Biomedical subjects
Publications and source records attributed to A Trouet.
A spirolactone with anti-tumor activity is formed during the reaction of paraformaldehyde with 17-deacetylvinblastine. The compound was identified by NMR techniques. The chemotherapeutic activity was assessed.
We have examined the effects of acidic pH, in the range of those prevailing within phagosomes and lysosomes, on the growth and the susceptibility to different antibiotics of several strains of Salmonella spp. The minimal inhibitory concentration and the minimal bactericidal concentration of several beta-lactams were increased considerably during culture at pH 5.2. The extent of the increase was a function of: (1) the beta-lactam structure and, more particularly, the hydrophobicity of the side-chain of the molecule; and (2) the bacterial serotype. This phenotypic resistance at acid pH was not due to beta-lactamase activity or to a lower growth rate. In contrast, rifamycin SV was more active at acidic pH than at neutral pH and chloramphenicol, another highly hydrophobic drug, was equally efficacious at both pH values. Membrane lipopolysaccharide mutants, but not porin mutants, cultivated at an acidic pH were inhibited by lower concentrations of the beta-lactams. This suggests that the increased resistance to beta-lactams, and the increased susceptibility to rifamycin SV, at acidic pH, could have resulted from modified permeability of the outer membrane to antibiotics.
The murine monoclonal antibody MA-15C5 binds to cross-linked fibrin but not to fibrinogen. Fab or F(ab')2 fragments of MA-15C5, labeled with iodine-123, were injected intravenously into nine rabbits with a nonocclusive 0.2-mL human plasma clot in the jugular vein and into eight control rabbits. Scintigrams obtained at hourly intervals after injection of the F(ab')2 fragment revealed a significantly higher accumulation of tracer in the clot region than in the contralateral region. The relative excess of tracer in the jugular vein region was significantly greater in the study animals than in the control animals throughout the study period. Blind reading of serial scans revealed no visible tracer accumulation in seven of eight control rabbits, whereas the scans of seven of nine study rabbits showed clearly visible accumulation (sensitivity = 77%, specificity = 87%). The Fab fragment of MA-15C5 had a lower affinity and bound less avidly to the blood clot in vivo. These results suggest that the F(ab')2 fragment has better imaging properties than the Fab fragment and that it may be useful for imaging blood clots in man.
The aim of this study was to analyze whether a monoclonal antibody to human milk fat globule membrane-associated antigens, recognized specifically and homogeneously by human breast carcinoma cells but also by normal epithelial cells active in secretion, could be used to restrict the access of antitumoral drugs to cells exposing the epitope. The drug-antibody conjugate to be used is constructed by means of a covalent peptidic linkage stable in extracellular medium but hydrolyzed by lysomal enzymes after endocytosis of the drug-carrier conjugate. This monoclonal antibody specifically immunoprecipitates radioactive material from MCF-7 cells biosynthetically radiolabeled with galactose, glucosamine, palmitic acid, or acetic acid but not with mannose, leucine, or methionine. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, the label migrates as two bands with apparent molecular weights of about 350,000 and 400,000. These bands disappear, or their molecular weight is affected, after treatment of the cells with cycloheximide or of cell lysates with trypsin, Pronase, or neuraminidase but not treatment of the immunoprecipitate with endoglycosidase F. This suggests that these antigens are glycoproteins with O-linked oligosaccharides containing sialic acid in the epitope. By analogy, they should be similar, if not identical, to those recognized by the monoclonal antibodies designated HMFG1 (H. Burchell, H. Durbin, and J. Taylor-Papadimitriou, J. Immunol., 131:508-513, 1983) and DF3 (H. Sekine, T. Ohno, and D.W. Kufe, J. Immunol., 135:3610-3615, 1985). Binding at 4 degrees C of the 3H-labeled antibody by MCF-7 cells indicates the specific attachment of about 1.2 X 10(6) IgG molecules per cells with a Kd of about 14 nM. At 37 degrees C, cells take up the 3H-labeled antibody in amounts much higher than the binding capacity. In addition to cell-associated material, labeled digestion products are released into the culture medium. Cell fractionation by differential centrifugation and isopycnic equilibration on sucrose gradient indicates that the bulk of cell-associated antibody is distributed like the marker enzyme of lysosomes. Although the total uptake of the antibody by the cells is unaffected by either 50 microM chloroquine or 3 micrograms/ml cycloheximide, the release of digestion products is completely inhibited by chloroquine. Antigen-antibody dissociation is pH dependent, since, respectively, 50 and 84% of membrane-bound antibody are released during washing at pH 4.6 and 4.1.(ABSTRACT TRUNCATED AT 400 WORDS)
When incubated with 3H-astemizole, a potent antagonist of H1 receptor, cultured rat hepatocytes, which do not express specific receptors for this ligand, avidly take up 3H-label proportionally to the drug concentration. HPLC analysis indicates that at 10 ng 3H-astemizole/ml, cells almost entirely deplete the culture medium of the drug within 4 hr of incubation. At 37 degrees, astemizole is metabolized and released into the culture medium mainly under the form of glucuronoconjugated metabolites. Differential centrifugation of homogenates from hepatocytes incubated with 3H-astemizole indicates that astemizole and unconjugated metabolites are found in the particulate fraction, whereas astemizole and conjugated metabolites are present in the cytosol. Isopycnic centrifugation on sucrose gradient shows that the major part of the 3H-label in the particulate fraction distributes like phospholipids and NADPH cytochrome c reductase, suggesting an association with membranes and, in particular, with the endoplasmic reticulum. Chloroquine, a drug accumulating within lysosomes and acidic endosomes, decreases the uptake of 3H-astemizole by hepatocytes and induces, during isopycnic centrifugation of a particulate fraction, a shift of the 3H-label towards lower densities where it closely accompanies cathepsin B. This suggests that a minor part of astemizole accumulated in the hepatocytes could be trapped within lysosomes. These results could support the hypothesis that aspecific binding of astemizole to cellular membranes and, to a lesser extent, trapping in lysosomes could play a role in the pharmacokinetics of the drug.
Cultured neurons from embryonic rat brain display central type benzodiazepine receptors characterized by high-affinity binding of [3H]flunitrazepam which is allosterically enhanced in the presence of gamma-aminobutyric acid (GABA). A 48 h treatment of the cultured neurons with 1 microM diazepam, 0.1 microM clonazepam or 0.1 microM beta-carboline ester derivatives did not change either Bmax or KD values of the [3H]flunitrazepam specific binding. A 48 h incubation in the presence of GABA (1 mM) or muscimol (0.1 mM) induced a 30% decrease of the Bmax value of [3H]flunitrazepam specific binding without change of the KD value. The down-regulation was dependent on GABA concentrations and temperature, and was partially inhibited by bicuculline but not by the benzodiazepine antagonist Ro 15-1788. The other subunits of the benzodiazepine-GABA-chloride channel receptor complex also seemed to be down-regulated by GABA since there was a decrease of the specific binding of [3H]muscimol and [35S]t-butylbicyclophosphorothionate (TBPS) to the GABAA and chloride channel sites respectively. The GABA-induced down-regulation of the GABA-benzodiazepine receptor seems to be selective since the specific binding of ligands to other receptors was not affected. Our results suggests that activation of the low-affinity GABA subunit which is involved in cellular electrophysiological responses, induced the receptor down-regulation.
DNR can be targeted to the liver by linking to galactosylated human serum albumin (AG). The linkage is stable in the blood stream and allows the release of active DNR after endocytosis of the conjugate by the target cells. Receptors for AG are present at the cell membrane of hepatocytes, primary human hepatoma cells and lung metastases. After i.v. administration of AG-DNR more than 70% of the drug is taken up by the liver and in rats 50% of DNR is eliminated in the bile after 16 h. Nude mice bearing human hepatoblastoma cells implanted s.c. were treated twice a week with a dose equivalent to 6.6 mg/kg of DNR either for the free drug or the conjugate AG-DNR. After 7 injections, tumor growth is inhibited in both case, however, the conjugate seems more active and is at least twice less toxic in terms of LD50. A phase I clinical trial of AG-DNR on 11 patients bearing hepatocarcinoma revealed that despite a transitory hyperthermia (37-38 degrees C) during the first day of the 4 day-perfusion, and modifications of hepatic enzymes in 3 cirrhotic patients, no hematologic and cardiac toxicity could be detected. A subjective response has been obtained in half of the patients with a decrease of plasmatic alpha-foetoprotein levels by more than 50% in 4 patients and one complete remission of more than 23 months with disparition of pulmonary metastases.
The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13-1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex. However, after treatment with low concentrations of digitonin the 3H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterol-rich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 degrees C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles. On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.
Vinblastine tryptophan ester (VTrpE) is a new vinca alkaloid derivative that achieves antitumor activity in a large variety of animal models. In this phase I study the drug was given as an i.v. injection over 5 min, once a week or once every 2 weeks. Twenty patients with advanced cancer were entered in this trial. The doses ranged from 2.5 mg/m2 to 35 mg/m2. Myelosuppression is the dose-limiting toxicity, with the risk of leukopenia being more serious than that of thrombocytopenia, but the myelosuppression is always reversible. Neurotoxicity, well documented when other vinca alkaloid derivatives are used, is insignificant. Two cases of disease stabilization have been observed in patients with non-small cell lung cancer. For VTrpE, a dose schedule of 30 mg/m2 per week may be recommended for phase II studies in non-small cell lung cancer.
The stability of doxorubicin (DOX) and daunorubicin (DNR) in rabbit and human plasma, bile and urine and in rabbit faeces was studied in the presence or absence of light, and at body, room and cold room temperatures. Fluorescence was determined by spectrofluorimetry after normal and reversed phase HPLC. Under each set of conditions, DOX and DNR fluorescence decreased with time; the decrease was more rapid with DOX. As the parent drugs were degraded, apolar compounds were formed which behaved like 7-deoxyaglycones and generally did not compensate for the loss in fluorescence of the parent drug. The degradation of anthracyclines occurred even in the absence of light, was not due to bacterial contamination and was faster at higher pH or temperature. The rapid degradation of DOX and DNR in biological fluids at body temperature may have implications on the disposition of anthracyclines in vivo. Prior to analysis, biological fluids and solutions containing anthracyclines should be processed quickly at 4 degrees C, in the absence of light, and at a pH no greater than 6 to avoid degradation.
Cortex from rat, dog, and human brain was submitted to subcellular fractionation using an analytical approach consisting of a two-step procedure. First, fractions were obtained by differential centrifugation and were analyzed for their content of serotonin S2 and muscarinic receptors, serotonin uptake, and marker enzymes. Second, the cytoplasmic extracts were subfractionated by equilibration in sucrose density gradient. In human brain, serotonin and muscarinic receptors were found associated mostly with mitochondrial fractions which contain synaptosomes, whereas in rat brain they were concentrated mainly in the microsomal fractions. Density gradient centrifugation confirmed a more marked synaptosomal localization of receptors in human than in rat brain, the dog displaying an intermediate profile. In human brain, indeed, more receptor sites were found to be associated with the second peak characterized in electron microscopy by the largest number of nerve terminals. In addition, synaptosomes from human brain are denser than those from rat brain and some marker enzymes reveal different subcellular distribution in the three species. These data indicate that more receptors are of synaptosomal nature in human brain than in other species and this finding is compatible with a larger amount of synaptic contacts in human brain.
Two radiolabelled derivatives of deferrioxamine B (DF) have been synthesized: methyl-DF and acetyl-DF. Both derivatives are non cytotoxic and stable in cell culture but they are degraded in human plasma and more extensively in rat plasma. Methyl-DF, acetyl-DF and DF mobilize radioiron to the same extent from hepatocytes loaded with 59Fe citrate in the same range of extracellular concentrations. The uptake and release of the 3H-labelled derivatives and their corresponding iron complexes have been measured and appear to represent a passive phenomenon resulting from the gradient of concentration between the cellular compartment and the extracellular medium. The results indicate that only a limited pool of cellular iron is accessible for chelation and that neither the permeability of the cellular membrane, nor the intracellular concentration of the chelators are the limiting factors for iron mobilization. On the basis of the subcellular distribution of the 3H-DF analogues, methylamine inhibition of iron chelation by siderophores in cell cultures and the positive effect of acidic pH and hydrolysis by lysosomal enzymes on in vitro iron mobilization from radiolabelled ferritin, we suggest that iron mobilization by DF and its derivatives occurs in lysosomes where they complex iron released from ferritin under the conjugate actions of acidic pH and lysosomal enzymes.
Nine children with poor-prognosis malignancies--seven with advanced neuroblastoma and two with metastatic Ewing's sarcoma--were given high doses of melphalan (HDM), 150 mg/m2 (3 patients) and 180 mg/m2 (6 patients), as a 'late intensification' agent combined with noncryopreserved autologous bone marrow transplants. Melphalan levels in the plasma decreased biphasically, with mean half-lives of 6.6 min and 3.0 h. At the time of marrow reinfusion (12-21 h after HDM) the melphalan plasma level was generally below 0.1 microgram/ml. The renal contribution to melphalan clearance was low, a mean of 5.8% of the injected dose being found in patients' urine over the 12 h following HDM administration. No significant difference was seen in pharmacokinetic parameters between patients undergoing and not undergoing forced diuresis.
The dimeric alkaloids vinblastine (VLB) and vincristine (VCR) differ structurally only in the functional group on the dihydroindole nitrogen. The semisynthetic derivative vindesine (VDS) differs slightly from VLB by having an amide group instead of an ester group. However, these minor distinctions are responsible for profound differences in the oncolytic spectrum, potency, and toxicity of these compounds. Vinblastin-23-oyl amino acid derivatives were synthesized by linking amino acid carboxylic esters to the vinblastin-23-oyl moiety through an amide linkage. Studies were extended to explore the influence of the nature of the amino acid, the ester alkyl chain lengths, the stereoisomerism of the amino acid, or the reacetylation of the hydroxyl group (position O-4) of the vindoline moiety. The present study deals with the synthesis of 21 vinblastin-23-oyl amino acid derivatives, some of their physicochemical data, the acute toxicity in mice, and therapeutic activities of these derivatives against the P388 and L1210 leukemias in comparison with VDS, VBL, and VCR.
The resistance of cultured mouse neuroblastoma cells, primary cultures of rat cerebellar neurons, and rat brain astrocytes to a block of aerobic metabolism was studied. Parameters such as lactate production and ATP content were measured in the presence of antimycin A and under various conditions of glucose, oxygen, and serum supply. The following conclusions can be drawn: (1) All cell types studied were characterized by an active production of lactate; (2) Incubation of the various cell types in the absence of glucose at normal oxygen tension did not affect ATP levels; (3) Respiration blocked by antimycin led to a Pasteur effect; (4) Neuroblastoma cells, but not the other cell types, were fully resistant to inhibition of respiration provided that sufficient glucose was supplied; (5) In the absence of glucose no stores of energy or utilizable substrate were present in the cell types studied when respiration was blocked; (6) In the presence of fetal calf serum anoxic neurons showed irreversible signs of degeneration.
Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells.
The stimulation of glycolysis by glucose and anoxia has been investigated in cultivated rat cerebellar neurons, mice neuroblastoma cells and rat brain astrocytes. It is shown that fructose 2,6-bisphosphate exerts no predominant role in: (1) the stimulation of glycolysis by glucose, and (2) the onset of anaerobic glycolysis.
Detorubicin (DET), a semi-synthetic analog of daunorubicin, releases at neutral pH doxorubicin (DOX) upon hydrolysis. DET enters faster than DOX into the cultured L1210 cells and reaches higher intracellular levels. When the cells are incubated for 120 min at pH 6.5, in spite of its rapid hydrolysis, one third of the intracellular fluorescence was due to undegraded DET. DET, like the other anthracyclines studied, is found associated intracellularly only to the lysosomes and to the nuclei. Unchanged DET is found mainly inside the lysosomes where the drug is stabilized, while DOX is found essentially associated to the nuclear DNA of L1210 cells. DET can therefore be viewed as an hydrophobic prodrug of DOX characterized, however, by a distinct subcellular localization in L1210 cells.