Predation by carabid beetles on the invasive slug Arion vulgaris in an agricultural semi-field experiment.

Arion vulgaris Moquin-Tandon 1855 is one of the most important invasive species in Europe, affecting both biodiversity and agriculture. The species is spreading in many parts of Europe, inflicting severe damage to horticultural plants and cultivated crops partly due to a lack of satisfactory and effective management solutions. Molluscicides have traditionally been used to manage slug densities, although the effects are variable and some have severe side-effects on other biota. Thus, there is a need to explore potential alternatives such as biological control. The nematode Phasmarhabditis hermaphrodita is the only biological agent that has been applied commercially so far. However, other biological control agents such as carabid beetles have also been found to be promising. In addition, some carabid species have been shown to feed on A. vulgaris in the field as well as in the laboratory. Two species in particular have been found to be important predators of A. vulgaris, and these species are also common in agricultural environments: Pterostichus melanarius and Carabus nemoralis. This study is the first to use semi-field experiments in a strawberry field, manipulating densities, to investigate how P. melanarius and C. nemoralis affect densities of A. vulgaris eggs and juveniles, respectively. Gut contents of C. nemoralis were analysed using multiplex PCR methods to detect DNA of juvenile slugs. Results show that both P. melanarius and C. nemoralis significantly affect densities of slug eggs and juvenile slugs under semi-field conditions and that C. nemoralis seems to prefer slugs smaller than one gram. Carabus nemoralis seems to be especially promising in reducing densities of A. vulgaris, and future studies should investigate the potential of using this species as a biological control agent.

Broad phase II and pharmacokinetic study of methoxy-morpholino doxorubicin (FCE 23762-MMRDX) in non-small-cell lung cancer, renal cancer and other solid tumour patients.

The aim was to perform a broad phase II and pharmacokinetic study of methoxymorpholino-doxorubicin (MMRDX), a drug active against multidrug-resistant tumour cells in vitro when given by i.v. bolus at 1.5 mg m(-2) every 4 weeks, in metastatic or unresectable solid tumour patients with known intrinsic drug resistance. Patients received a maximum of six cycles. Plasma, urine and leucocyte MMRDX and its 13-dihydro metabolite pharmacokinetic analysis was performed in patients without liver metastases. Patients (n = 48, 21 NSCLC, 19 renal cell, three head and neck tumour, three cervical cancer and two adenocarcinoma of unknown primary) received 132 cycles of MMRDX. Common toxicity criteria (CTC) grade III/IV thrombocytopenia (12% of cycles) and neutropenia (27% of cycles) occurred with median nadir on day 22. Transient transaminases elevation > grade III/IV was observed in 7% of cycles, late and prolonged nausea > or = grade II in 34% and vomiting > or = grade II in 39%. In two patients, the left ventricular ejection fraction was reduced > or = 15%. Of 37 evaluable patients, one out of 17 NSCLC had a partial response. Mean (+/- s.d.) MMRDX AUC0-infinity calculated up to 24 h after dosing was 20.4 +/- 6.2 microg h l(-1) (n = 11) and t(1/2, gamma) was 44.2 h. Mean plasma clearance (+/- s.d.) was 37.2 +/- 7.3 l h(-1) m(-2) and volume of distribution 1982 +/- 64 l m(-2). MMRDX leucocyte levels 2 and 24 h after infusion were 450 to 600-fold higher than corresponding MMRDX plasma levels. In urine, 2% of the MMRDX dose was excreted unchanged, and 2% as metabolite. The main side-effects of 1.5 mg m(-2) every 4 weeks of MMRDX are delayed nausea and vomiting and haematological toxicity. MMRDX is characterized by extensive clearance and rapid and extensive distribution into tissues. A low response rate was observed in patients with tumours with intrinsic chemotherapy resistance.

Determination of turosteride, a new inhibitor of 5 alpha-reductase, in human plasma by high-performance liquid chromatography with ultraviolet detection.

A sensitive and specific HPLC method for the determination of turosteride in human plasma was developed and validated. Turosteride was extracted from plasma with diethyl ether. Further purifications of the fraction extracted were performed sequentially by solid-phase extraction using a CN cartridge and by liquid-liquid partition between n-hexane and acetonitrile. Finally the acetonitrile solution containing the test compound was evaporated to dryness and the residue dissolved in the mobile phase, then injected onto the HPLC system. The chromatographic separation was performed isocratically by a reversed-phase column filled with ODS using a water-acetonitrile-methanol mixture. The eluate was monitored at 210 nm. No peak interfering with that of turosteride was observed when blank human plasma was assayed. Linearity was obtained in the turosteride concentration range of 5-1000 ng/ml plasma. Six calibration curves in plasma prepared and run on six different days showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C.V. = 4.3%). The intra-day precision, evaluated at three concentrations (in the low, mid and high range of the standard curve) and expressed as C.V., ranged from 0.81 to 13.25%. The inter-day precision evaluated at the same concentrations was better than 10.7%. The inter-day accuracy evaluated in the same samples and expressed as the ratio of found/added amount of turosteride ranged from 97.66 to 98.38%. The limit of quantitation was 5 ng/ml plasma. The HPLC method described was successfully employed for the determination of turosteride in plasma samples obtained during a phase I clinical trial with the test compound.

Determination of FCE 26644, a new polysulphonated derivative of distamycin A, in monkey plasma by reversed-phase ion-pair high-performance liquid chromatography with ultraviolet detection.

A sensitive and selective ion-pair high-performance liquid chromatographic method for the determination of 7,7'-carbonylbis[imino-N-methyl-4, 2-pyrrolecarbonylimino(N-methyl-4,2-pyrrole)carbonylimino]¿- bis(1,3-naphthalenedisulphonic acid), tetrasodium salt in monkey plasma has been developed. The compound and internal standard (bromphenol blue) were extracted from plasma samples were methylene chloride (twice) after deproteination with acetonitrile and addition of the ion-pairing agent (tetrabutylammonium hydroxide). The combined organic phases were dried, the residue dissolved in the mobile phase and then analysed by reversed-phase ion-pair liquid chromatography under isocratic conditions. The HPLC analysis time was about 20 min. Quantitation was achieved by UV detection at 323 nm. The linearity, precision and accuracy of the method were evaluated. The limit of quantitation was 0.3 microgram/ml plasma. No interference from blank monkey, mouse, rat, dog and human plasma was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from three male cynomolgus monkeys that had received a 20 mg/kg single i.v. dose of the test compound.

Determination of ethambutol in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective HPLC method for the determination of ethambutol in human plasma and urine was developed. Ethambutol was extracted from basified plasma samples (0.2 ml) with diethyl ether, back-extracted into 0.01 M phosphoric acid and derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. After 30 min at 80 degrees C and elimination of the reactive excess, the compound was determined by reversed-phase liquid chromatography. urine was analysed for ethambutol after dilution 1:200 with distilled water and derivatization as described for plasma. Quantification in plasma and urine was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human plasma and urine was observed. The limit of quantification was 10 ng/ml in plasma and 10 micrograms/ml in urine. The suitability of the method for in vivo samples was checked by analysis of plasma and urine samples drawn from healthy volunteers who had received a 1200-mg oral dose of the test compound.

Determination of tallimustine in human plasma by high-performance liquid chromatography.

A sensitive and reproducible HPLC method for the determination of tallimustine (I) in human plasma has been developed and validated. Compound I was extracted from plasma by solid-phase extraction using a C18 cartridge from which the test compound was eluted with a methanol-formic acid mixture. The methanol solution was evaporated to dryness and the residue dissolved in a 0.2 M formic acid in methanol-water (1:1, v/v) mixture, then injected onto the HPLC column. The chromatographic separation was performed isocratically by a reversed-phase column filled with ODS, using a 50 mM KH2PO4-acetonitrile mixture as the mobile phase. The flow-rate was 1 ml/min. The eluate was monitored at 314 nm. No peak interfering with that of I was observed when blank human plasma was assayed. Linearity was established in the concentration range 0.5-85.5 nanograms of I per millilitre of plasma. Four calibration curves in plasma, prepared and run on four different days, showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C.V. = 4.5%). The intra-day precision, evaluated at three concentrations (in the low, mid and high range of the standard curve) and expressed as C.V. ranged from 0.9 to 14.4%. The inter-day precision evaluated at the same concentrations was better than 10.2%. The inter-day accuracy evaluated in the same samples and expressed as the ratio of found/added amount of I, ranged from 86.2 to 108.5%. The limit of quantitation was 0.5 ng/ml plasma. The HPLC method described here was successfully employed for the determination of I in some plasma samples obtained during a phase I clinical trial with the test compound.

An investigation of the pharmacokinetics and autoinduction of rifabutin metabolism in mice treated with 10 mg/kg/day six times a week for 8 weeks.

Plasma, lung and spleen concentrations of rifabutin have been measured in mice after single and multiple oral administrations of the drug. Rifabutin concentrations in spleen were similar to those measured in plasma, whereas concentrations in lung were higher. No significant autoinduction of rifabutin metabolism was observed.

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection.

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.

Pharmacokinetics, pharmacodynamics, and tolerability of cabergoline, a prolactin-lowering drug, after administration of increasing oral doses (0.5, 1.0, and 1.5 milligrams) in healthy male volunteers.

Cabergoline (CAB) a long-acting dopaminergic ergoline derivative, was given orally, in single doses of 0.5, 1.0, and 1.5 mg, to 12 healthy men in order to evaluate its PRL-lowering effect as well as its plasma pharmacokinetics and urinary excretion. Drug administrations were separated by 5-week washout periods. Blood samples for PRL and CAB determination were taken at baseline and for 840 h thereafter (every 1 h up to 4 h, every 4 h up to 12 h, every 24 h up to 168 h, and weekly up to 5 weeks). Fractional urine collections for CAB excretion were taken immediately before drug administration, every 4 h up to 12 h, and every 12 h up to 168 h. During the study period, blood pressure and heart rate were monitored at the same time periods of plasma sampling for CAB, and electrocardiographic tracings and hematological evaluations were performed before and after each treatment period. All CAB doses (0.5, 1.0, and 1.5 mg) produced in all subjects a complete PRL suppression (PRL < 1.0 micrograms/L), that occurred earlier and persisted longer with the two higher doses. PRL secretion areas [area under the curve (AUC) 0-48 h and 48-840 h] were higher after 0.5-mg than after 1.0- and 1.5-mg doses. In particular, in the first portion of the area, the difference between 0.5 mg and both 1.0 and 1.5 mg was highly statistically significant (P < 0.01) without significant differences between the two highest doses. Mean CAB maximal plasma concentrations (Cmax) were 33.3 +/- 3.69, 40.3 +/- 2.49, and 67.0 +/- 9.79 ng/L after 0.5, 1.0, and 1.5 mg CAB, respectively; time to Cmax was 2 h (median) for all doses; CAB AUC(0-168 h) after 0.5 mg CAB was significantly lower (P < 0.01) than after 1.5 mg CAB. The percentages of the administered doses of CAB excreted in urine were 1.1 +/- 0.1%, 1.1 +/- 0.1%, and 1.2 +/- 0.1% for the 0.5-, 1.0-, and 1.5 mg doses, respectively (P = NS). CAB AUCs(0-168 h) and Cmax normalized to the 1.0-mg dose were compared by two-way analysis of variance; no significant differences were found for CAB AUCs(0-168h); Cmax after 0.5 mg was significantly higher (P < 0.01) than after 1.0 and 1.5 mg CAB. A progressive decrease of systolic and diastolic blood pressure was observed, and symptomatic hypotension after the 1.0-mg dose did not allow one subject to receive the 1.5-mg dose. Other mild to moderate adverse events occurred only after 1.0 and 1.5 mg CAB. These results indicate that, in the dose range of 0.5-1.5 mg, the pharmacokinetics of CAB are dose independent, and that the pharmacodynamic data and the frequencies of adverse events of CAB are dose related, with no significant differences in the PRL-lowering effect of the 1.0- and 1.5-mg doses.

Pharmacodynamics and relative bioavailability of cabergoline tablets vs solution in healthy volunteers.

The effect of formulation on the urinary pharmacokinetics, pharmacodynamics, and relative bioavailability of cabergoline was investigated. Twelve healthy female volunteers, aged 23-35 years, were treated, according to an open, randomized, crossover design, with cabergoline (1-mg single oral dose) both as tablets and as a solution. The two administrations were separated by a 4-week wash-out period. Cabergoline and prolactin were measured in urine and plasma, respectively, by specific radioimmunoassays. Blood samples were collected before and up to 30 days after dosing. Urine was collected before and up to 8 days after dosing. Cabergoline elimination half-lives calculated from urinary data were 68 and 63 h after administration of the tablets and the solution, respectively. Urinary excretion of unchanged cabergoline accounted, on average, for 1.92% (range, 0.14-3.26) and 1.80% (range, 0.67-3.09) of the dose after administration of the tablets and the aqueous solution, respectively. Relative bioavailability of tablets vs solution was 99% (geometric mean with the 90% confidence intervals of 68-144%). Prolactin levels in 10 out of 12 subjects fell below the detection limit of the assay (1.5 micrograms/L) after both treatments. The mean maximum prolactin decrease (ca. 70%) was achieved by 2 or 3 h after dosing; the effect persisted up to 9 days, being completely exhausted 23-28 days after dosing. The analysis of variance performed on the pharmacodynamic effects of the two cabergoline formulations indicated that the percent decreases of plasma prolactin levels were not significantly different for tablets and solution. These results indicate that the pharmacodynamics and relative bioavailability of cabergoline are not influenced by formulation, as tablets or solution.

Sensitive high-performance liquid chromatographic method for the determination of the three main metabolites of selegiline (L-deprenyl) in human plasma.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination in human plasma of the three main metabolites of selegiline (L-deprenyl): amphetamine, methamphetamine and norselegiline. The HPLC separation of the analytes was performed under isocratic conditions, after extraction from plasma and precolumn derivatization with 9-fluorenylmethyl chloroformate. The linearity, precision and accuracy of the method were evaluated; the limit of quantification for all three metabolites in plasma was 0.5 ng/ml.

Radioimmunoassay for the testosterone 5 alpha-reductase inhibitor turosteride in biological fluids.

An antiserum against turosteride (code name FCE 26073), a potent testosterone 5 alpha-reductase inhibitor, has been raised in rabbits by immunization with an immunogen produced by conjugation of a derivative of FCE 26073 (FCE 27424) to bovine serum albumin. The antiserum was able to distinguish FCE 26073 from its derivatives modified at the 17 beta position and from all the endogenous steroids tested. A radioimmunoassay for the determination of FCE 26073 in human plasma and urine was developed using this antiserum and tritium labeled turosteride. FCE 26073 was extracted from 50 microliters of plasma or 25 microliters of urine using ethyl-ether with a recovery greater than 90%. Using this procedure it was possible to achieve a final limit of quantitation of 142 pg/ml in plasma and 284 pg/ml in urine. The assay was validated in terms of reproducibility, accuracy and precision in the range 3.9-250 pg/50 microliters of plasma and 25 microliters of urine. The plasma concentration of FCE 26073 in a healthy male volunteer who received 0.2 mg of the drug was measured using the radioimmunoassay.

High-performance liquid chromatographic determination of FCE 24928, a new aromatase inhibitor, in human plasma.

A sensitive and selective high-performance liquid chromatographic method for the determination of FCE 24928 (4-amino-androsta-1,4,6-triene-3,17-dione) in human plasma is reported. The drug was extracted from buffered (pH = 8) plasma samples with methylene chloride-isooctane, then analysed by reversed-phase liquid chromatography. Quantitation was achieved by ultraviolet detection of the eluate at 238 nm. Blank plasma samples from humans, dog and rat assayed as described showed no significant peak at the retention time of the compound of interest. The suitability of the method for in vivo samples was tested by measuring the plasma levels of FCE 24928 in rats that received oral doses of the test compound.

Sensitive procedure for the determination of reboxetine enantiomers in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive and selective high-performance liquid chromatographic method for the determination of reboxetine enantiomers in human plasma was developed. Although two chiral centres are present in reboxetine, its stereospecific synthesis leads to two rather than four possible enantiomers. After extraction from plasma and reaction with (+)-1-(9-fluorenyl)ethyl chloroformate, reboxetine enantiomers were separated as diastereoisomeric derivatives by reversed-phase high-performance liquid chromatography (HPLC) and determined by fluorimetric detection. The HPLC analysis time was about 90 min. The linearity, precision, accuracy and limit of quantification of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was assessed by the analysis of plasma samples obtained from a healthy male volunteer who had received a single oral dose of 4 mg of reboxetine in tablet form.

Determination of exemestane, a new aromatase inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of 6-methylen-androsta-1,4-diene-3,17-dione (exemestane) and its 17-dihydro metabolite in human plasma has been developed. The analytes and internal standard (Norgestrel) were extracted from plasma samples with a methylene chloride-iso-octane mixture; the organic phase was dried and the residue was reconstituted with an acetonitrile-water mixture, then analyzed by reversed-phase liquid chromatography. Quantification was achieved by ultraviolet detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human blank plasma was observed. The lower limit of quantification was 10 ng/ml plasma. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from healthy male volunteers who had received a 200-mg single oral dose of the test compound.

Determination of FCE 23884, a new ergolene derivative, and its possible metabolite, the 6-nor-derivative in plasma by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of FCE 23884 and its 6-nor-derivative (FCE 26506) in plasma has been developed. After buffering the plasma samples, the compounds and the internal standard were extracted with ethyl ether-n-octanol (9:1, v/v), back-extracted into 0.01 M phosphoric acid and then analysed by reversed-phase liquid chromatography. Quantification was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the biological matrix was observed. The assay was adequate for the quantification of plasma levels of the two compounds after a single oral dose of 1 mg of FCE 23884 in humans.

Stereoselectivity of iododoxorubicin reduction in various animal species and humans.

1. The excretion of iododoxorubicin in urine as the 13-dihydro derivative, iododoxorubicinol, is much greater in man than in rat, dog, rabbit and monkey. Iododoxorubicinol epimers in urine from man and the animal species after i.v. administration of iododoxorubicin, were quantified by h.p.l.c. 2. (13R)-Iododoxorubicinol was not detectable in rat, dog and human urine and accounted for no more than 0.15% dose in urine of 1/3 rabbits and 3/3 monkeys. (13S)-Iododoxorubicinol in human urine amounted to 5.6% dose, whereas in rat only 0.01% dose and in monkey only 0.91% dose was found. 3. As for idarubicin, the in vivo reduction of iododoxorubicin is highly stereoselective, giving the (13S)-epimer almost exclusively. Erythrocyte ketone reductases may account for the higher formation of (13S)-iododoxorubicinol in man.

Phase I and endocrine study of exemestane (FCE 24304), a new aromatase inhibitor, in postmenopausal women.

Aromatase inhibitors are a useful therapeutic option in the management of endocrine-dependent advanced breast cancer. A single-dose administration of exemestane (FCE 24304; 6-methylenandrosta-1,4-diene-3,17-dione), a new irreversible aromatase inhibitor, was investigated in 29 healthy postmenopausal female volunteers. The compound, given at p.o. doses of 0.5, 5, 12.5, 25, 50, 200, 400, and 800 mg (n = 3-4), was found to be a well tolerated, potent, long-lasting, and specific inhibitor of estrogen biosynthesis. The minimal dose which produced the maximum suppression of plasma estrogens was 25 mg, reducing plasma estrone, estradiol, and estrone sulfate to 35, 28, and 39% of basal values, respectively. This maximum suppression, observed at 3 days, persisted for at least 5 days after administration of a single dose. However, there was no interference on cortisol, aldosterone, 17-hydroxyprogesterone, or dehydroepiandrostenedione sulfate plasma levels. Peak plasma exemestane concentrations of 27, 221, 343, and 414 ng/ml were reached within 2 h after administration of 50, 200, 400, and 800 mg, respectively. Plasma concentrations declined rapidly and fell under the detection limit (10 ng/ml) at 4 (50 mg) or 24 h (200 and 400 mg). No clinically significant adverse events which could be attributed to the drug were reported. Apart from transient eosinophilia in 3 patients, all biochemical and hematological laboratory parameters were within 1.25-fold of the normal ranges.

Determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin, a new morpholinyl anthracycline, in plasma by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin and its possible 13-dihydro metabolite in human plasma has been developed. The plasma samples were buffered and the drugs and internal standard (doxorubicin) were extracted with diethyl ether-n-butanol, back-extracted into 0.3 M phosphoric acid, then analysed by reversed-phase liquid chromatography. Quantitation was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from female rats that had received repeated intravenous doses of the test compound.