Quantitation of SU1 1248, an oral multi-target tyrosine kinase inhibitor, and its metabolite in monkey tissues by liquid chromatograph with tandem mass spectrometry following semi-automated liquid-liquid extraction.

SU11248 is a potent inhibitor of PDGFR, VEGFR, KIT, and Flt3, and is currently under Phase I clinical evaluation as an anticancer drug. A sensitive and specific analytical method for the quantitation of SU11248 and its metabolite in several monkey tissues (liver, kidney, brain and white fat) using LC-MS-MS following semi-automated liquid-liquid extraction (LLE) was developed and validated. Amounts of 50 mg of tissue were homogenized using an ultrasonic processor. After addition of the stable labelled internal standard (IS) and ammonium hydroxide (0.3%), samples were extracted with 2.5 ml of tert-butyl methyl ether. Following centrifugation, aliquots of 1.8 ml of the organic phase were transferred into a 96-well plate. The Packard Multiprobe II robotic liquid handler was used to perform all steps mentioned above. The organic phase was dried and the residue was reconstituted with 800 microl of 15 mM ammonium formate buffer solution (pH 3.25) using a Tomtec Quadra 96 workstation. Aliquots of 10 microl of the resulting solution were injected into the LC-MS-MS system. A Symmetry Shield C8 column (50 mm x 2.1 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase was 15 mM ammonium formate buffer solution (pH 3.25)-acetonitrile (74:26 (v/v)) with a flow-rate of 0.35 ml/min. Retention times of the metabolite and SU11248 were about 2.5 and 3.5 min, respectively. Total cycle time was 5 min. MS detection used the Applied Biosystems-MDS Sciex API 3000 with TurbolonSpray interface and multiple reaction monitoring (MRM) operated in positive ion mode. The method was validated for both compounds over the calibration range of about 2 and 2000 ng/g. The suitability and robustness of the method for in vivo samples were confirmed by analysis of monkey tissues from animals dosed with SU11248.

Enantioselective determination of FCE 28833, a new potential antiischemic agent, in gerbil plasma using column switching HPLC with UV detection.

A sensitive and selective high performance liquid chromatographic method using an automated column switching technique for the determination of FCE 28833 enantiomers in gerbil plasma was developed. After solid-liquid extraction using a Supelcosil C18 cartridge, FCE 28833 was eluted on a clean-up column (Spherisorb CN) and the enantiomers were separated using an analytical chiral column (Crownpack CR(+)). The mobile phase (15% methanol in HClO4 1 mM) was directed through the columns at a flow rate of 1 ml/min and the fraction eluted between 13 and 40 min was transferred from the clean-up column into the analytical column. FCE 28833 enantiomers were monitored at 257 nm. The limit of quantitation of the method was 20 ng/ml plasma for both enantiomers and proved to be linear, precise, and accurate for the assay of both enantiomers in the 20-6,000 ng/ml concentration range. No interference from the blank gerbil plasma sample was observed. The suitability of the method was assessed using plasma samples obtained from male gerbils treated with a single oral dose (400 mg/kg) of FCE 28833.

Effect of escalating doses of d-fenfluramine on the content of indoles in brain.

The neurochemical effects of a large dose challenge (5 mg/kg, i.p.) of d-fenfluramine (d-F) in rats, given saline or gradually escalating doses of d-F (0.1-2.5 mg/kg, i.p.), were examined with regard to regional sensitivity and the time-course of recovery. The indole-depleting effect after the large dose of d-F to saline-pretreated animals appeared to differ, depending on the areas of brain considered (cortex greater than hippocampus greater than striatum), despite the fact that the drug and its main metabolite, d-norfenfluramine (d-NF) distributed almost uniformly in the regions of brain examined. The depletion in all these regions of the brain was reversible within 6 weeks, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) being back to control levels in the hippocampus and striatum but not 5-HT in the cortex. However, when rats were exposed to gradually escalating doses of d-F the recovery of indoles in the brain, after injection of the large dose challenge, appeared to be faster. Indoles were markedly less reduced 1 week later in the cortex, hippocampus and striatum, with content of indole in the striatum showing complete recovery and the long-term depletion of 5-HT and 5-HIAA, by the subsequent large dose challenge was almost completely reversed in all regions. Analysis of the concentrations of d-F and its main metabolite d-fenfluramine (d-NF) in brain excluded any pharmacokinetic tolerance. These results suggest that during therapeutic treatment with d-F, the use of escalating doses may attenuate the potential for the long-lasting decrease of 5-HT in brain.

Solid-phase extraction of piribedil and its metabolites from plasma and urine without and after deconjugation, by high performance liquid chromatography.

A new high performance liquid chromatographic procedure has been developed for the simultaneous quantification of piribedil (PD) and its three main basic metabolites in rat plasma and urine, without and after hydrolysis. The procedure relies on isolation of the compounds from plasma and urine constituents using the Sep-Pak C18 cartridge, with satisfactory recovery and specificity, and resolution by acetonitrile gradient elution on a C18 reversed phase column coupled to a UV detector monitored at 240 nm. The assay was linear over a wide range of concentrations for all compounds in both body fluids with mean within-day and day-to-day coefficient of variation (CV) and relative error (RE) generally below 10%. Plasma concentrations of PD and its metabolites at selected intervals and urinary recoveries of all compounds before and after enzymatic hydrolysis are presented.

Effects of short- and long-term administration of fluoxetine on the monoamine content of rat brain.

The effects of repeated doses of fluoxetine over time and dose-responses of the content of indoles and catecholamines and metabolism, were examined in rats in relation to the concentrations of the parent compound and its active metabolite norfluoxetine in brain. Brains were removed for assays of the regional content of monoamines and concentrations of drugs 24 hr after the last dose on days 1, 7 and 21 of a twice-daily schedule of fluoxetine (15 mg/kg, i.p.). Measurements were also taken 1 week after the last dose (7.5 and 15 mg/kg, b.i.d.) of the 21-day regimen. On day 1 fluoxetine did not change the content of serotonin (5-HT) but reduced the concentrations of 5-hydroxyindolacetic acid (5-HIAA) in the hippocampus and cortex, compatible with the action of a blocker of the uptake of 5-HT. Continued injections of fluoxetine, however, significantly reduced 5-HT in the brain of the rat, the depletion being significant on days 7 and 21 in the hippocampus and cortex, respectively. The content of indoles remained significantly decreased for at least a week after the last dose of fluoxetine in the 21-day regimen, although the concentrations of 5-HIAA (but not 5-HT) totally recovered at the smaller dose (7.5 mg/kg) in all regions of the brain (cortex, hippocampus and striatum). In spite of slight changes in the concentrations and metabolism of dopamine (DA) in the striatum, 24 hr after the last dose (15 mg/kg), treatment with drug had no significant long-term effects on the content of catecholamines in these regions of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of tyrosine on the potentiation by aspartame and phenylalanine of metrazol-induced convulsions in rats.

Male rats were treated by oral intubation with tyrosine (Tyr), at doses of 0.5 and 1.0 g/kg body weight, alone or together with 1 g aspartame (APM)/kg body weight, or an equivalent dose of phenylalanine (Phe; 0.5 g/kg body weight); the effects on seizures induced by an effective dose of metrazol (ED50) were observed. Tyr (0.5 g/kg body weight) had a protective effect against the Phe-potentiation of metrazol-induced clonic-tonic convulsions. At the same dose Tyr had no effect on the seizure-promoting activity of APM, but at 1 g/kg it reduced the proconvulsant potential of the sweetener. Analysis of the brain and plasma amino acid concentrations indicated that the Tyr to Phe ratio tended to be enhanced in Tyr-Phe treated rats compared with those treated with Phe alone. This ratio remained essentially constant in the brain of APM-treated rats, compared with those treated with APM plus 1 g Tyr/kg body weight, whereas an increase in this ratio in the plasma was observed. These results confirm that Tyr antagonizes the proconvulsant effect of Phe and APM and they further suggest that no simple relationship exists between the relative brain concentrations of the two amino acids and the response to metrazol convulsions.

Determination of piribedil and its basic metabolites in plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of piribedil and its p-hydroxylated, catechol and N-oxide metabolites in plasma is described. After addition of an internal standard (buspirone), the plasma samples were subjected to a three-step extraction procedure. The final extracts were evaporated to dryness under nitrogen, and the residues were reconstituted in 100 microliters of mobile phase (0.01 M phosphate buffer-acetonitrile, 50:50, v/v) and chromatographed by acetonitrile gradient elution on a C18 reversed-phase column coupled to an ultraviolet detector set at 240 nm. The method was selective for piribedil and its metabolites, and sufficiently sensitive and precise for studies aimed at elucidating the role of the metabolites in the parent drug's pharmacological effects.

Kinetics of piribedil and effects on dopamine metabolism: hepatic biotransformation is not a determinant of its dopaminergic action in rats.

The importance of hepatic metabolism in relation to the central (dopaminergic) effects of piribedil (PD) is still not really known. Plasma and brain kinetics and the effects on striatal dopamine (DA) metabolism of the parent drug and its known metabolites were therefore evaluated in rats, a species widely used in neurochemical studies of this drug. PD injected intraperitoneally (IP, 15-60 mg/kg) and centrally (ICV, 100-200 micrograms/rat) lowered striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) content and the intensity and time-course of the neurochemical effects were route- and dose-relatedly dependent on brain PD kinetics. The catechol (M1), p-hydroxylated (M2) and N-oxide (M3) metabolites of the drug were detectable only in trace amounts in rat brain and only at the highest IP dose tested; when administered ICV at doses equimolar to PD they caused no decrease in striatal DA metabolites, although striatal concentrations were higher than after IP PD, being comparable to or higher than those of the ICV parent drug. These data suggest that metabolites do not contribute to the dopaminergic effects of PD in rats.

Propranolol does not alter flutoprazepam kinetics and metabolism in the rat.

The influence of propranolol on the disposition of flutoprazepam, a benzodiazepine derivative extensively biotransformed by hepatic microsomal oxidation, was evaluated in the rat. Propranolol was infused subcutaneously with osmotic minipumps (5 mg/day) to obtain steady-state concentrations of about 200 ng/ml. Flutoprazepam (5 mg/kg) was given intraperitoneally on the third day of propranolol infusion. There was some variability in flutoprazepam disposition, consistent with the concept of an extensive first-pass metabolism of high-extraction drugs. Propranolol had no significant effects on the kinetics of flutoprazepam or norflutoprazepam, an active metabolite possibly accounting for a substantial part of the parent compound's pharmacological and clinical effects. It was concluded that there is no evidence of any pharmacokinetic interaction between this beta-adrenoceptor blocker and flutoprazepam in the rat.