc-sestamibi uptake parallels that of 3H-daunomycin in cells treated with multidrug resistance (MDR) reversing agents. Since we have detected in a previous work a moderate typical MDR phenotype in rat cardiac cells, a model of cultured myocardial cells was used. METHODS: Newborn-rat cultured myocardial cells were incubated 120 min with the MDR-reversing agent verapamil 50 microM, PSC833 1 microM or S9788 10 microM alone or in combination, and the cellular retention of 3H-daunomycin and 99mTc-sestamibi was counted. RESULTS: Hydrogen-3-daunomycin cellular accumulation was never modified by more than 15% when compared to control values, while 99mTc-sestamibi decreased to 75% +/- 32% (m +/- s.d.) of controls in the presence of S9788 and to 44% +/- 19% when S9788 was associated with verapamil. CONCLUSION: The variations of 99mTc-sestamibi and 3H-daunomycin cellular accumulation induced by MDR-reversing agents in cultured myocardial cells can be dramatically different. While some MDR-reversing agents can significantly increase the 3H-daunomycin retention in cardiac cells, they have unexpected effects on that of 99mTc-sestamibi.

Comparative 99mTc-sestamibi and 3H-daunomycin uptake in human carcinoma cells: relation to the MDR phenotype and effects of reversing agents.

UNLABELLED: Because 99mTc-sestamibi (MIBI) appears to be a potent candidate for multidrug resistance (MDR) evaluation in tumors, its cellular uptake should be similar to that of 3H-daunomycin in a variety of conditions of expression and inhibition of MDR activity. METHODS: We used a human rhinopharyngeal carcinoma cell line (KB-3-1) and its MDR variant (KB-A1). Cells were incubated 2 h with 99mTc-MIBI and 3H-daunomycin under control conditions or in the presence of a reversing agent such as verapamil (10 pmol/L), PSC833 (1 micromol/L) or S9788 (5 micromol/L). RESULTS: Relative to the KB-3-1-sensitive cells, accumulations of 99mTc-MIBI and 3H-daunomycin were reduced to 31% +/- 5% and 36% +/- 11% (P < 0.001 for both) in KB-A1-resistant cells. In sensitive cells, accumulation of both agents was increased by verapamil and PSC833 (range 115%-140%; P < 0.05) but not by S9788. In KB-A1 cells, only S9788 significantly increased the cellular uptake of 99mTc-MIBI (138% +/- 25%; P < 0.01), whereas the intracellular uptake of 3H-daunomycin was markedly increased with the three reversing agents (up to 311% +/- 37% with S9788; P < 0.001). With this last treatment, uptake of 3H-daunomycin in KB-A1 cells nearly returned to its basal level in sensitive cells. CONCLUSION: 99mTc-MIBI monitors the MDR phenotype of tumor cells effectively but responds to reversing agents differently than 3H-daunomycin.

Uptake of technetium-99m-teboroxime in cultured myocardial cells: comparison with thallium-201 and technetium-99m-sestamibi.

The effects of metabolic inhibition on the uptake of 99mTc-teboroxime were assessed in cultured myocardial cells and compared with 201Tl and 99mTc-sestamibi. Metabolic impairment was induced by cyanide (CN), a blocker of the mitochondrial respiratory chain, iodoacetate (IAA), an inhibitor of the glycolytic pathway, and ouabain, an inhibitor of Na(+)-K+ sarcolemmal ATPase. Cellular viability was appreciated by the trypan blue exclusion method. The effects of low temperature and of cellular death resulting from osmotic lysis were also assessed. Net cellular uptake of the radiotracers and the amount of proteins in the culture dishes were measured. All experiments were performed in parallel with control conditions and the results were expressed relatively to the control values. Teboroxime uptake was clearly decreased at low temperature (29.6% +/- 2.2% at 0 degree C, p < 0.001), while metabolic inhibition or osmotic lysis had no definite effect. The uptake of 201Tl and sestamibi was severely diminished in the presence of a mixture of 5 mM CN and 0.1 mM IAA, but 201Tl was less resistant than sestamibi (13.7% +/- 0.3% and 73.5% +/- 3.3%, respectively, after 1 hr of preincubation, p < 0.001 for both). Uptake of both tracers was very low in the presence of dead cells (12.1% +/- 1.3% for 201Tl and 4.1% +/- 0.2% for sestamibi, p < 0.001 for both). Ouabain had a detrimental effect only on 201Tl uptake at doses higher than 100 microM. Of these three currently available coronary blood flow imaging agents, teboroxime shows the lowest sensitivity to metabolic impairment.

Effects of grisorixin on the distribution of thallium-201 and on the oxidative metabolism in cultured myocardial cells.

Previous experiments in the dog and guinea-pig have shown that grisorixin, a monocarboxylic ionophore, can significantly increase the coronary blood flow and the myocardial uptake of 201Tl, as well as have a stimulant effect on the heart. In this study, cultures of myocardial cells were used in order to isolate the cells from the vascular and extracardiac influences so that any ionophorous effect on 201Tl could be evidenced. The effects of grisorixin on the oxidative metabolism were simultaneously studied. The technique described by Harary was used to prepare the cultures. The activity of the 14CCO2 produced by oxidation of [14C]glucose and [14C]octanoate added to the medium of culture and the intra/extracellular ratio of 201Tl concentrations (Tl i/e) were measured. In the controls (n = 8), the Tl i/e was 40 +/- 10 while it was 17 +/- 6 (p less than 0.05) in the cells that received 201Tl and grisorixin at the same time (n = 4), and 19 +/- 5 (p less than 0.05) in the flasks where 201Tl was injected after grisorixin (n = 7). A significant decrease of the [14C]octanoate oxidation was found in the flasks treated with grisorixin (n = 4, -50 +/- 16%, p less than 0.01) while the [14C]glucose oxidation was not significantly lowered (n = 3; -11 +/- 12%). The conclusion is that grisorixin decreases both the intracellular concentration of 201Tl and the fatty-acids oxidation in cultured myocardial cells. The beneficial effects previously observed in vivo were probably the consequence of the strong coronary dilatation and of an indirect stimulation.

Comparison of verapamil and bepridil, two slow channel inhibitors, in protection against calcium-induced arrhythmias.

Verapamil and bepridil share the common property of antagonizing the slow inward calcium-mediated current, but bepridil has some additional antiarrhythmic properties. The efficacy of these two compounds against CaCl2-induced arrhythmias has been compared in rats. CaCl2 was administered i.v. by continuous infusion until death (25 mg X kg-1 X min-1 or 40 mg X kg-1 X min-1) or by bolus injection (160 mg X kg-1). Bepridil (5, 10 mg X kg-1) or verapamil (2.5, 5 mg X kg-1) were injected 10 min before CaCl2. Bepridil (10 mg X kg-1) or verapamil (5 mg X kg-1) prolong the survival time during CaCl2 infusion. After pretreatment, the injection of 160 mg X kg-1 CaCl2 is less toxic: 25% of animals are protected by bepridil (5 mg X kg-1), 41% by bepridil (10 mg X kg-1) or verapamil (5 mg X kg-1). At death the myocardial Ca2+ level is not different in controls and pretreated animals, thus, the ratio myocardial Ca2+/total injected Ca2+ is significantly lowered by bepridil (10 mg X kg-1) or verapamil (5 mg X kg-1). The efficacy of the two drugs on this model appears related solely to inhibition of slow inward current despite the additional antiarrhythmic profile of bepridil.

Synthesis, radiolabeling, and preliminary evaluation in mice of some (N-diethylaminoethyl)-4-iodobenzamide derivatives as melanoma imaging agents.

N-(2-Diethylaminoethyl)-4-iodobenzamide (BZA) is a radiopharmaceutical recently developed in our laboratory for the scintigraphic detection of melanoma and metastases. Optimal time for imaging was between 18-24 h p.i. of [123I] BZA. With a view to selecting compounds able to provide quality images shortly after the injection, synthesis of an initial series of BZA derivatives and their evaluation in B16 melanoma bearing mice have been carried out. The [125I] radiolabeled products were obtained by a simple isotopic exchange procedure with high radiochemical yields (85-95%). After i.v. administration of the compounds we observed a good tumoral targeting ability. Tumoral activity peaked at 2.6 to 7.70% injected dose per g within 1 h post-injection. One of the benzamides with a blood clearance faster than that of BZA--0.06 vs. 0.2% I D/g--6 h p.i. gave the same tumor to blood and to organ ratios as BZA at 12-18 h p.i. Based on these preclinical data we hope to obtain good tumoral images 6 h p.i. in scintigraphic studies in man.

Synthesis, characterization and comparative biodistribution study of a new series of p-iodine-125 benzamides as potential melanoma imaging agents.

Iodobenzamides are reported to possess some affinity for melanoma. In order to identify the compound having the most appropriate pharmacokinetic properties as a potential melanoma imaging agent, thirteen new [125I]radioiodobenzamides with a butylene amide-amine spacer and various substituents on the terminal amino group were investigated. Their synthesis, radioiodination and biodistribution in B16 melanoma bearing C57BL6 mice are described and compared to [125I] labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), our reference compound. Changes in the terminal amino constituents induced modifications of lipophilicity, tumor uptake and organ distribution. The dimethylaminobutyl iodobenzamide appeared to be the most promising radiopharmaceutical imaging agent for the detection of melanoma and its metastases.

Melanin affinity of N-(2-diethylaminoethyl)-4-iodobenzamide, an effective melanoma imaging agent.

The cellular uptake and incorporation in macromolecules of iodine-125 labelled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), a melanoma imaging agent, was studied using human melanoma cells M3Dau (amelanotic) and M4Beu (melanotic). The interaction between [125I]BZA and synthetic melanin was examined in various conditions of incubation. The results showed that uptake was high only for M4Beu, whereas the incorporation in trichloroacetic acid-precipitable proteins was very low for both model cell lines, with no correlation with melanin content. Experiments with synthetic melanin showed that BZA binding to melanin was saturable and reversible, and involved several types of interaction. The influence of the ionic environment indicated that electrostatic forces play a role in the affinity, and the decrease in binding produced by the presence of an alcohol in the medium suggested that hydrophobic interactions may be involved in the binding mechanism. This was supported by the Scatchard analysis, which revealed two classes of binding sites, and the determination of two association constants (K1 = 3.9 +/- 1.9 x 106/M and K2 = 2.9 +/- 0.9 x 104/M). The affinity of BZA for melanin might explain the good results obtained in a phase II clinical trial for the diagnosis of malignant melanoma metastases, in which the specificity was 100%.

Establishment of IPC 227 cells as human xenografts in rabbits: a model of uveal melanoma.

This study was designed in order to evaluate the feasibility of establishing an animal model of human uveal melanoma. IPC227, a cell line established from the biopsy of a patient with a spindle cell ciliary body melanoma, was transplanted into the anterior chamber of the eye in immunosuppressed New Zealand rabbits. In a second step, a tumour fragment from the anterior chamber was implanted transclerally into the posterior choroid. Complete ophthalmological examinations were then performed on the animals. Characteristic growth patterns were noted depending on the location of implantation. In the anterior chamber, diffuse, flat, heavily pigmented tumours appeared 8 days after the injection of the cell suspension that covered the iris and the angle by day 25, with a success rate of 100%. Nodular, lightly pigmented tumours were obtained 6-7 weeks after subchoroidal implantation, with a 25% success rate. Clinical examination, including fundus photography, ultrasound and magnetic resonance imaging, demonstrated the same characteristics as those of human uveal melanoma, confirming the value of this model for the evaluation of new therapeutic and diagnostic methods in the management of uveal melanoma.

Effects of MDR reversing agent combinations on the 3H-daunomycin accumulation in drug-sensitive and drug-resistant human cancer cells.

BACKGROUND: As multidrug resistant (MDR) tumour cells generally exhibit a drug accumulation deficit, the effects of three prototype modulators and their combinations were investigated by studying the modulation of 3H-dounomycin cellular accumulation. MATERIALS AND METHODS: Two cell lines derived from a rhino-pharingeal human carcinoma, either sensitive (KB-3-1) or selected as MDR (KB-A1) were used. Verapamil (10mumol.L-1), PSC 833 (lmumol.L-1) and S9788 (5mumol.L-1) were tested alone or in association two by two. The cells were characterized by reverse transcriptase polymerase chain reaction (RT-PCR) in terms of pleiotropic resistance gene expression. RESULTS: A strong mdr1 and a light LRP gene expression were found in KB-A1 resistant cells compared to KB-3-1, whereas MRP expression was found to a similar extent. Relative to the KB-3-1, cells, accumulation of 3H-daunomycin was reduced to 31 +/- 5% in the KB-A1 cells. In these KB-A1 cells, the three agents tested significantly increased the 3H-daunomycin intracellular concentration, S9788 being the most active (311 +/- 37%) and inducing a near complete reversion to the basal level of the sensitive cells. Verapamil and PSC 833 demonstrated an additive effect (252 +/- 69% compared to 188 +/- 33% and 126 +/- 27%, respectively). On KB-3-1 sensitive cells, S9788 had no effect, while verapamil or PSC 833 moderately increased the 3H-daunomycin accumulation, without additive effect. CONCLUSION: These results show a strong MDR reversing effect of S9788, which appears specific to P-glycoprotein (Pgp) and an additive effect between verapamil and PSC 833, suggesting a better therapeutic efficiency if used in well defined combinations.

Relationship between 201Tl uptake and oxidative metabolism in cultured myocardial cells.

The uptake of thallium-201 (201Tl) by myocardial cells in cultures was assessed in the presence of 10(-3) M potassium cyanide (KCN), an inhibitor of mitochondrial respiration, and 10(-4) M dinitrophenol (DNP), an uncoupler of oxidative phosphorylation. The cultures were incubated with 14C-glucose or 14C-octanoate, allowing the measurement of the oxidative metabolism and beta-oxidation from the production of 14CO2. The results demonstrated a moderate decrease in the ratio between the intra/extracellular concentration of 201Tl (Tl i/e) in the presence of KCN (28.9 +/- 8.1 versus 35.6 +/- 9.7 in the controls, n.s.) and no change with DNP (37.6 +/- 9.7). Glycolysis and fatty acid oxidation were lowered with KCN (-28 +/- 15 and -45 +/- 22% respectively, p less than 0.05 in both cases) and were non significantly increased with DNP (+37 +/- 23 and +10 +/- 52% respectively). These results show that 201Tl intracellular uptake is not related directly, but is not totally independent of glycolysis and fatty acid oxidation.

[Phase 2 clinical study of 123I-N-(2-diethylaminoethyl)-2-iodobenzamide in the diagnostic of primary and metastatic ocular melanoma]. / Etude clinique en phase 2 de la BZA2 (123IN-(2-diéthylaminoéthyl)-2-iodobenzamide) dans le diagnostic du mélanome malin oculaire et de ses métastases.

PURPOSE: Iodobenzamides are reported to possess an affinity for melanoma. A first selected compound, BZA, was studied in a phase 2 clinical trial on 159 patients as an imaging agent for the detection of primary melanoma and metastases with good results. We report the results of a second phase 2 clinical trial on 40 patients with a new radiopharmaceutical BZA2 (an orthoiodinated BZA analog), which was expected to provide quality images sooner after injection and with better imaging contrast. PATIENTS AND METHODS: Performance was evaluated in 40 patients classified with primary ocular lesions (12), suspicion of metastases of ocular or cutaneous origin (15), or with no known secondary lesion (13), and results were compared with conventional investigation techniques (ophthalmoscopy, ultrasonography, and angiography for ocular melanoma, whole-body CT scan and ultrasonography for metastases). RESULTS: No adverse events were recorded. The overall results on a per patient basis showed a sensitivity of 78% and a specificity of 95%. The four false negatives observed were ocular lesions (three with a thickness<3mm and one achromic), but all the proven secondary lesions were imaged. Moreover, negative BZA2 scintigraphy in cases of suspicious lesions led to the correction of two diagnoses: the prostatic origin of bone metastases and the endocrine tumor origin (APUD system) of an ocular lesion. DISCUSSION: BZA2 scintigraphy is an easy test with good tolerance. In the diagnosis of ocular primary melanoma, the sensitivity of the test is 64%, although limited by the thickness (3mm) and the pigmentation of the lesion. However, the BZA2 scintigraphy is a very useful test for the detection of melanoma metastases, with a sensitivity of 100% and a specificity of 95%. CONCLUSION: BZA2 scintigraphy showed good tolerance in patients and it appears promising for differential diagnosis, staging, and restaging of melanoma.

Internal dosimetry through GATE simulations of preclinical radiotherapy using a melanin-targeting ligand.

The GATE Monte Carlo simulation platform based on the Geant4 toolkit is under constant improvement for dosimetric calculations. In this study, we explore its use for the dosimetry of the preclinical targeted radiotherapy of melanoma using a new specific melanin-targeting radiotracer labeled with iodine 131. Calculated absorbed fractions and S values for spheres and murine models (digital and CT-scan-based mouse phantoms) are compared between GATE and EGSnrc Monte Carlo codes considering monoenergetic electrons and the detailed energy spectrum of iodine 131. The behavior of Geant4 standard and low energy models is also tested. Following the different authors' guidelines concerning the parameterization of electron physics models, this study demonstrates an agreement of 1.2% and 1.5% with EGSnrc, respectively, for the calculation of S values for small spheres and mouse phantoms. S values calculated with GATE are then used to compute the dose distribution in organs of interest using the activity distribution in mouse phantoms. This study gives the dosimetric data required for the translation of the new treatment to the clinic.

Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution.

Targeted internal radionuclide therapy (TRT) could be an efficient, specific way to treat disseminated melanoma. Based on a previous pharmacomodulation study, we selected a quinoxaline-derived molecule (ICF01012) for its melanin specificity and kinetic properties suitable for TRT. Here, we determined the efficacy of [(131)I]ICF01012 radiotherapy in vitro and in vivo in relation to melanogenesis using human melanoma models. [(125)I]ICF01012 uptake was first assessed in relation to melanin content. We found that melanin distribution in different models was representative of pathology seen in human tumours: melanin content was high in the extracellular space of SKMel3 tumours, and accumulated primarily in melanophages in M4Beu tumours. Targeted [(131)I]ICF01012 radiotherapy had a strong anti-tumoural efficacy in pigmented versus unpigmented tumours, regardless of target distribution and content. This study supports the use of melanin targeting with (131)I-labelled iodoquinoxaline for effective treatment of melanoma.