Pharmacokinetic equivalence of 5(ethyl(2H)5)- and unlabelled phenobarbitone.

The present study shows the absence of in vivo pharmacokinetic isotope effect on phenobarbitone (PB) C5-ethyl deuteration (PBd5) following oral administration to man of equimolar PB/PBd5 mixtures (0.40 mmol each). Plasma PB and PBd5 (17 days) and urine PB, PBd5 and parahydroxy-metabolites (PBOH, PBHOd5) levels were determined by GC-MS. Isotope effect research includes comparison of pharmacokinetic parameters, study of time-dependence of isotope ratios (IRs) in plasma and urine (linearity test), comparison of IRs between samples and administered mixtures (Mann Whitney's test) and comparison of PBOH/PBOHd5 ratios before and after urine enzymatic hydrolysis (Student's two tailed t-test). No significant isotope effect was observed on pharmacokinetic parameters, PB hydroxylation or PBOH conjugation (x less than or equal to 5%); which the absence of pentadeuteration-induced alteration in PB's HSA binding parameters (binding mode, Ka, N) corroborates (x less than or equal to 5%). These results establish bioequivalence of PB and PBd5; the latter can be used with benefit in stable-isotope clinical pharmacology (steady state pharmacokinetics, drug interactions...) investigations as well as bioavailability studies of PB preparations.

Determination of ketoprofen in biological fluids by reversed-phase chromatography.

A sensitive and specific high-performance liquid chromatographic method was developed for the determination of ketoprofen [2-(3-benzoylphenyl)propionic acid] in plasma and urine. The method includes an extraction of the drug and the internal standard [2-(4-benzoylphenyl)butyric acid] into ether from acidified plasma. The organic phase is evaporated, and the residue is dissolved in the mobile phase (acetonitrile-0.02 M phosphate buffer, pH 3) (45:55). A 20-microliter aliquot is analyzed on a reversed-phase column. The accuracy is within 1.5% for therapeutic concentrations, and the coefficients of variation are 5.5 and 3.4% for 2 and 10 micrograms/ml, respectively. For the urine assay, the accuracy is within 3%, and the cofficients of variation are 1.9 and 1.7% for 3 and 50 micrograms/ml, respectively. This method was applied successfully to the determination of ketoprofen in humans for pharmacokinetic studies.

Effects of capsaicinoids on oxidative metabolism of caffeine in isolated rat hepatocytes.

The metabolism of caffeine was studied in isolated rat hepatocytes, in the absence and presence of capsaicinoids. Caffeine and four primary metabolite fractions were identified by high performance liquid chromatography: 1,7-dimethylxanthine, 3,7-dimethylxanthine, 1,3-dimethylxanthine and 1,3,7-trimethyluric acid. The incubation with the lowest concentrations (0.1 and 1 microM) of capsaicinoids (natural extract, capsaicin, dihydrocapsaicin) showed a stimulatory effect on caffeine metabolism, which was further enhanced with capsaicin. At 10 microM, capsaicin stimulated the two pathways of metabolism of caffeine (N-demethylation and C-8 oxidation). In contrast, dihydrocapsaicin and the natural extract seem to inhibit the N-demethylation pathways without affecting the C-8 oxidation route. The inhibitory activity on the N-demethylation pathways and especially the N-7 demethylation pathway was pronounced at the first 30 min of incubation. These results suggest that the two pathways (N-demethylation and C-8 oxidation) are mediated by different isozymes of cytochromes P-450. This is in agreement with recent findings.

Methods of theophylline assay and therapeutic monitoring of this drug.

The purpose of this article is to review various analytical methods of monitoring plasma theophylline. This article was investigated by the "Drug Commission" of SFBC (Société Française de Biologie Clinique). The primary objective is to provide the "know-how", particular for this analysis, which allows the choice between various analytical methods available: immunochemical or physiochemical ones. The techniques described are not necessarily the best, they are approved and tested methods which are the most frequently used in routine practice. The proposed immunochemical methods are: absorption spectroscopy methods: Enzyme ImmunoAssay (EIA), Enzyme Multiplied ImmunoAssay Technique (EMIT); Reflectance spectroscopy method: Apoenzyme Reactivation Immunoassay System (ARIS); Fluorometry spectroscopy method: Substrate Labeled FluoroImmunoAssay (SLFIA); Fluorometry spectroscopy on solid base; Polarization fluorescence spectroscopy ImmunoAssay (FPIA); Turbidimetric measurements: Particle Enhanced Turbidimetric Inhibition ImmunoAssay (PETINIA); Nephelometric measurement: Nephelometric Inhibition ImmunoAssay (NIIA). And the proposed physicochemical methods are: High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC). The second objective is a review of pharmacological properties of theophylline, necessary for a good understanding of therapeutic drug monitoring: intestinal resorption, distribution, metabolism and elimination, drug interactions, dose/response relationship, physiopathological variations and proposed "predictive" "theophylline test". The authors conclude that because of the multiplicity of methodologies used in theophylline therapeutic monitoring the choice of one of them is not easy. The best way to compare different techniques available would be the use of a "reference material" for theophylline monitoring and a quality control network between different clinical pharmacological laboratories.

Simultaneous quantitation of phenobarbitone and p-hydroxyphenobarbitone and their perdeuteroethyl analogues in biological fluids by combined gas chromatography-mass spectrometry.

In order to develop 5-pentadeuteroethyl-5-phenyl barbituric acid as an alternative tracer in pharmacokinetic and metabolic studies of phenobarbitone, and to search for possible isotope effects associated with such labelling, we propose a gas chromatographic-mass spectrometric assay for simultaneous measurement of phenobarbitone, p-hydroxyphenobarbitone and their perdeuteroethyl analogues, using [1,3-15N2,2-13C] phenobarbitone as internal standard. These compounds were extracted from plasma (50 microliter) or urine (500 microliter) and pentylated according to Greeley's method. Linear calibration curves were obtained in the concentration range from 0.5 to 3 micrograms/ml. The interday precision, mean accuracy and detection limit were 0.77-5.28%, 99.99-100.80% and 0.03-0.05 microgram/ml, respectively. Results for plasma and urine concentrations, and pharmacokinetic parameters in humans, are presented to illustrate this method.

Gas chromatographic-mass spectrometric quantitation of tri-, di- and monomethylxanthines and uric acids from hepatocyte incubation media.

A gas chromatographic-mass spectrometric method is described for the measurement of the concentration of fourteen methylxanthines and methyluric acid metabolites of methylxanthines, especially caffeine, from cell incubation media. The method shows linearity, accuracy and recovery suitable for metabolic studies. The reproducibility of relative retention times is satisfactory (less than 0.07%) and allows rapid and conclusive identification of chromatographic peaks corresponding to metabolites. Moreover, this method enables the simultaneous determination of 3.7-methylxanthine and its 1.7-isomer, which are not chromatographically resolved. This method can be successfully applied when molecules labelled with stable isotopes are used as tracers for metabolic studies.

[Determination of ketoprofen in plasma using high-performance liquid chromatography. Comparison with gas--liquid chromatography (author's transl)]. / Dosage du kétoprofène dans le sang par chromatographie liquide haute performance. Comparaison avec la chromatographie en phase gazeuse.

A new method of determination of ketoprofen 2-(3-benzoyl phenyl) propionic acid in plasma using high-performance liquid chromatography (HPLC) is described. After extraction by diethyl either in acidic medium, ketoprofen and the internal standard, 2-(4-benzoyl phenyl) butyric acid, are methylated with gaseous diazomethane and their concentrations measured by HPLC using in LiChrosorb Si 60 (5 micrometer) column and dichloromethane-hexane (60:40) as the mobile phase. The absolute retention times of the internal standard and ketoprofen are 11.6 and 12.8 min, respectively. The precision of the methods is +/- 4% and the lower detection limit ranges from 0.06 to 0.10 microgram/ml. The results obtained by HPLC show a very good correlation with those obtained by gas--liquid chromatography. The proposed method is sensitive, reproducible and rapid and very suitable for ketoprofen determination in pharmacokinetic studies.

[Pharmacokinetics of injectable phenobarbital in the premature infant. Study of a new lyophilized form]. / Pharmacocinétique du phénobarbital injectable chez le prématuré. Etude à partir d'une nouvelle forme lyophilisée.

A lyophilized preparation of phenobarbital was studied in newborns without cerebral palsy. Plasma levels were determined using gas chromatograph fitted with thermo ionic probe after either an intra-muscular (IM) injection in premature infants or an intravenous (IV) injection over single dose of phenobarbital 10 mg/kg within 6 hours after birth. Five term babies were included in the study as controls and received an IM injection. The results showed rapid increase in plasma concentration after IM injection in 10 of 13 subjects with a peak concentration reached 60 minutes after injection. The mean ratio (maximal concentration/dose) was 1.25 and 1.10 for term infants and preterm infants respectively. In all cases, the drug was well tolerated. In 15 preterm infants (n: 7 IM and n: 8 IV) the plasma concentrations were followed over a period of 15 days. The disappearance curve was biphasic; it varied the first 7 days, then remained constant for the following week (apparent half life 106 hours).

Study of theophylline metabolism in premature human newborns using stable isotope labelling.

A new metabolic pathway of theophylline has been investigated in premature human newborns using the ion cluster technique of stable isotope labelling combined with gas chromatography mass spectrometry. Labelled caffeine, paraxanthine and theobromine have been found in plasma and urine of two preterm newborns receiving [1,3-15N], [2-13C] theophylline for the treatment of primitive apneas. Theophylline is converted to caffeine by N-7 methylation. In adults, the inverse process exists wherein caffeine is demethylated to give theophylline.

Plasma xanthine levels in low birthweight infants treated or not treated with theophylline.

The use of theophylline in the management of apnoea in the newborn was studied in 33 preterm infants. Infants received a dose of 3 mg/kg, 13 of them every six hours, the remaining 20 every eight hours. All the infants had significantly fewer apnoeic episodes. In a pharmacokinetic study, the half life of theophylline was 30.3 +/- 7.2 hours and the clearance rate was 23.9 +/- 5.06 ml/kg per hour (means and SD). The plasma theophylline level remained constant at between 13 and 15 mg/l from the 5th day of treatment but, at the same time, the plasma levels of caffeine rose to a mean level of 4.4 mg/l. Caffeine was detectable in plasma at birth, and in preterm infants not receiving theophylline; plasma levels of caffeine tended to be similar to the levels in their mothers' milk. These observations have led to clear conclusions on the optimum timing and dosage of theophylline, and on the need to monitor plasma levels of both theophylline and caffeine in newborn infants treated with theophylline.

In vivo N7 methylation of theophylline to caffeine in premature infants. Studies with use of stable isotopes.

We studied the metabolism of theophylline in premature neonates by the use of molecules labelled with stable isotopes. 2 prematures received from birth up to 8th day of life 3 mg/kg/8 h of a mixed solution containing 46% of labelled and 54% of unlabelled theophylline. Plasma levels of caffeine and theophylline, measured by gas chromatographic mass spectrometric analysis, demonstrated the in vivo biotransformation of theophylline to caffeine in prematures.