Phenylisothiocyanate and dansyl chloride precolumn derivatizations for the high-performance liquid chromatography analysis of the antitumoral agent ES-285 in dog plasma.

Chromophor and fluorophor addition reactions involving phenylisothiocyanate (PITC) and dansyl chloride (DC) were optimized to adapt two high-performance liquid chromatography (HPLC) procedures designed for the accurate determination of the novel antitumoral agent ES-285 in Beagle dog plasma. ES-285 was reacted with PITC at 60 degrees C for 15 min in the presence of triethylamine. The dansyl derivative was obtained by reaction of ES-285 with dansyl chloride in a basic medium at 80 degrees C for 20 min. Both reactions also worked for ES-299, a compound structurally related to ES-285 which was used as internal standard. The treatment of ES-285 and ES-299 spiked plasma samples with a basic phosphate buffer and MeOH permitted the extraction of the drug from the matrix. The purification of the analytes was carried out by solid-phase extraction followed by precolumn derivatization with PITC and DC. The phenylisothiocyanate adducts were analyzed by isocratic HPLC with UV detection at 254 nm. The ES-285 and ES-299 derivatives were eluted from a C(18) column at approximately 6.9 and approximately 8.1 min, respectively. The eluent ACN-water (95:5, v/v) was delivered to the column at a flow-rate of 1 ml/min and the analysis was completed in 15 min. The dansyl derivatives were analysed by a two-HPLC column system with fluorescence detection and gradient elution. The column temperature was maintained at 40 degrees C. The analysis lasted 55 min with the elution of ES-285 and ES-299 derivatives at approximately 35.2 and approximately 37.9 min, respectively. The PITC- and DC-based procedures were characterized by limits of quantification of 20 and 15 ng/ml, respectively. Both procedures were validated according to the ICH and FDA guidelines. They were selective for ES-285 and provided accurate, precise and reproducible results. ES-299 was shown to be a suitable internal standard. The HPLC procedure involving derivatization with DC was more sensitive and permitted to process plasma sample volumes as low as 100 microl. On the other hand, the PITC-based procedure characterised by a quite similar LOQ permitted a higher throughput but implied the processing of a 500-microl plasma volume.

Validation of an analytical procedure for the determination of the fluoroquinolone ofloxacin in chicken tissues.

A novel analytical procedure was developed for the determination of the fluoroquinolone ofloxacin in chicken kidney, liver, muscle and fat plus skin tissues. The procedure involved a preliminary extraction with 0.15 M HCl followed by solid-phase extraction clean-up. The purification step was performed using a polymeric sorbent coated cartridge. Ofloxacin was analyzed by reversed-phase HPLC using UV detection at 295 nm. The mobile phase used was water-acetonitrile-triethylamine (83:14:0.45, v/v, pH 2.30). The use of triethylamine and the acidic pH modulated the retention of ofloxacin and avoided chemical tailing. The amine modifier and acetonitrile content of the mobile phase were optimized to provide the best selectivity. A flow-rate of 1 ml/min was used and ofloxacin eluted at approximately 5.1 min. HPLC analysis of the tissue samples was performed in 12 min. The procedure was validated according to the International Conference on Harmonisation guidelines across the concentration ranges (100 microg/kg-1.7 mg/kg for kidney and liver tissues and 50 microg/kg-1.0 mg/kg for muscle and fat plus skin tissues). The linearity, the intra- and inter-day accuracies and precisions were determined. The limits of quantification were 50 microg/kg for muscle and fat plus skin tissues and 100 microg/kg for liver and kidney tissues. The procedure was specific and the accuracy values were lower than 20% at the limit of quantitation of spiked samples. The recovery values ranged from 80 to 100%. The limits of detection were established at 60 microg/kg for liver and kidney tissues and at 25 microg/kg for muscle and fat plus skin tissues. Finally, ofloxacin was found to be stable in acidic conditions. The developed procedure is simple, sensitive, accurate and adapted to routine sample analyses such as those carried out for residue depletion studies.

Bioavailability of S(+)-ketoprofen after oral administration of different mixtures of ketoprofen enantiomers to dogs.

Recent reports have disagreed on whether the bioavailability of S(+)-ketoprofen is affected by the presence of R(-)-ketoprofen. To examine this directly, we designed a randomized crossover study in beagle dogs. [14C]-S(+)-ketoprofen trometamol and R(-)-ketoprofen trometamol were administered in the following percentage ratios: A, 99:1; B, 95:5; C, 90:10; D, 70:30; E, 50:50. Treatments were administered as a single oral dose of 1 mg/kg tromnetamol salt. Each of eight dogs received all five combinations in random order with a 1-week wash-out period between doses. Blood samples were taken before drug administration and at regular intervals for 240 min after dosing. A progressive increase in the plasma concentration of [14C]-S(+)-ketoprofen was observed on going from treatment E (lowest dose of Senantiomer) to treatments containing the highest doses of [14C]-S(+)-ketoprofen. When the pharmacokinetic calculations were normalized to the dose of [14C]-S(+)-ketoprofen, we found no statistically significant differences among the normalized AUC and Cmax values of the five treatments. Therefore, S(+)-ketoprofen absorption was linear and was not influenced by the presence of R(-)-ketoprofen. Furthermore, there were no significant differences in tmax values among treatments, indicating that the rate of S(+)-ketoprofen absorption was also unaffected by the presence of R(-)-ketoprofen.

Pharmacokinetics of ketoprofen enantiomers in monkeys following single and multiple oral administration.

Pharmacokinetic studies are reported after single oral administration of 3 mg/kg of stereochemically pure (S)-ketoprofen [(S)-KP] and (R)-ketoprofen [(R)-KP] to three male Cynomolgus monkeys and after repeated administration for 6 months of 3, 15 and 75 mg/kg/day of (S)-KP to both male and female monkeys. A high-performance liquid chromatographic (HPLC) analysis was performed without derivatization of the samples, using a chiral column. The pharmacokinetic parameters for (S)-KP after administration of (S)-KP and for (R)-KP after administration of (R)-KP were, respectively, elimination half-life 2.32 +/- 0.36 and 1.64 +/- 0.40 h; oral clearance 3.50 +/- 0.66 and 7.50 +/- 3.20 ml/min/kg; apparent volume of distribution 0.74 +/- 0.24 and 1.16 +/- 0.76 liter/kg; mean residence time 1.79 +/- 0.77 and 1.41 +/- 0.65 h; area under the concentration/time curve 14.16 +/- 2.93 and 7.31 +/- 2.98 micrograms.h/ml. Forty-nine percent unidirectional bioinversion of (R)-KP to (S)-KP was observed in this species and the pharmacokinetic parameters for the (S)-KP resulting from this inversion were also calculated. In the study of 6-month repeated administration of (S)-KP, linear pharmacokinetic behavior and no evidence of drug accumulation were observed at the three dose levels.

Percutaneous absorption and skin distribution of [14C]flutrimazole in mini-pigs.

The percutaneous absorption and skin distribution of a skin cream containing 1% 14C-labelled 1-[(fluorophenyl) (4-fluorophenyl) phenylmethyl]-1H-imidazole (flutrimazole, UR-4056, CAS 119006-77-8) was studied in minipigs. The same dose of flutrimazole was administered i.v. and topically (as a cream) on scarified skin according to a crossover protocol. Samples of urine and faeces were taken at various intervals after administration, and radioactivity was measured. The percentage of radioactivity accumulated in urine after topical and intravenous administration were 1.46% and 41.7%, respectively. In faeces, the percentage of radioactivity observed was 6.0% after intravenous administration, and none was detected after topical application. In order to study the distribution and penetration of [14C]flutrimazole, the cream was applied to intact and scarified skin. At various intervals after administration, skin samples were taken. The samples for the autoradiographic studies were cut transversely, and for the measurement of the levels of radioactivity at different skin depths, slices were cut parallel to the cutaneous layers. The results obtained indicate that [14C]flutrimazole penetrates quickly into the different epidermic layers and is retained mainly in the strata spinosum, granulosum and basale. The stratum basale possibly acts as a selective barrier preventing the penetration of the compound into the dermis. The percentage of radioactivity in the stratum corneum is lower than that detected in all the other epidermic layers taken together. The stratum corneum offers low resistance to penetration by the flutrimazole, which very probably crosses the epidermic strata by a transcellular route.

Pharmacokinetic study of [14C]flutrimazole after oral and intravenous administration in dogs. Comparison with clotrimazole.

This trial involved a comparative study using 6 Beagle dogs on the pharmacokinetics of 14C-labelled 1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole (flutrimazole, CAS 119006-77-8) and [14C]clotrimazole labelled in the imidazole ring. On the basis of a cross-over trial, each animal received a dose of 5 mg/kg (approx. 100 microCi) [14C]flutrimazole and [14C]clotrimazole, both intravenously and orally. The levels in plasma, urine and faeces of the total radioactivity, unchanged drug and the [14C]imidazole formed by metabolization of the unchanged drug were determined. Flutrimazole presented a biological half-life (t1/2) of 14.4 +/- 3.8 h and a clearance (Cl) of 6.7 +/- 0.8 l/h, while the values for clotrimazole were very different: t1/2 4.6 +/- 0.8 h and Cl: 13.6 +/- 1.0 l/h. After oral administration a fraction of absorbed dose (f) of 78 +/- 21% and bioavailability of 8.9 +/- 6.1% were calculated for flutrimazole. For clotrimazole, these were: 52 +/- 10% and 4.9 +/- 1.9%, respectively. Both drugs showed a significant first-pass effect, with 90% of the absorbed dose being metabolized before reaching the systemic circulation. The total recovery of radioactivity in faeces and urine 5 days after i.v. and oral administration was 58% and 68%, respectively, for [14C]flutrimazole, and 81% and 79% for [14C]clotrimazole. In both cases, most of the radioactivity was recovered in the faeces. The high radioactivity obtained in faeces after i.v. administration of both drugs confirms biliary elimination. For both flutrimazole and clotrimazole, less than 1% of the total recovered in the urine after i.v. administration was recovered as unchanged drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Diuretic effect of ITA 529.

The diuretic and saluretic activity of ITA 529 (Ethyl-beta-[(5-tert-butyl-3-chloro-2-hydroxy)benzylamino]crotonate++ +) in rat, rabbit and monkey is described in this study. In rat, the diuretic ED50 of ITA 529 was 1.23 mg/kg p.o. and its ED100 1.69 mg/kg p.o. The diuretic ED50 of furosemide was 18.48 mg/kg p.o. and its ED100 22.38 mg/kg p.o. Their effect lasted approximately 3 hours. In monkey, the diuretic ED50 of ITA 529 was 2.29 mg/kg p.o. and its ED100 6.54 mg/kg p.o. while for furosemide its ED50 was 2.44 mg/kg p.o. and its ED100 5.72 mg/kg p.o. In rabbit the ED50 of ITA 529 was 1.83 mg/kg p.o. and its ED100 3.37 mg/kg p.o. For furosemide, its ED50 was 2.25 mg/kg p.o. and its ED100 7.13 mg/kg p.o. The natriuretic activity of ITA 529 was reduced by indomethacin.

Anti-ulcer activity and other pharmacological properties of lozilurea.

As a result of trials on a large series of compounds, one of these, N' -3-chlorobenzyl-N'-ethylurea (lozilurea, ITA 312) has shown marked anti-ulcer activity. It has shown itself to be active against chemically and neurogenically induced gastric and duodenal lesions in various experimental animal models. It has no major anti-secretory action. The experimental data obtained suggest that the mechanism of action of lozilurea consists in increasing the protective function of the mucus barrier. In the screening trials carried out in order to detect the side effects of lozilurea, it has shown sedative, antipyretic and vasodilatory actions.

Activity of plafibride on erythrocyte deformability.

N-2-(p-Chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, Idonor, Perifunal, Plafibrinol) is an acid derivative of morpholinomethylurea and clofibric acid. In these studies the effects of plafibride on haemorheological properties have been observed. The studies have been carried out in different animal species (rat, rabbit and dog), and in human volunteers. Under these circumstances, plafibride has shown itself to be highly active in all the animal species and in humans, as well as with the two laboratory methods used for determination of erythrocyte deformability, these being Teitel's method, which evaluates the deformative capacity of erythrocytes when forming compact groups, and the method of Schmid-Schoenbein in which the deformability of individual erythrocytes is measured by passage through fine capillaries. In trials in volunteers, the increase in erythrocyte deformability is notable even 5 days after the last administration. In addition to its microhaemorheological activity, plafibride has been shown to have effects on platelet aggregation and hyperlipaemia.

Endothelial damage induced by polyethylene catheter in rat.

A method is described for obtaining endothelial lesions in the aorta by implanting polyethylene catheters in the aorta of rats for two days. Three types of lesions were observed and were quantified by measuring their lengths: (a) the simple disendothelization (b) raised lesions, and (c) tunnel lesions. The effect of a number of antiplatelet drugs on these types of lesions was investigated, and it was observed that these are particularly active in inhibiting tunnel lesions. Using this method, endothelial lesions can be obtained in a short period of time. Moreover, a large number of results can be obtained rapidly, which is important in these types of experiments due to the high dispersion obtained.

Influence of plafibride, an antiplatelet and hypolipemic agent, on prostacyclin and thromboxane synthesis, 3',5'-cyclic AMP phosphodiesterase activity and serum clearance of a lipid emulsion.

The activity of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) on arachidonic acid metabolism, the 3',5'-cyclic AMP-phosphodiesterase and the serum clearance of a lipid emulsion is reported in order to clarify its mechanism of action on platelet aggregation and lipid metabolism. Plafibride did not act on the arachidonic acid metabolism as far as platelet aggregation was concerned, since it did not modify the generation of prostaglandin endoperoxides nor prostacyclin. Neither did it act on the generation of thromboxane A2. Plafibride inhibited the activity of 3',5'-cyclic AMP-phosphodiesterase, which is one of the principal mechanisms of inhibition of platelet aggregation. The serum c clearance of a commercial lipid emfy the generation of prostaglandin endoperoxides nor prostacyclin. Neither did it act on the generation of thromboxane A2. Plafibride inhibited the activity of 3',5'-cyclic AMP-phosphodiesterase, which is one of the principal mechanisms of inhibition of platelet aggregation. The serum clearance of a commercial lipid emulsion was increased by plafibride which also possessed a strong hypotriglyceride activity. The correlation between platelet antiaggregant and hypolipemic activity of plafibride is discussed in this paper.

Synthesis and pharmacological evaluation of N'-morpholinomethylurea derivatives with platelet antiaggregant activity.

In the attempt to prolong and stabilize the known antiaggregant activity of morpholinomethylurea (MMU) derivatives were prepared with carboxylic acids possessing antiaggregant, anti-inflammatory or hypolipemic activity. The results of the experiments showed the important anti-aggregant and hypolipemic activity of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104). The results obtained seem to indicate that the activity of these new N-acyl-N'-MMU is due initially to the molecule per se although it cannot be totally ruled out that part of the pharmacodynamic effect of the MMU may depend on a prodrug-like behaviour.

Platelet antiaggregant activity of plafibride ex vivo in rat, dog and rabbit.

N-2-(p-Chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) is an acyl derivative of morpholinomethylurea (MMU) with clofibric acid that was found to be active as a hypotriglyceridemic increasing the serum alpha-lipoproteins and decreasing drastically the serum turbidity after olive oil ingestion. This paper reports the antiaggregating activity ex vivo of plafibride in experimental animals. In ADP-induced platelet aggregation in the rat, plafibride at double dose was as active as acetylsalicylic acid (ASA), dipyridamole was less active and clofibrate practically inactive. In the rabbit, plafibride either administered p.o. or i.v. was as active as ASA. In ADP-, collagen- or adrenaline-induced aggregation in the dog, plafibride showed marked platelet antiaggregant activity after a single dose of 100 mg/kg p.o. In all the experiments, the antiaggregating activity of plafibride was similar in intensity to that of ASA but more retarded. Inhibition by plafibride of arachidonic acid-induced platelet aggregation was of a competitive type whereas the inhibition by ASA was unspecific. In the rat, plafibride inhibited significantly the spontaneously formed circulating platelet aggregates. In vitro plafibride appeared as an effective antiaggregant agent although less powerful than morpholinomethylurea, one of its presumed metabolites. The ex vivo activity of MMU was nevertheless too short-lasting to be of any therapeutic interest. The kinetics of platelet antiaggregant activity and the correlation between hypolipemic and platelet antiaggregant activity of plafibride is discussed in this paper. It is evident that plafibride at above dose is active as an antiaggregant and hypolipemic agent.

Pharmacokinetic approach of plafibride in rat.

The serum concentration of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) and its metabolites was measured in the rat after oral and intravenous administration of the drug. Plafibride was distributed in the rat according to an open two-compartment model. Moreover, 2-(p-chlorophenoxy)isobutyrylurea and clofibric acid were detected. In the urine, 81.7% of the administered dose was recovered in the form of free clofibric acid, 0.8% as plafibride itself and 1.1% as 2-(p-chlorophenoxy)isobutyrylurea. The amount of plafibride and its metabolites present in the feces was practically negligible. A mechanistic scheme for the degradation of plafibride is proposed, which agrees with the observed pharmacological and pharmacokinetic data.

Hypolipemic activity of plafibride.

This paper reports on the hypolipemic activity of N-2-(p-chlorophenoxy)-isobutyryl-N' morpholinomethylurea (plafibride, ITA 104) in experimental animals. In Triton hypercholesterolemic animals, the activity of plafibride was greater than that of clofibrate, and was more evident in the total lipids and reducing the serum lactacidemia than the cholesterol serum levels. Plafibride protected the rats and the mice almost completely against the increase in serum lactacidemia due to the administration of olive oil. In normolipemic rats the activity of plafibride was greater than that of clofibrate on triglycerides but not on cholesterol. Plafibride increased the serum alpha-lipoprotein fraction, too.

Toxicological studies of plafibride. Part 4: Interaction of plafibride with other drugs.

The possible pharmacological interaction was studied between N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) and a series of products with analgesic, anticoagulant, antidepressant, antidiabetic, Beta-blocking, diuretic, hypotensive, nootropic, tranquillizing and vasodilator activities. In the experiment the LD50 of each product was compared with that found for the joint administration of the product and plafibride. Plafibride increased the acute toxicity of reserpine and potentiated its hypothermic effect in the mouse; it did not alter its effect on arterial pressure in dog and rat nor its effect on palpebral ptosis in the mouse. Although plafibride did not potentiate the acute toxicity of warfarin, its anticoagulant activity was potentiated, possibly due to the movement of warfarin from its binding sites to the plasma proteins.

Bioavailability of plafibride in healthy volunteers.

The pharmacokinetics of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) in rats, and its intestinal absorption, plasmatic biotransformation, urinary elimination and fecal excretion were previously studied. 1200 mg of plafibride orally were administered to 5 healthy volunteers and then the presence of the drug and its metabolites was studied in the serum. The presence of unchanged plafibride and clofibric acid was detected, whereas that of 2-(p-chlorophenoxy) isobutyrylurea was not. At the same time the platelet antiaggregating activity of plafibride was investigated in the 5 volunteers. In this study plafibride presented a good and long-lasting platelet antiaggregating activity.