Molecular identification of iridoviruses infecting various sturgeon species in Europe.

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low-to-high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74-88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus-European (AcIV-E). Moreover, three samples infected with AcIV-E showed genetic heterogeneity, with the co-existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV-E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV-E and an NV-related virus.

Stereoselective determination of verapamil and norverapamil by capillary electrophoresis.

An analytical method has been developed to determine simultaneously the verapamil and norverapamil enantiomers in human plasma using capillary electrophoresis. Among the cyclodextrins tested as chiral selector, only trimethyl-beta-cyclodextrin was suitable to resolve the four enantiomers. The analysis was achieved in less than 10 min. Selectivity, linearity, precision and accuracy were evaluated before the chiral method was successfully implemented for routine use to simultaneously determine the four enantiomers in several thousands of human plasma samples. The robustness of the capillary electrophoretic method and its suitability were demonstrated by the coefficients of variation which were lower than 11%, even at the limit of quantification (2.5 ng/ml), for the analysis of more than one hundred quality control samples.

Comparative metabolism of SC-42867 and SC-51089, two PGE2 antagonists, in rat and human hepatocyte cultures.

1. The metabolism of SC-42867 and SC-51089, two PGE2 antagonists, was studied in cultured rat and human hepatocytes. Both compounds possess an 8-chlorodibenzoxazepine moiety, but differ from each other by the nature of the side chain connected to the nitrogen atom. SC-42867 and SC-51089 and their in vitro metabolites were separated by reversed-phase hplc. The major metabolites of both compounds were identified by mass spectrometry (ms) analysis. 2. SC-42867 was metabolized on the tricyclic moiety only. Oxidative N-dealkylation with opening of the oxazepine ring was the major metabolic pathway obtained in rat hepatocytes. The metabolic profile obtained in cultured human hepatocytes was comparable with that of cultured rat hepatocytes. However, the compound was metabolized to a much lower extent by the human cells. 3. SC-51089 was extensively metabolized by both cultured rat and human hepatocytes. Human cells metabolized this compound quite differently than cultured rat hepatocytes. Aromatic hydroxylation with consequent glucuronidation and sulphation were the main metabolic pathways observed in cultured human hepatocytes. Oxidative N-dealkylation with opening of the oxazepine ring and consequent glucuronidation was the major metabolic pathway observed in rat hepatocytes. Further metabolism occurred, in contrast with the human hepatocytes, mainly on the side chain. 4. The present in vitro results are compared with data of previous in vivo studies performed in rat.

Irreversible binding to proteins after single and repeated daily oral administration of 4-(2,2-diphenylethyl) imidazole (SC-46264) to the Cynomolgus monkey.

SC-46264 is an antagonist of the alpha 2-adrenergic receptor. Distribution and excretion of [14C]-SC-46264 were studied after single and repeated daily oral administrations to the Cynomolgus monkey at a 1.5 mg/kg dose. After a single oral administration, more than 95% of the administered dose was recovered within 48 h in the urine (+/- 60%) and faeces (+/- 40%). Approximately 1.7% remained in the gastro-intestinal (GI) tract and 2% in the animal body. However, the radioactivity remaining in the animal body decreased very slowly from 2 to 1% between 48 and 144 h. An accumulation of very small amounts of radioactivity could be suspected in the plasma, the liver, the thyroid, the adrenals and the kidneys. In a 2 week daily oral administration of [14C]-SC-46264, the amount of total radioactivity remaining in the animal body 24, 48 and 216 h after the last administration was approximately 21, 11 and 5% of the daily administered dose, respectively. It confirmed the accumulation of [14C]-SC-46264 related compound in the plasma, the liver, the thyroid, the adrenals and the kidneys. The minimum plasma concentrations of total radioactivity observed before each administration increased during the treatment and apparently did not yet reach an equilibrium after 14 days. In these plasma samples obtained throughout the study, an increasing fraction of the total radioactivity could not be extracted and was recovered with precipitable material. These observations lead to the hypothesis of an irreversible binding of some material to the proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the pharmacokinetics and pharmacodynamics of torasemide and furosemide in healthy volunteers.

The pharmacodynamic effects and the pharmacokinetic parameters of torasemide (1-isopropyl-3- ([4-(3-methyl-phenylamino)pyridine]-3-sulfonyl)urea) 20 mg and furosemide 40 mg were compared after oral and intravenous administration in 6 healthy volunteers. The plasma elimination half-life for i.v. and oral torasemide was 2.2 h and 2.8 h, its bioavailability after oral administration was 91%, about 25% of the total body clearance was due to renal excretion both after iv. or oral application. For furosemide, a plasma elimination half-life of 0.6 h for i.v. and 0.8 h for oral application was found. The bioavailability was 40%, and about 62% of the drug was excreted via the kidney. Both drugs produced a similar diuretic and natriuretic effect. However, torasemide showed an increased duration of action compared to furosemide and a higher relation between urinary Na and K excretion, both after i.v. and oral administration, suggesting less loss of potassium after To. Both agents were well tolerated.

The quantification of gamma-aminobutyric acid in the cerebrospinal fluid by a radioreceptorassay.

The development of a radioreceptor assay designed to measure gamma-aminobutyric acid (GABA) in CSF is described. The method is based on the presence of high affinity and selective GABA binding sites obtained from rat brain membrane preparations treated with 0.05% Triton X-100. The optimum protein concentration in the incubation medium, the duration of incubation to reach equilibrium, the temperature and the optimum pH are discussed. The standard curve permits measurements in the range 35 to 2250 nmol/l GABA. The imprecision of the method calculated from three different concentrations: 1125, 562 and 281 nmol/l shows coefficients of variation for 'within' and 'between' assays, between 5.2% and 9.3% and between 7.4% and 12.4%, respectively. The percentage of recovery is 102 +/- 3.3% (n = 4). This radioreceptorassay has a sensitivity of 14 nmol/l. The method is easy, rapid and not expensive. It enables analysis of small volumes of CSF (200 microliters). Several samples (greater than 20) can be analysed in the same run in about one hour.

Benzodiazepine receptors are involved in tabernanthine-induced tremor: in vitro and in vivo evidence.

Tabernanthine, an indol alkaloid, is structurally related to carbolines (harmane, harmaline) which, in vitro, displace specific flunitrazepam binding to brain benzodiazepine receptors. In vivo, both tabernanthine and carbolines cause a fine general tremor, suggesting that a possible interaction with benzodiazepine receptors could be involved in the activity of tabernanthine. This hypothesis was validated by the in vitro and in vivo antagonism of benzodiazepine by tabernanthine. In vitro, tabernanthine inhibited specific flunitrazepam binding in a competitive manner with an affinity (IC50 150 microM) in the same range as harmane. Tabernanthine appeared as a benzodiazepine receptor inverse agonist in a discriminant in vitro binding assay. In vivo, the time course of tremorigenic activity was related to the tabernanthine concentration in brain (half-life = 2 h). Moreover, tabernanthine-induced tremor was inhibited reversibly by flunitrazepam or by Ro-15 1788 (an antagonist of benzodiazepine-receptors). These results suggest that part of the action of tabernanthine may be mediated by an interaction at the benzodiazepine receptor level.

New automated high-performance liquid chromatographic analysis of cyclosporin A and G in human serum.

An automated isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of cyclosporin A and G in human serum. This method involves the use of an automated solid-liquid extraction procedure following rapid protein precipitation with acetonitrile. The use of a disposable C8 extraction cartridge allows a good recovery of cyclosporine (87%) from serum and a detection limit of 20 ng/ml with good reproducibility using 0.5 ml of sample. This method can also be adapted to whole blood measurements. The choice of a 3-micron cyano analytical column and of the mobile phase hexane-isopropanol (85:15) permitted a low column temperature (50 degrees C), a low flow-rate (0.6 ml/min) and a short run time (14 min). This method allows the accurate and fast routine monitoring of cyclosporine by HPLC, which is particularly important in hepatic transplantations.

Interaction of carbolines and some GABA receptor ligands with the GABA and the benzodiazepine receptors.

The binding of different drugs were investigated on benzodiazepines and GABA receptors using P2 fraction or 0.05% Triton X-100 treated membrane preparation (TX-100 P) obtained from rat's CNS. Bicuculline and picrotoxin have the ability to modulate the specific 3H-flunitrazepam binding to its receptor present in P2 fraction; this modulation decreases for bicuculline and disappears for picrotoxin when a TX-100 P was used. Bicuculline at the opposite of picrotoxin displaces 3H-GABA binding on TX-100 P. The beta-CCE binds to the brain benzodiazepines receptor with a high affinity (IC50 = 8.5 nM on TX-100 P) and does not inhibit the binding of 3H-GABA on TX-100 P. On the contrary, other related carbolines such as harmine, harmaline, harmane and harmalol have a much lower ability to inhibit flunitrazepam binding (IC50 between 28 and 130 microM with TX-100 P) and can also inhibit 3H-GABA binding (IC50 between 75 and 186 microM with TX-100 P). But the inhibition of 3H-GABA binding by those related carbolines can't be reversed by a benzodiazepine like flunitrazepam or a benzodiazepine antagonist like Ro 15-1788.

Modulation of ligands binding to the benzodiazepine receptor by an endogenous inhibitor (GABA-modulin) and bicuculline.

We have prepared and purified an endogenous protein (brain neuropeptide) by extraction with 0.025 N acetic acid and precipitation with a saturated ammonium sulfate solution followed by column chromatography. This protein was isolated and partially purified (approx. 500 times). When added to Triton X-100 treated synaptic membrane preparation obtained from rat CNS, this modulates specific high affinity GABA binding site without affecting low affinity site. This protein was called GABA-modulin (GM). GM like bicuculline (competitive GABA antagonist) modulates the affinity of benzodiazepine receptor ligands. But the effect of bicuculline is more important than that of GM. Bicuculline increases or decreases respectively the binding of 3H-beta-CCM or 3H-flunitrazepam by approximately 60%. In the same conditions, at a concentration of 12 micrograms/ml GM increases or decreases respectively the binding of 3H-beta-CCM or 3H-flunitrazepam by approximately 35% and no more modulation was obtained even the concentration of GM was increased. Bicuculline and GM doesn't modify the specific binding of 3H-Ro 15-1788 to the BZD receptor. It is concluded that GM exhibit a lower ability than bicuculline to modulate the binding of the BZD receptor ligands because GM interacts only with the GABA high affinity binding site without affecting the low affinity binding site. These observations suggest that the two GABA binding sites probably interact with the BZD receptor.

Selective affinity of one enantiomer of suriclone demonstrated by a binding assay with benzodiazepine receptors.

The binding of racemic 3H-suriclone on the BZD receptor was performed in special conditions in order to discriminate between the affinity of the two enantiomers: a rebinding method was used. In the first incubation 40% of 3H-suriclone was specifically bound to the BZD receptor. In the second incubation performed with the supernatant coming from the first incubation, less than 1% of the radiolabelled suriclone was specifically bound to the BZD receptor. These results suggest that most probably only one of the two enantiomers of suriclone may bind the BZD receptor. It appears that this enantiomer has the greatest affinity constant ever found for a BZD receptor agonist (Kd = 20 pM at 37 degrees C in presence of GABA and 70 pM in absence of GABA).

Classification of benzodiazepine receptor agonists, inverse agonists and antagonists using bicuculline in an in vitro test.

The mechanism by which a substance that binds to the benzodiazepine receptor acts as an agonist, an inverse agonist (e.g. methyl-beta-carboline-3-carboxylate (beta-CCM] or an antagonist (e.g. Ro 15-1788) was investigated. For this purpose, we studied the influence of bicuculline, an antagonist of gamma-aminobutyric acid (GABA), on the binding of these substances in crude synaptosomal preparation (P2 fraction) containing high levels of endogenous GABA. Displacement curves were performed, using 3H-flunitrazepam in the absence and in the presence of a high concentration (7.10(-5) M) of bicuculline. The ratios of IC50 values with and without bicuculline were significantly higher than 1 for all benzodiazepine agonists investigated (e.g. 1.91 +/- 0.11 (n = 3) for diazepam), about 1 for Ro 15-1788 (0.94 +/- 0.06 (n = 4)) and lower than 1 for beta-CCE (0.55 +/- 0.05 (n = 4)). Statistically significant differences were also observed among benzodiazepine agonists e.g. between flunitrazepam (a sedative-hypnotic drug) and clonazepam (an anticonvulsant drug) or lorazepam (an anxiolytic drug). These data indicate that the ratios of IC50 values with and without bicuculline might provide the basis for an in vitro, pharmacologically relevant, classification of drugs acting on the benzodiazepine receptor. This procedure does not require extensive washing of the membrane preparation, in contrast to the method in which the ratios of IC50 values were determined with and without addition of GABA.

Doxorubicin and daunorubicin plasmatic, hepatic and renal disposition in the rabbit with or without enterohepatic circulation.

The pharmacokinetics, metabolism and disposition of doxorubicin and daunorubicin were studied for periods up to 100 hr in rabbits with (group II) or without a biliary fistula (groups I and III) and with (group I) or without (groups II and III) ligatured ureters using high-performance liquid chromatography to separate parent drug and metabolites. The plasma decay of doxorubicin and daunorubicin was triexponential. Metabolites appearing in the plasma after doxorubicin and daunorubicin bolus i.v. injection were respectively doxorubicinol and daunorubicinol, the latter being the major compound after daunorubicin injection. The elimination of daunorubicin was faster than that of doxorubicin. No differences in the elimination were observed between the 3 groups. In bile, 21% of the injected dose of doxorubicin were excreted mainly as the parent drug and 60% of the injected dose of daunorubicin were excreted, mainly as daunorubicinol. Enterohepatic circulation did not affect the biliary excretion of both doxorubicin and daunorubicin. Ligature of ureters increased slightly the biliary excretion of doxorubicin. The hepatic clearance of daunorubicin was greater than that of doxorubicin. The total urinary excretion was not different between the II and III groups and amounted to 11.6 and 12.8% of the injected dose of doxorubicin and daunorubicin, respectively. Metabolic ratios of doxorubicinol/doxorubicin and daunorubicinol/daunorubicin were similar in bile and urine.

Pharmacokinetics of daunorubicinol in the rabbit: comparison with daunorubicin.

The pharmacokinetics of daunorubicinol (DOL), the main metabolite of daunorubicin (DNR), was studied in rabbits and compared to that of daunorubicin after an 8 mg/kg dose. High-performance liquid chromatography was used to separate parent drug and metabolites. The plasma disappearance of DNR and DOL was triexponential. DOL was the major species detected in plasma and urine. Both drugs had large volumes of distribution. About 70% of DNR or DOL were bound to plasma proteins and mainly to albumin. Pharmacokinetic parameters of DOL obtained after injection of DOL were different from those calculated for DNR and those calculated for DOL after injection of DNR. The total urinary excretions of DNR or DOL were similar and amounted to 25% of the dose. No conjugates were identified in urine after enzymatic treatment. No fluorescent drug was identified in the feces. Anthracyclines were degraded in vitro in rabbit feces. The rabbit seems to be a good model for the study of anthracycline pharmacokinetics as our results in rabbits after DNR injection were similar to those in human studies.

Site of action of torasemide in man.

The effect of torasemide, a new orally and parenterally active diuretic agent, on the renal mechanisms of dilution and concentration was studied in 6 healthy volunteers. The experimental conditions included water and osmotic diuresis. Torasemide caused maximal chloruresis and natriuresis during the 20-40 min after administration. The effect was more pronounced under osmotic diuresis and persisted throughout the 100 min of those experiments. A distinct effect both on free water clearance (CLH2O) during water diuresis and tubular reabsorption of solute free water (TcH2O) during osmotic diuresis strongly suggests that the major site of action of torasemide is the ascending limb of the loop of Henle. Comparison with furosemide under osmotic diuresis indicates longer abolition of TcH2O/GFR by torasemide in keeping with the fact that its half-life is 2- to 3-times longer than that of furosemide.