Klaus D. Haegele

Kinetics of α‐difluoromethylornithine: An irreversible inhibitor of ornithine decarboxylase

We gave α‐difluoromethylornithine (DFMO), a selective, irreversible inhibitor of ornithine decarboxylase, to six healthy men in single intravenous doses of 5 and 10 mg/kg body weight and oral doses of 10 and 20 mg/kg. Plasma concentrations were monitored during the 24 hr after each dose. Urine was collected from 0 to 24 hr after drug and amount of unchanged drug excreted was determined. Peak plasma concentrations were reached within 6 hr after oral doses. The decay of the plasma concentrations followed first‐order kinetics with a mean half‐life (t½) for all four doses studied of 199 ± 6 min (±SD). Mean total body clearance (ClT) for the four doses was 1.20 ± 0.06 ml · min−1 · kg−1. Mean renal clearance was determined as 0.99 ± 0.03 ml · min−1 · kg−1, accounting for 83% of drug elimination. Mean apparent volume of distribution (aVD) was 0.337 ± 0.031 l/kg−1, corresponding to 24 l for 70 kg of body weight. The amount of unchanged drug in 24‐hr urine samples was 47 ± 7% and 40 ± 11% after 10 and 20 mglkg orally, and 78% and 81 ± 8% after 5 and 10 mglkg intravenously. Bioavailability of the 10 mg/kg dose was estimated as 58% from the urinary recoveries and as 54% from the areas under the plasma concentration curves (AUC0→∞). Since doubling of the dose resulted in a doubling of the mean AUC0→∞ and since other kinetic parameters, such as aVD, t½, ClT, and the urinary recovery of unchanged drug, were essentially the same at all doses, DFMO kinetics follow a dose‐linear model.

Determination of the R(―)- and S(+)-enantiomers of γ-vinyl-γ-aminobutyric acid in human body fluids by gas chromatography―mass spectrometry

Abstract An analytical procedure, which allows the determination and quantitation of the R(−)- and S(+)-enantiomers of γ-vinyl-γ-aminobutyric acid (γ-vinyl-GABA; MDL 71.754) in body fluids was developed. The method is based on a combined gas chromatographic—mass spectrometric technique. A glass capillary column coated with a chiral phase enabled the separation of the enantiomers of γ-vinyl-GABA as their N-trifluoroacetyl-O-methyl ester derivatives. This was followed by quantitation using a selected ion monitoring technique in the electron-impact mode of ionization. The internal standard,

Effect of Long-Term Vigabatrin Therapy on Selected Neurotransmitter Concentrations in Cerebrospinal Fluid

γ-acetylenic GABA, was included throughout the work-up of the samples. The assay was shown to be reproducible, specific and sensitive. No interferences were encountered from plasma, urine or cerebrospinal fluid constituents. The method was applied to the analysis of plasma samples obtained from a human volunteer who had received racemic γ-vinyl-GABA. Significant differences in the plasma concentrations and plasma half-lives of the two enantiomers were seen, clearly illustrating the need for a specific assay technique capable of distinguishing between the enantiomers of this drug.

Analysis of 2-difluoromethyl-dl-ornithine in human plasma, cerebrospinal fluid and urine by cation-exchange high-performance liquid chromatography

Ten patients, suffering from drug-resistant complex partial seizures were treated for a period of up to 3 years with vigabatrin (Sabril). Vigabatrin is a novel antiepileptic agent, whose action is based on the inhibition of γ-aminobutyric acid (GABA) aminotransferase, the enzyme responsible for the catabolism of the neurotransmitter GABA. Samples of lumbar cerebrospinal fluid were obtained from the patients prior to commencing vigabatrin therapy, and thereafter at 6 months, 1 year, 2 years, and up to 3 years following the initiation of vigabatrin treatment. The influence of vigabatrin on the cerebrospinal fluid concentrations of free and total GABA, homocarnosine, homovanillic acid, 5-hydroxyindoleacetic acid, and 3-methoxy-4-hydroxyphenylethylene glycol, as well as of the drug itself, was assessed. All patients demonstrated a clinical response to vigabatrin, and the drug was well tolerated over the entire observation period. Mean (±SD) reduction of seizure frequency was 65% ± 23% (range, 26% to 100%) when comparing the end of the treatment period to the previgabatrin baseline. The cerebrospinal fluid concentrations of both free and total GABA and of the dipeptide homocarnosine showed approximately 2- to 5-fold increases over baseline values, with free GABA and homocarnosine being the more sensitive variables. Cerebrospinal fluid concentrations of homovanillic acid, 5-hydroxyindoleacetic acid, and 3-methoxy-4-hydroxyphenylethylene glycol were not altered in a significant manner over the observation period. These findings support the concept that the effects of vigabatrin are restricted to an effect on GABA catabolism and do not extend to the neurotransmitters dopamine and norepinephrine. Clinical efficacy and elevation of GABA and homocarnosine concentration were sustained over the period of observation. (J Child Neurol 1991;6(Suppl):2S11-2S16).

pNAT and CYP2D6 gene polymorphism in epileptic patients

An analytical method has been developed based on cation-exchange liquid chromatography for the measurement of 2-difluoromethyl-DT-ornithine (DFMO) in human plasma, cerebrospinal fluid (CSF) and urine. Fluorescence detection at excitation/emission wavelengths of 340/440 nm is followed by postcolumn derivatization with o-phthalaldehyde-2,mercaptoethanol. All calibration ranges yielded linear relationships with correlation coefficients better than 0.999. In each case the limit of quantitation was equal to the lowest value of the standard curve. The variability of the assay, expressed as relative standard deviations, was less than 7.1%, 15.3% and 7.1% for plasma, CSF and urine, respectively. The accuracy of the assay (expressed as relative errors) ranged between 4.3% and 2.0% for plasma analysis, between -0.1% and 14.0% for CSF analysis and between -8.0% and 2.0% for urine analysis. Plasma, CSF and urinary DFMO concentrations were measured in samples obtained from patients undergoing treatment for trypanosomiasis. The method was found to be applicable for the measurement of DFMO levels in human body fluids for the determination of pharmacokinetic parameters in clinical studies.

beta- and alpha-Adrenoceptor antagonism by medroxalol in healthy volunteers: relationship to dose and plasma concentration.

Certain anticonvulsant drugs require N-acetylation as a major route of metabolic clearance. Single point mutations of the polymorphic N-acetyltransferase gene (pNAT) are the primary cause for impaired drug acetylation. Pharmacokinetic parameters are altered in slow acetylator phenotypes and this may compromise drug safety. Genetic analysis of allelic frequencies of individual pNAT genotypes point to significant increases in carriers of the S1/wt and S3/wt (P < 0.05) allele and a significant reduction in carriers of the S2/S2 (P < 0.01) allele, when control and epileptic patients are compared. Furthermore, the presumed link between the cytochrome P450 CYP2D6 polymorphism and the pathogenesis of Parkinsons disease led us to investigate, whether a similar relationship can be expected for other CNS disorders. Our findings indicate that poor metabolizers are more frequent (P < 0.05) amongst epileptic patients, when compared with a control population. An estimate of the odds ratio may suggest an increased risk [95% CI (confidence interval) 1.043-4.734] of up to 5-fold in epileptic patients carrying this mutation. This provides further evidence for a potential link between the debrisoquine hydroxylase gene polymorphism and CNS disorder and therefore warrants further study.

Simultaneous measurement of the major metabolites of dolasetron mesilate in human urine using solid-phase extraction and high-performance liquid chromatography

Summary We studied β- and α-adrenoceptor blockades by medroxalol in eight normal, male volunteers using exercise and isoproterenol tests for β-antagonism and a phenylephrine test for α-antagonism. Each volunteer received three oral doses (400–1,200 mg) and two intravenous doses (range 0.5–1.5 mg/kg) at 1− to 6-week intervals. Venous blood was sampled for determination of the medroxalol plasma concentration (Cp) at fixed intervals up to 25 h after each dose. Ergometric exercise and adrenergic agonist injections (isoproterenol) or infusions (phenylephrine) were repeated at 1, 4, and 24 h after medroxalol and results compared to pretreatment control values. In addition, isoproterenol and phenylephrine tests were performed 5 min after the intravenous infusion of medroxalol. After oral administration, peak Cp of medroxalol occurred between 2 and 3 h and the mean t1/2 elimination was 11.1 ± 0.6 h. Heart rate at 75% maximum load (HR 75) decreased significantly after the three oral doses with a peak dose-dependent effect at 4 h. At this time, the dose of isoproterenol which raised heart rate by 25 beats/minute (CD 25) was increased 27 times by 400 mg of medroxalol, 51 times by 800 mg, and 94 times by 1,200 mg. The dose of phenylephrine necessary to increase mean blood pressure by 15 mm Hg (PD 15) increased 1.3-fold and 2.4-fold after 400 mg and 1,200 mg of medroxalol, respectively. β-Blockade remained significant for at least 24 h after medroxalol. Reduction in HR 75 and increase in CD 25 were both significantly correlated with log Cp of medroxalol, whereas the increase in PD 15 did not correlate with log Cp. After intravenous administration of medroxalol. β-blockade was maximal at the end of infusion and was proportional to log Cp. but there was no significant change in PD 15. Medroxalol administration was well tolerated, and minor gastrointestinal disturbances appeared 1 - 2 h only after the 1,200 mg oral dose in five out of seven subjects. We conclude that medroxalol (a) has a long plasma elimination half-life, (b) exerts a marked and prolonged β-adrenoceptor blockade, and (c) has significant but less marked α-adrenoceptor blocking properties, which appear only after oral administration of the drug.

Kinetics and metabolism of p-tyramine during monoamine oxidase inhibition by mofegiline.

A method based on solid-phase extraction and high-performance liquid chromatography (HPLC) has been developed for the simultaneous quantitation of the principal active metabolites of dolasetron mesilate [i.e. MDL 74,156 (II), MDL 102,382 (III) and MDL 73,492 (IV)] in human urine. The method has been validated over the concentration range of 200-5000 pmol/ml for all three metabolites. Within-day and day-to-day coefficients of variation were less than 9 and 14%, respectively, for the three metabolites. The method allowed the simultaneous quantitation of III, IV and II and the evaluation of the urinary excretion of these metabolites in human urine following the administration of dolasetron mesilate.

Analysis of (R)-4-oxo-5-phosphononorvaline (MDL 100,453) in rat plasma and brain dialysate using liquid chromatography after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

The effects of monoamine oxidase B (MAO‐B) inhibition by mofegiline on the pharmacokinetics of p‐tyramine and its major metabolite, p‐hydroxyphenylacetic acid, were investigated in 24 healthy male volunteers. p‐Tyramine doses were administered before and after a 14‐day treatment period of 1, 12, or 24 mg mofegiline or placebo. Normalized p‐tyramine for area under the plasma concentration–time curve after treatment were not significantly different from their respective before‐treatment values for any of the dose groups. The relative bioavailability of p‐tyramine after treatment was not significantly different from before treatment, although a tendency to a greater bioavailability was seen with the 12 and 24 mg doses. There were no significant differences between pharmacokinetic parameters for p‐hydroxyphenylacetic acid. The data suggest that mofegiline maintains its selectivity for MAO‐B in the intestine and liver at doses up to and including 24 mg. Therefore these doses would not be expected to be associated with the hypertensive crises normally associated with the “cheese effect.”

Kinetics of medroxalol, a β‐ and α‐adrenoceptor antagonist

A liquid chromatographic (LC) method with precolumn derivatization and fluorescence detection has been developed for the quantitation of (R)-4-oxo-5-phosphononorvaline (MDL 100,453), which is a selective antagonist of N-methyl-D-aspartate receptor, in rat plasma and brain dialysate. The plasma samples were deproteinized with acetonitrile and then derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The brain dialysis samples were dried in vacuum, reconstituted with borate buffer, and derivatized with AQC. The derivatized MDL 100,453 was analyzed by LC with a Nova-Pak C18 column at 32 degrees C using a gradient mobile phase. Detection was accomplished by fluorescence with excitation at 250 nm and emission at 395 nm. This analytical method was used to follow the time course of drug concentrations in rat plasma and brain dialysate after intravenous (i.v.) bolus injection of MDL 100,453 or a combination of i.v. bolus injection and i.v. infusion.