Absolute bioavailability of midazolam after subcutaneous administration to healthy volunteers.

AIMS: Midazolam is given intravenously for induction of anaesthesia and conscious sedation and by subcutaneous infusion in patients in palliative care units. The objective of the present study was to determine the absolute bioavailability of subcutaneous midazolam and its pharmacokinetics in young, healthy, male volunteers. METHODS: Eighteen volunteers were given single doses of 0.1 mg kg-1 midazolam i.v. and s.c. after a wash-out period of 7-15 days in an open-label, randomized, cross-over study. Blood samples were collected up to 12 h post-infusion. Plasma concentrations of midazolam and of its two metabolites, 1'-OHM and 4-OHM, were assessed using an h.p.l.c.-MS method (LOQ 0.5 ng ml-1 for each analyte). Vital signs, cardiac parameters and oximetry were monitored. Local tolerance was determined and adverse events were also monitored. RESULTS: After s.c. infusion t(max) and C(max) were 0.51 +/- 0.18 h and 127.8 +/- 29.3 ng ml-1 (mean +/- s.d.), respectively. No statistically significant difference was detected in AUC(0, infinity ) after i.v. and s.c. administration. The mean (+/- s.d.) absolute bioavailability of subcutaneous midazolam was 0.96 (+/- 0.14) (CI 0.84, 1.03). Mean (+/- s.d.) t1/2 was similar after s.c. (3.2 (+/- 1.0) h) and i.v. infusion (2.9 (+/- 0.7) h), although a statistically significant difference was reached (P < 0.05). Mean CL and V of i.v. midazolam were 4.4 +/- 1.0 ml min-1 kg-1 and 1.1 +/- 0.2 l kg-1 (mean +/- s.d.), respectively. Plasma concentrations of 1'-OHM were higher than those of 4-OHM. Few mild and transient adverse events were noted and there were no clinically significant effects on EEG, blood pressure and laboratory parameters. CONCLUSIONS: This study has shown that subcutaneous midazolam has excellent bioavailability and that administration of midazolam by this route could be preferable when the intravenous route is inappropriate.

Enantio-specific induction of apoptosis by an endogenous neurotoxin, N-methyl(R)salsolinol, in dopaminergic SH-SY5Y cells: suppression of apoptosis by N-(2-heptyl)-N-methylpropargylamine.

Endogenous N-methyl(R)salsolinol, which caused parkinsonism in rats by injection in the striatum, was found to induce apoptosis in dopaminergic neuroblastoma SH-SY5Y cells. After 12-h incubation with 500[microM N-methyl(R)salsolinol, almost all the cells died with apoptosis and necrotic cell death was negligible. N-Methyl(R)salsolinol was much more potent to induce apoptosis than the (S)-enantiomer. The mechanism of apoptosis was studied in relation to changes in mitochondrial membrane potential, deltapsi(m), using a fluorescent indicator, JC-1. Red fluorescence of J-aggregates representing hyperpolarized deltapsi(m) was found to decrease significantly within 60 min after incubation with N-methyl(R)salsolinol, but not by the (S)-enantiomer at the same concentration. It suggests that mitochondria may recognize the stereo-chemical structure of N-methyl(R) salsolinol. Aliphatic propargylamines, (R)-N-(2-heptyl)-N-methylpropargylamine and (R)-N-(2-heptyl)propargylamine, were found to prevent deltapsim loss and subsequent apoptosis induced by N-methyl(R)salsolinol. These results suggest that mitochondria play a key role in the induction of apoptosis by the neurotoxin and the prevention by aliphatic propargylamines.

Dopaminergic neurotoxins, 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, cause different types of cell death in SH-SY5Y cells: apoptosis was induced by oxidized papaverolines and necrosis by reduced tetrahydropapaverolines.

Dopamine-derived 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, papaverolines and tetrahydropapaverolines, have been proposed to be neurotoxin candidates related to the pathogenesis of Parkinson's disease. In this paper, the cytotoxicity of papaverolines and their N-methyl derivatives was examined using human dopaminergic neuroblastoma SH-SY5Y cells as a model of dopamine neurons. Apoptotic and necrotic cell death were assessed by morphological observation of cells after staining with propidium iodide and Hoechst 33342. Papaveroline and N-methyl-papaveroline induced apoptosis in almost all the cells with typical features of condensed and fragmented nuclei. On the other hand, (R)- and (S)-tetrahydropapaveroline caused necrosis in cells. Tetrahydropapaverolines markedly reduced adenosine triphosphate (ATP) level, whereas papaverolines did not, suggesting that the types of cell death induced by these isoquinolines, necrosis and apoptosis, depend on ATP concentrations in the cells.

Enantio-selective occurrence of (S)-tetrahydropapaveroline in human brain.

Tetrahydropapaveroline is an endogenous complex alkaloid derived from dopamine through the oxidation by monoamine oxidase. This alkaloid is considered to be involved in the pathogenesis of alcoholism and to act as a false neurotransmitter. Recently the (S) enantiomer was proposed to be a precursor of morphine biosynthesis in the opium poppy. In this paper stereo-chemical characteristic of tetrahydropapaveroline in human brains was examined. In all four control human brains examined, only the (S)-tetrahydropapaveroline was detected. The concentrations were 0.12-0.22 pmol/g wet weight of brain tissue, and the presence of alcohol in blood did not affect the concentration. The results suggest that (S)-tetrahydropapaveroline may be enantio-selectively synthesized in human brain and it may be an intermediate of the de novo synthesis of morphine analogues.

Species-differences in disposition and reductive metabolism of methoxymorpholinodoxorubicin (PNU 152243), a new potential anticancer agent.

Plasma pharmacokinetics, excretion balance and urinary metabolites of methoxymorpholino doxorubicin (MMDX) were investigated in male and female rats and in female dogs after i.v. administration of the(14)C-labelled drug. The mean total recovery of radioactivity in 96 h (urine plus faeces) was approximately 74 and 60% dose in male and female rats, respectively, while in female dogs approximately 72% dose was recovered in 336 h. Most of the radioactivity was present in faeces, with the urinary elimination accounting for only 3-4% dose in rats and dogs. These data suggest that biliary excretion is an important route of elimination of MMDX and/or its metabolites in both species. No differences were observed in the urinary metabolic profile of male and female rats. Two main peaks were present in radiochromatograms of urine from rats and dogs, i.e. MMDX and its 13-dihydro metabolite (MMDX-ol), accounting for approximately 25 and 20% of total radioactivity in 0-24-h urine in rats and 30 and 36% in dogs. The MMDX-ol/MMDX ratio in dog urine was higher than that observed in rat urine. No aglycones were detected in the urine samples from either species. In the rat, the plasma concentration-time profile suggested that the disposition of MMDX, MMDX-ol and total radioactivity is not sex-dependent. MMDX was the major species present in the systemic circulation; its AUC (0-96 h) accounted for 70% of total plasma radioactivity with the sum of AUC (MMDX) plus AUC (MMDX-ol) accounting for 77% of total radioactivity. In the dog, the sum of AUC (MMDX) plus AUC (MMDX-ol) amounted to 8% of radioactivity AUC(0-t(z) indicating that an important proportion of other(s) unknown metabolite(s) is present in dog plasma. Plasma levels of MMDX-ol in the rat were approximately 10-fold lower than those of the parent compound, whereas they were three times higher than those of MMDX in the dog. These data show that the reduction of the 13-keto group of MMDX is species-dependent, and occurs preferentially in the dog compared to the rat.

Some peculiar aspects of monoamine oxidase inhibition.

CN- ions enhance the inhibition of monoamine oxidase by the hydrazine derivatives, phenelzine [2-phenylethylhydrazine] and pheniprazine [(1-methyl-2-phenylethyl)hydrazine]. This involves partial competitive activation of the initial noncovalent enzyme-inhibitor complex with no significant effect on the subsequent reaction to give the irreversibly inhibited species. Whereas the maximum effects on pheniprazine inhibition of rat liver MAO-B occurred at about 5 microM cyanide, concentrations of 5 mM were necessary for maximum stimulation of MAO-A inhibition. A comparison of the behaviour of rat and ox MAO revealed considerable differences in their sensitivities to pheniprazine and the potentiating effects of cyanide. Species differences were also evident in the interactions derivatives of milacemide [2-n-pentylaminoacetamide] as substrates and mechanism-based inhibitors of MAO-B. In one case there was evidence for apparently large difference in inhibitor sensitivities between human brain MAO-B from different individuals.

Dose proportionality of reboxetine enantiomers in healthy male volunteers.

Reboxetine is a racemic mixture of FCE 22071 and FCE 21684 enantiomers. The pharmacokinetics of the enantiomers of reboxetine were observed to be linear in male healthy subjects (n = 6) after the administration of 1.5, 3, 4.5 mg dose of reboxetine as solutions. Kinetic analysis was based on chiral HPLC assay of the enantiomers in plasma collected up to 72 h after each administration. C(max) and AUC were more than double for FCE 22071 (C(max): 38.3+/-13.5, 76. 6+/-26.3, 99.8+/-24.1 ng/mL and AUC(infinity): 605.8+/-233.2, 1288. 3+/-796.4, 1780.7+/-669.3 ng. h/mL for 1.5, 3, 4.5 mg, respectively) than for FCE 21684 (C(max): 15.2+/-5.3, 34.6+/-14.0, 43.1+/-12.3 ng/mL and AUC(infinity): 247.0+/-103.9, 529.1+/-278.4, 773.0+/-355.3 ng. h/mL), whatever the administered dose. The half-lives of the enantiomers were similar (FCE 22071: 13.1, 11.0, 12.6 h and FCE 21684: 12.8, 11.2, 12.2 h after 1.5, 3, 4.5 mg, respectively) and not substantially affected by the dose level.

Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes.

The aim of this study was to evaluate rifabutin (RBT) and rifampicin (RIF) capabilities in inducing various xenobiotic metabolizing enzymes such as cytochromes P450 (CYPs) and UDP-glucuronosyl-transferases (UGTs) in cultured fresh and cryopreserved human hepatocytes. Enzyme induction was assessed through the use of several diagnostic markers, i.e. testosterone, midazolam (MDZ), diazepam (DZP) and 7-ethoxyresorufin for CYP-dependent enzyme reactions; and AZT for UGT-dependent enzyme reactions. RBT concentrations (0.118, 0.708 microM) were selected according to previously published pharmacokinetic data in patients. The known CYP3A4 inducer in humans, RIF, was used as a positive control. At the concentrations used, no sign of cytotoxicity was evidenced. Both compounds were able to dose-dependently induce the overall metabolism of testosterone (approximately 2-fold for RBT, 4-fold for RIF) and the formation of the 6beta-hydroxylated-derivative (up to approximately 4-fold over control for RBT and approximately 10-fold for RIF), which is CYP3A4 dependent. The other hydroxylated metabolites (16alpha-OH and 2alpha-OH) were also enhanced. The metabolism of MDZ, which is specifically metabolized by CYP3A4 in humans, was also investigated following drug's exposure to hepatocytes. DZP one, which is governed by various CYPs, including CYP3A, was also investigated. RBT was shown to increase the biotransformation of both benzodiazepines (approximately 1.9-fold over control). Moreover, the effects of both drugs on ethoxyresorufin O-deethylase activity (EROD), which is representative of CYPIA1/2 isoforms, were tested. Results showed only a moderate induction of this marker (approximately 2-fold over control) when compared to the high effect observed after hepatocyte exposure to 3-methylcholantene (approximately 14-fold over control). Finally, the action of RBT and RIF on UGTs expression was investigated by using AZT as diagnostic substrate: glucuronides formation was not significantly affected by the two rifamycin derivatives. On the whole, exposure of fresh or cryopreserved human hepatocytes to RBT dose-dependently affected the levels of drug metabolizing enzymes in a dose-dependent manner. However, as already demonstrated by in vivo pharmacokinetic studies, its inducing properties towards CYPs, CYP3A in particular, are less pronounced than RIF.

Effects of various tetrahydroisoquinoline derivatives on mitochondrial respiration and the electron transfer complexes.

We report effect of various tetrahydroisoquinoline derivatives on mitochondrial respiration and the electron transfer complexes. Generally these compounds were potent inhibitors of NADH-linked mitochondrial state 3 respiration and complex I. Presence of a phenyl group at the C1 position or oxidation of N-methylated isoquinones into N-methylisoquinolinium ion augmented the potency to inhibit mitochondrial respiration and complex I. Many of these compounds have been identified in human brains. In view of the mitochondrial and oxidative stress hypothesis, our results suggest involvement of these neurotoxins as potential causes of mitochondrial failure in Parkinson's disease.

Oxidation of N-methyl(R)salsolinol: involvement to neurotoxicity and neuroprotection by endogenous catechol isoquinolines.

1(R), 2(N)-Dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl(R)salsolinol, is a potent dopaminergic neurotoxin to induce parkinsonism in rats. The cytotoxicity of N-methyl(R)salsolinol proved to be ascribed to its oxidation into cytotoxic 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion with generation of hydroxyl radical. The isoquinolinium ion caused massive necrosis in the striatum, whereas N-methyl(R)salsolinol depleted selectively dopaminergic neurons in the substantia nigra without necrotic tissue reaction. N-Methyl(R)salsolinol induced DNA damage to human neuroblastoma SH-SY5Y cells, which could be prevented by anti-oxidants and cycloheximide. These results suggest that oxidative stress through oxidation of N-methyl(R)salsolinol induces apoptotic cell death. On the other hand, (R)salsolinol proved to scavenge hydroxyl radical produced by oxidation of dopamine. The neurotoxicity and neuroprotection of catechol isoquinolines may be ascribed to their oxidation and scavenging of radicals.

Synthesis and anticonvulsant activity of a new class of 2-[(arylalky)amino]alkanamide derivatives.

Although most epilepsies are adequately treated by conventional antiepileptic therapy, there remains an unfulfilled need for safer and more effective anticonvulsant agents. Starting from milacemide, a weak anticonvulsant, and trying to elucidate its mechanism of action, we discovered a structurally novel class of potent and preclinically safe anticonvulsants. Here we report the structure-activity relationship (SAR) study within this series of compounds. Different parts of the structural lead 2-[[4-(3-chlorobenzoxy)benzyl]amino]acetamide (6) were thus varied (Figure 1), and many potent anticonvulsants were found. As an outcome of this study, 57 ((S)-2-[[4-(3-fluorobenzoxy)benzyl]amino]propanamide methanesulfonate, PNU-151774E) emerged as a promising candidate for further development for its potent anticonvulsant activity and outstanding therapeutic indexes (TIs) in different animal tests.

Quantitative determination of cabergoline in human plasma using liquid chromatography combined with tandem mass spectrometry.

A liquid chromatography/mass spectrometry (LC/MS) method using electrospray ionization (ESI) is described for the quantitative determination of cabergoline (N-[3-(dimethylamino)propyl]-N-(ethylamino)-carbonyl-6-(2-propenyl)- ergoline-8 beta-carboxamide) in human plasma. The method consists of liquid-liquid extraction after addition of deuterated internal standard, and reverse-phase liquid chromatography with electrospray ionization combined with tandem mass spectrometry (MS/MS). Using selected reaction monitoring, the method provides a quantitation limit of 1.86 pg/mL. Calibration curves acquired on five different days showed good linearity (r > 0.99) in the range 1.86-124 pg/mL and reproducibility of the slope (% relative standard deviation, RSD = 10.0). The intra-day precision, determined by assaying plasma containing four different concentrations of cabergoline processed in replicate, was found to range from 2.4 to 17.0% (RSD). The inter-day precision, evaluated for the same concentrations, ranged from 7.9 to 10.7% (RSD). The accuracy of the method, expressed as the percent ratio between found to added amount, was 99.1 +/- 10.2% (RSD = 10.3%, n = 78).

An endogenous dopaminergic neurotoxin, N-methyl-(R)-salsolinol, induces DNA damage in human dopaminergic neuroblastoma SH-SY5Y cells.

Recently, an endogenous neurotoxin, 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl-(R)-salsolinol], was found to elicit parkinsonism in rats with selective depletion of dopamine neurons in the substantia nigra without necrotic tissue reaction. The mechanism of the cell death was examined by detection of DNA damage using a single-cell gel electrophoresis (comet) assay in human dopaminergic neuroblastoma SH-SY5Y cells. Only N-methylsalsolinol was found to induce DNA damage, whereas other catechol isoquinolines, such as (R)-salsolinol, (S)-salsolinol, and 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion, did not. The (R)-enantiomer of N-methylsalsolinol damaged DNA much more profoundly than the (S)-enantiomer. Cycloheximide protected the cells from DNA damage, suggesting that an apoptotic process may account for the DNA damage. Morphological changes indicating apoptotic cell death were also confirmed. Antioxidants and deprenyl reduced DNA damage, indicating that the damage was initiated by oxidative stress and that neuroprotection by deprenyl may be partially ascribed to its prevention of DNA damage. Apoptosis induced by neurotoxins may be a mechanism underlying the cell death of dopamine neurons in the substantia nigra of Parkinson's disease.

Review of the pharmacokinetics and metabolism of reboxetine, a selective noradrenaline reuptake inhibitor.

The pharmacokinetics and metabolism of reboxetine, a selective noradrenaline reuptake inhibitor, in humans and animal models are reviewed here. Reboxetine has potent antidepressant activity, low affinity for alpha-adrenergic and muscarinic receptors and low toxicity in animals. It is a mixture of (R,R) and (S,S) enantiomer, the latter being more potent but no qualitative differences in pharmacodynamic properties are observed between the two. Humans rapidly absorb reboxetine (tmax about 2 h) with a terminal half-life of elimination (t1/2) of 13 h, allowing twice-daily administration. Animal models also rapidly absorb reboxetine (tmax 0.5-2 h) but t1/2 was 1-2 h. Food does not affect bioavailability. There were no major inter-species differences in the metabolic profile of reboxetine. Elimination is principally renal in humans and monkeys. Reboxetine has linear pharmacokinetics in young, healthy males for single doses of 1-5 mg and in elderly, female depressed patients (up to 4 mg b.i.d.). Multiple dosing, gender or liver insufficiency had no significant effects on the pharmacokinetics. Elderly (particularly frail elderly) patients and patients with severe renal impairment may need dose reduction. Reboxetine shows no clinically relevant interaction with lorazepam and has no inhibitory effects on the major enzymes involved in drug metabolism. It may be possible to use reboxetine in combination with monoamine oxidase inhibitors as it has no inhibitory effect on this enzyme; in addition, it may protect patients against tyramine-induced reactions. In conclusion, reboxetine seems to be an antidepressant with negligible interference with the pharmacokinetics of other drugs thus fewer drug-drug interactions are expected.

A dopaminergic neurotoxin, 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl(R)salsolinol, and its oxidation product, 1,2(N)-dimethyl-6,7-dihydroxyisoquinolinium ion, accumulate in the nigro-striatal system of the human brain.

N-Methyl(R)salsolinol was found to be an endogenous dopaminergic neurotoxin inducing parkinsonism in rodents and to increase in the cerebrospinal fluid of parkinsonian patients. The amounts of N-methyl(R)salsolinol and related compounds in the human brain regions were quantitatively analyzed. Only the (R)-enantiomer of salsolinol derivatives were detected, which suggests their enzymatic synthesis in situ. In the nigro-striatal system, the concentration of N-methyl(+)salsolinol was higher than in the frontal cortex, and its oxidized catechol isoquinolinium ion was detected only in the substantia nigra significantly. The accumulation of these neurotoxins in the nigro-striatal region might account for selective cell death of dopamine neurons in the substantia nigra of Parkinson's disease.

Assay for the (R)- and (S)-enantiomers of salsolinols in biological samples and foods with ion-pair high-performance liquid chromatography using beta-cyclodextrin as a chiral mobile phase additive.

A chromatographic procedure was devised for the quantitative determination of the enantiomers of salsolinol and N-methylsalsolinol, which are biologically important alkaloids. The enantiomers of salsolinol and N-methylsalsolinol were completely separated using beta-cyclodextrin in a reversed-phase ion-pair system. The HPLC method was sensitive enough to detect the isoquinolines at a concentration less than 0.1 pmol per injection. The presence of (R)- and (S)-salsolinol was confirmed in fermented foods and beverages, while N-methylsalsolinol was not detected. On the other hand, the (R)-enantiomers of both salsolinol and N-methylsalsolinol were found to predominate in the human brain.

Pharmacokinetics of reboxetine enantiomers in the dog.

Reboxetine, (RS)-2-[(RS)-alpha-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. The pharmacokinetics of reboxetine enantiomers were determined in a crossover study in three male beagle dogs. Each animal received the following oral treatments, separated by 1-week washout period: 10 mg/kg reboxetine, 5 mg/kg (R,R)- and 5 mg/kg (S,S)-. Plasma and urinary levels of the reboxetine enantiomers were monitored up to 48 h post-dosing using an enantiospecific HPLC method with fluorimetric detection (LOQ: 1.1 ng/ml in plasma and 5 ng/ml in urine for each enantiomer). After reboxetine administration mean tmax was about 1 h for both enantiomers. Cmax and AUC were about 1.5 times higher for the (R,R)- than for the (S,S)-enantiomer, mean values +/- SD being 704 +/- 330 and 427 +/- 175 ng/ml for Cmax and 2,876 +/- 1,354 and 1,998 +/- 848 ng.h/ml for AUC, respectively. No differences between the (R,R)- and (S,S)-enantiomers were observed in t1/2 (3.9 h). Total recovery of the two enantiomers in urine was similar, the Ae (0-48 h) being 1.3 +/- 0.7 and 1.1 +/- 0.7% of the enantiomer dose for the (R,R)- and the (S,S)-enantiomers, respectively. No marked differences in the main plasma pharmacokinetic parameters were found for either enantiomer on administration of the single enantiomers or reboxetine. No chiral inversion was observed after administration of the separate enantiomers, as already observed in humans.

Cytotoxicity of endogenous isoquinolines to human dopaminergic neuroblastoma SH-SY5Y cells.

Endogenous isoquinolines with and without catechol structure have been proposed to be neurotoxins specific for dopamine neurons. In this paper they were examined for the cytotoxicity of human dopaminergic neuroblastoma SH-SY5Y cells. The cytotoxicity was quantitatively determined using Alamar Blue assay, by which the reduction-oxidation potency in the living cells can be measured spectrometrically. 1,2-Dimethyl-6,7-dihydroxyisoquinolinium ion [1,2-DMDHIQ+], an oxidation product of a parkinsonism-inducing isoquinoline, 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahroisoquinoline [N-methyl-(R)salsolinol, NM(R)Sal] was found to be the most potent toxin among isoquinolines examined. In general, catechol isoquinolines were more toxic than isoquinolines without catechol structure. With and without catechol structure, the oxidized isoquinolinium ion having methyl groups at C-1 and N-2 positions proved to be more cytotoxic than the simple isoquinolines. The involvement of 1,2-DMDHIQ+ to the neurotoxicity of NM(R)Sal was suggested and discussed.

N-methyl-(R)salsolinol as a dopaminergic neurotoxin: from an animal model to an early marker of Parkinson's disease.

A dopamine-derived 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydrosioquinoline [N-methyl-(R)salsolinol] was found to occur enantioselectively in human brain. This isoquinoline induced parkinsonism in rat after injection in the striatum, and the behavioral, biochemical and pathological changes were very similar to those in Parkinson's disease. N-Methyl-(R)salsolinol depleted dopamine neurons in the rat substantia nigra without necrotic tissue reaction, which may be due to the apoptotic death process, as proved by its induction of DNA damage in dopaminergic neuroblastoma SH-SY5Y cells. N-Methyl-(R)salsolinol was found to increase significantly in the cerebrospinal fluid of parkinsonian patients. All these results suggest that N-methyl-(R)salsolinol may be an endogenous neurotoxin to cause Parkinson's disease and the enzymes involved in its biosynthesis and catabolism may be endogenous factors in the pathogenesis of this disease.

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.