Liquid chromatographic determination of urea in water-soluble urea-formaldehyde fertilizer products and in aqueous urea solutions: collaborative study.

Water soluble urea-formaldehyde (UF) fertilizers, manufactured by complex reaction of urea and formaldehyde, typically contain varying amounts of unreacted urea. A liquid chromatography method for the analysis of urea in these products, and in aqueous urea solutions, was collaboratively studied. An amine chromatography column was used to separate the unreacted urea from numerous UF reaction products present in these liquid fertilizers. Unreacted urea was determined by using external urea standards with UV detection at 195 nm. The standards and test samples were prepared in the mobile phase of 85% (v/v) acetonitrile in water. Ten laboratories analyzed 5 different UF-based commercial products containing unreacted urea in the range of 6 to 17% by weight, and 5 different concentrations of urea in water equivalent to commercial products of that nature. The aqueous urea solutions contained 2-20% urea (w/w). The range of s(R) values for the 5 UF-based commercial fertilizers was 0.49-1.02 and the %RSD(R) was 1.94-6.14. The s(R) range for the 5 urea solutions was 0.10 to 0.79 and the %RSD(R) range was 2.54 to 4.88. The average recovery of urea from the aqueous urea solutions was 96-103%. Therefore, this method is capable of monitoring urea nitrogen manufacturers' label claims and total nitrogen claims in those cases where urea is the sole source of plant food nitrogen. Based on the collaborative study data, the authors recommend this method be approved for AOAC Official First Action status.

Antibacterial activities and pharmacokinetics of E-4767 and E-5065, two new 8-chlorofluoroquinolones with a 7-azetidin ring substituent.

E-4767 [(-)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid] and E-5065 [(-)-7-(3-amino-1-azetidinyl)-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid] are two new chlorofluoroquinolones with an azetidine moiety at position 7. Their in vitro activities were evaluated in comparison with those of ciprofloxacin, ofloxacin, fleroxacin, and tosufloxacin, while ciprofloxacin was used as a reference for in vivo studies. Against gram-positive organisms, E-4767 and E-5065 were, in general, eight- and fourfold more active than tosufloxacin, which is the most potent of the reference compounds. E-4767 and E-5065 were also more potent than the reference compounds against all species of enteric bacteria tested. The MICs of E-4767 and E-5065 at which 90% of the isolates tested were inhibited (MIC(90)s) were 0.007 to 0.5 microg/ml and 0.03 to 2 microg/ml, respectively, for gram-positive organisms and

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate.

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.

[Experimental autoimmune uveitis. Characterization of retina infiltrating cells]. / Experimentelle Autoimmun-Uveitis. Charakterisierung der die Retina infiltrierenden Zellen.

UNLABELLED: The chronic model of murine EAU induced by interphotoreceptor retinoid binding protein represents a disease similar to clinical chorioretinitis. In this study we characterized the kinetics of retina infiltrating T-cells, macrophages and expression of the adhesion molecules ICAM-1 and ICAM-2. METHODS: B10.A mice were immunized subcutaneously with IRBP, and the eyes were analyzed on days 10, 18, 24 and 28. The infiltrating cells were characterized by mAbs recognizing T-cell receptors (TCR) Vss6 and Vss8, T-cell markers, macrophages and ICAM-1 and ICAM-2. RESULTS: While CD8+ T-cells and ICAM-2 were detectable from day 10 (retina is intact) until day 28, CD4+ T-cells, macrophages and ICAM-1 appear with the onset of retinal destruction. Starting at day 10 the dominating TCR was Vss6; Vss8 was noticed from day 18 on. CONCLUSION: CD8+ T-cells infiltrating the intact retina and stimulating the expression of high endothelial venules (HEVs) could be responsible for the onset of uveitis.

E-4695, a new C-7 azetidinyl fluoronaphthyridine with enhanced activity against gram-positive and anaerobic pathogens.

E-4695, (-)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-1-cyclopropyl-1,4- dihydro-6-fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid, is a new fluorinated naphthyridine with an azetidine moiety. The MICs of E-4695 at which 90% of the isolates were inhibited (MIC90s) were 0.06 to 0.5 microgram/ml for gram-positive cocci, including species of the genera Staphylococcus, Streptococcus, and Enterococcus, and the MIC90s against gram-negative pathogens such as members of the family Enterobacteriaceae (with the exception of Providencia spp. [MIC90, 8 micrograms/ml]) and Pseudomonas aeruginosa were 0.015 to 0.5 microgram/ml. E-4695 inhibited 90% of the Clostridium perfringens and Bacteroides fragilis isolates at 0.25 and 4 micrograms/ml, respectively. Against gram-positive cocci the potency of E-4695 was 2- to 8-fold higher than that of ciprofloxacin, 4- to 8-fold higher than that of ofloxacin, and 8- to 16-fold higher than that of fleroxacin. Against enteric bacteria and P. aeruginosa the potency of E-4695 was, in general, similar to that of ciprofloxacin and eightfold higher than those of ofloxacin and fleroxacin. E-4695 was four- and eightfold more potent than ciprofloxacin against C. perfringens and B. fragilis isolates, respectively. E-4695 and ciprofloxacin showed similar properties when the effects of pH or magnesium concentration were tested on them. E-4695 and ciprofloxacin had substantial reductions of activity only when pH decreased below 4.8. E-4695 and ciprofloxacin activities were not markedly affected by the presence of 5 or 10 mM Mg2+. The presence of serum and human urine at pH 7.2 decreased the activity of E-4695 between two- and fourfold. After an oral dose of 50 mg/kg of body weight, the maximum level in serum, the biological half-life, and the area under the concentration-time curve from 0 to 10 h for E-4695 were 13.2 microgram/ml, 3.3 h, and 45.6 microgram . h/ml, respectively. The area under the concentration-time curve from 0 to 4 h for ciprofloxacin was 2.3 microgram . h/ml at the same dose. Fifty-percent effective doses (ED50S) against Staphylococcus aureus HS-93 infections in mice were 4.5 mg/kg with E-4695 and 37.6 mg/kg with ciprofloxacin. Infection with Streptococcus pneumoniae 29206 was more effectively treated with E-4695 (ED50, 41,2 mg/kg) than with ciprofloxacin (ED50, 200 mg/kg). The ED50 of E-4695 for infections with Streptococcus pneumoniae 1625 was 132.2 mg/kg; ciprofloxacin was ineffective at 400 mg/kg against this strain. E-4695 was also more potent than ciprofloxacin in treatment of infections caused by gram-negative organisms such as Escherichia coli HM-42 (ED50S, 1.0 and 3.9 mg/kg, respectively). The ED50S of E-4695 and ciprofloxacin were 33.0 and 145.5 mg/kg against P. aeruginosa HS-116 and 9.6 and 18.9 mg/kg against P. aeruginosa B-120, respectively. The therapeutic efficacy of E-4695 may depend not only on its in vitro activity but also on its improved pharmacokinetic properties.

In vitro and in vivo antibacterial activities of E-4868, a new fluoroquinolone with a 7-azetidin ring substituent.

E-4868, (-)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-1-(2,4- difluorophenyl)-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid, is a new fluoroquinolone with an azetidine moiety at the 7 position. The in vitro activity of E-4868 has been compared with those of ciprofloxacin, ofloxacin, and fleroxacin, while the activity of ciprofloxacin was used as reference for in vivo studies. The MICs of E-4868 for 90% of the isolates tested (MIC90s) were 0.06 to 0.5 microgram/ml against gram-positive organisms, including Staphylococcus, Streptococcus, and Enterococcus spp. In general, the in vitro potency of E-4868 against gram-positive bacteria was higher than those of all of the other fluoroquinolones tested. MIC90s against members of the family Enterobacteriaceae between 0.03 and 1 microgram/ml were observed, with the exception of those against Serratia marcescens and Providencia spp., and a MIC90 of 2 micrograms/ml against Pseudomonas aeruginosa was obtained. E-4868 inhibited 90% of the Clostridium spp. and Bacteroides spp. at 2 micrograms/ml and was twofold more active than ciprofloxacin. An increase in the Mg2+ concentration from 1 to 10 mM increased the MIC between two and three times. Human urine caused a significant decrease in activity of E-4868, which was more pronounced at pH 5.5 than at pH 7.2. The presence of serum also decreased the activity of E-4868. Fifty percent effective dose (ED50) values against experimental Escherichia coli HM-42 infections in mice were 3.9 mg/kg of body weight with E-4868 and 3.5 mg/kg of body weight with ciprofloxacin. Corresponding ED50 values against P. aeruginosa HS-116 were 93.2 and 107.8 mg/kg, respectively, and those against Staphylococcus aureus HS-93 were 6.5 and 44.6 mg/kg, respectively. In experimental infections with Streptococcus pneumoniae 84551, the ED50 value of E-4868 was 154.4 mg/kg, while ciprofloxacin proved totally inactive at a dose of 400 mg/kg. When E-4868 was administered orally at a dose of 50 mg/kg in mice, the area under the concentration-time curve (0 to 4 h) value was 28.4 microgram . h/ml, while an area under the concentration-time curve value of 2.3 microgram . h/ml was observed for ciprofloxacin at the same dose. In these studies, levels of the two agents in blood 1 h postadministration were 7.6 and 1.2 microgram/ml, respectively.

The effect of antacid and ranitidine on droxicam pharmacokinetics.

Droxicam is a nonsteroidal anti-inflammatory drug that is a pro-drug of piroxicam. The influence of concomitant administration of antacid or ranitidine on droxicam pharmacokinetics has been investigated. On three separate phases, 15 healthy volunteers received a single oral 20-mg dose of droxicam either alone, with antacid (400 mg aluminum hydroxide + 400 mg magnesium hydroxide, three times/day), or with ranitidine (300 mg, two times/day) for 6 days. Piroxicam, the active substance from droxicam, was quantified by high-performance liquid chromatography. The pharmacokinetic parameters for droxicam given alone were: maximum peak plasma concentration (Cmax) = 1.53 +/- .21 micrograms/mL (mean +/- SD), time to peak concentration (Tmax) = 7.5 +/- 2.1 hr, t1/2a = 1.38 +/- .82 hour, t1/2el = 53.3 +/- 11.9 hr, Cl/F = 2.98 +/- .71 mL/min, volume of distribution (Vd/F) = 13.2 +/- 1.8 L and area under the curve (AUC) = 117.6 +/- 26.8 micrograms/hour/mL. The subject effect was significant for all the pharmacokinetic parameters except for the absorption half-life (P < .05). Concomitant antacid or ranitidine administration had no significant effect on any of the droxicam pharmacokinetic parameters. The results of this study suggest that antacid or ranitidine do not significantly alter the oral absorption or pharmacokinetic disposition of single-dose droxicam.

Cross-over study of the bioavailability of a new NSAID (droxicam) versus piroxicam in healthy volunteers following single and multiple dose administration.

Droxicam is a new anti-inflammatory drug which is a pro-drug of piroxicam and possesses delayed absorption kinetics. In this study, the comparative bioavailability of the two compounds was investigated. The study was performed following a cross-over design with single (20 mg) and multiple (20 mg/day for 30 consecutive days) administration in 25 healthy volunteers. The peak plasma concentrations of piroxicam, obtained following administration of droxicam, were lower than those calculated for administration of piroxicam, and the time taken to reach these peak concentrations was increased by approximately 5-7 h. There was no significant difference in either the elimination kinetics of piroxicam or the AUC values found following administration of the two products. Bioavailability of droxicam is equal to that of piroxicam, with a slower rate of absorption.

A pharmacokinetic study of E-4441, a new quinolone, in the rat, mouse and cynomolgus monkey.

The purpose of this present work has been to study the pharmacokinetical profile of E-4441 in the rat, mouse and cynomolgus monkey. Analytical determination of the levels in plasma, urine and organs was affected by two different techniques: a microbiological agar diffusion assay with Bacillus subtilis, and HPLC with preliminary extraction in chloroform (pH 8.5). The kinetic behaviour of the unchanged substance was similar in the three animal species studied. Absorption and excretion took place rapidly (T1/2a = 2-20 min, T1/2el = 25-225 min). In mouse there is a linear relationship between administered dose and the area under the curve (AUC) of plasma levels. The absolute bioavailability of unchanged E-4441 is of 20% and the urinary excretion represents 1 to 2% of the administered oral dose. The organs in which the highest concentrations of substance were found were bile, liver and kidney, while the substance could not be detected in brain, testis, ovary or adipose tissue.

Comparative in vitro and in vivo activities of six new monofluoroquinolone and difluoroquinolone 3-carboxylic acids with a 7-azetidin ring substituent.

E-4502, E-4501, E-4500, E-4480, E-4474, and E-4441 are new monofluorinated or difluorinated quinolone agents that are chemically characterized by the presence of an azetidin ring, with different C'-3 substituents, at position 7 of the molecular structure. The MICs of the difluorinated compounds E-4501, E-4474, and E-4441 for 90% of isolates were 0.06 to 1, 0.06 to 1, and 0.12 to 1 microgram/ml, respectively, against gram-positive organisms (staphylococci, streptococci, and Enterococcus faecalis); 0.0015 to 0.12, 0.015 to 0.12, and 0.03 to 0.12 microgram/ml, respectively, against members of the family Enterobacteriaceae except Providencia spp.; and 1, 1, and 2 micrograms/ml, respectively, against Pseudomonas aeruginosa. E-4501, E-4474, and E-4441 inhibited all anaerobic bacteria at concentrations of 1, 2, and 4 micrograms/ml, respectively. Difluorinated compounds were significantly more active than the corresponding monofluorinated analogs E-4502, E-4500, and E-4480 against aerobic and facultatively anaerobic organisms, as well as against anaerobes. Considering monofluorinated and difluorinated compounds, activity in moderate ascending order was observed in quinolones containing an amine and a methyl group (E-4441 and E-4480), an amine group (E-4474 and E-4500), and a methylamine group (E-4501 and E-4502) in the C'-3 position of the azetidin ring. E-4501, E-4474, and E-4441 were more active than norfloxacin and DR-3355 [S-(-)-ofloxacin], had activities comparable to or slightly lower than that of ciprofloxacin against gram-negative bacteria, and were more active than all the reference quinolones against gram-positive organisms and anaerobes. E-4502 and E-4501, which were used to determine the effect of pH, were less active in acidic medium. In general, E-4502, E-4501, E-4500, E-4480, E-4474, and E-4441 activities were not affected or increased in medium containing serum but decreased in the presence of 10 mM Mg2+ or in human urine at pH 5.5. The protective effect of E-4501, E-4474, and E-4441 after oral administration against systemic infections with Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa in mice was greater than that of ciprofloxacin.

Single and multiple dose pharmacokinetics of a new NSAID (droxicam) in healthy volunteers.

The pharmacokinetics of droxicam, both as a single 10 mg dose and as a multidose regimen of 10 mg/day for 20 consecutive days, have been studied in healthy volunteers. The study was performed in two separate groups of volunteers. Following a single dose the Cmax was 0.82 +/- 0.15 micrograms/ml, the Tmax was achieved at 6.1 +/- 3.5 h, the elimination half life was 65.7 +/- 17.6 h, the Clt/F was 2.04 +/- 0.53 ml/min, the Vd/F was 11.0 +/- 1.7 l and the AUC infinity was 86.9 +/- 24.6 mugh/ml, which was similar to results reported in other study from piroxicam (10 mg). Following multiple doses the Cmed(ss) was 2.06 +/- 0.42 microgram/ml, the Tmax(ss) was 8.2 +/- 6.0 h, the elimination half life was 41.4 +/- 12.4 h, the Clt/F was 3.30 +/- 0.63 ml/min, the Vd/F was 11.8 +/- 4.3 l and the AUC infinity was 52.4 +/- 11.3 mugh/ml. The differences encountered between single and multiple dose administration in elimination kinetics are due to the wide interpersonal variation described for the elimination half life of piroxicam. It may be concluded from these results that absorption, elimination and bioavailability kinetics of droxicam are independent of the administered dose.

The influence of gastric emptying on droxicam pharmacokinetics.

Droxicam is a new nonsteroidal anti-inflammatory drug that is a pro-drug of piroxicam. The influence of gastric emptying rate on droxicam pharmacokinetics has been investigated in eight healthy male volunteers. A single, 20 mg dose was administered p.o. together with 1500 mg of paracetamol. Gastric transit was experimentally modified by administration of propantheline (45 mg, p.o.) or metoclopramide (10 mg, i.v.) simultaneously with the droxicam and the paracetamol. Plasma levels of paracetamol were used as markers of gastric transit. The plasma concentrations of piroxicam, the active substance from droxicam, were determined by a high-performance liquid chromatographic method. The pharmacokinetic parameters of droxicam were: Cmax = 1.03 +/- 0.16 micrograms/mL (mean +/- SD). Tmax = 11.1 +/- 5.7 hr, AUC = 115.7 +/- 29.6 micrograms hr/mL, T 1/2 a = 2.64 +/- 0.72 hr. T 1/2 el = 73.6 +/- 16.7 hr, CL/F = 3.06 +/- 0.80 mL/min and MRT = 111.1 +/- 23.5 hr. Following modification of gastric emptying, only Tmax (droxicam + metoclopramide = 25.0 +/- 10.8 hr and droxicam + propantheline = 20.8 +/- 8.8 hr) underwent significant change (P less than 0.05). These results indicate that absorption rate of droxicam has been modified but bioavailability does not suffer modification in conditions of altered gastric emptying.

A study of reference parameters in the CFY (remote Sprague-Dawley) rat.

The reference parameters for 10- and 19-week old CFY (remote Sprague-Dawley) rats fed powdered feed have been studied: growth curves, food consumption, ophthalmoscopy, results of urinalyses and hematological and biochemical assays, and absolute and relative organ weights. Detailed information is given of instrumentation and methods employed, of the animals, strain and environment, and of blood-sampling technique. In general, the results obtained were similar to those previously reported in literature.