Polymorphic hydroxylation of perhexiline in vitro.

AIMS: The aims of this study were to examine the in vitro enzyme kinetics and CYP isoform selectivity of perhexiline monohydroxylation using human liver microsomes. METHODS: Conversion of rac-perhexiline to monohydroxyperhexiline by human liver microsomes was assessed using a high-performance liquid chromatography assay with precolumn derivatization to measure the formation rate of the product. Isoform selective inhibitors were used to define the CYP isoform profile of perhexiline monohydroxylation. RESULTS: The rate of perhexiline monohydroxylation with microsomes from 20 livers varied 50-fold. The activity in 18 phenotypic perhexiline extensive metabolizer (PEM) livers varied about five-fold. The apparent Km was 3.3 +/- 1.5 micro m, the Vmax was 9.1 +/- 3.1 pmol min-1 mg-1 microsomal protein and the in vitro intrinsic clearance (Vmax/Km) was 2.9 +/- 0.5 micro l min-1 mg-1 microsomal protein in the extensive metabolizer livers. The corresponding values in the poor metabolizer livers were: apparent Km 124 +/- 141 micro m; Vmax 1.4 +/- 0.6 pmol min-1 mg-1 microsomal protein; and intrinsic clearance 0.026 micro l min-1 mg-1 microsomal protein. Quinidine almost completely inhibited perhexiline monohydroxylation activity, but inhibitors selective for other CYP isoforms had little effect. CONCLUSIONS: Perhexiline monohydroxylation is almost exclusively catalysed by CYP2D6 with activities being about 100-fold lower in CYP2D6 poor metabolizers than in extensive metabolizers. The in vitro data predict the in vivo saturable metabolism and pharmacogenetics of perhexiline.

Method for the analysis of perhexiline and its hydroxy metabolite in plasma using high-performance liquid chromatography with precolumn derivatization.

A high-performance liquid chromatographic method for the analysis of perhexiline and its monohydroxy metabolite in plasma has been developed. After a simple extraction procedure, the analytes are derivatized over a 30-min period with trans-4-nitrocinnamoyl chloride. The derivatized products are monitored at 340 nm following separation on a 5-micron phenyl reversed-phase column under isocratic conditions. The limits of detection for perhexiline and its hydroxy metabolite are 0.03 and 0.02 mg/l, respectively. The between-day and within-day assay coefficients of variation for perhexiline and its hydroxy metabolite at concentrations of 0.2 and 1.0 mg/I were less than 10%. The method has proved robust and suitable for the routine monitoring of perhexiline and hydroxyperhexiline.

Single oral dose pharmacokinetics of erythromycin and roxithromycin and the effects of chronic dosing.

Roxithromycin is a semisynthetic macrolide antibiotic having similar in vitro antibacterial profile and potency to erythromycin but possibly greater in vivo potency. The single and multiple oral dose pharmacokinetics of roxithromycin and erythromycin were studied in 12 healthy volunteers. Plasma concentrations of the two compounds were measured by a sensitive and specific high-performance liquid chromatographic method using electrochemical detection. After single doses, roxithromycin 150 mg gave a Cmax 3.3-fold higher and an area under the curve (AUC) 16.2-fold higher than erythromycin 250 mg. The half-life for roxithromycin was 12.42 +/- 3.94 h compared with 1.53 +/- 0.42 h for erythromycin. On multiple dosing, the AUC over a dosing interval for erythromycin (250 mg, six hourly doses) was increased 2.3-fold compared with the single dose, whereas that for roxithromycin (150 mg, 12 hourly) was decreased by 25.4%. Because of these opposing changes during chronic dosing, the average plasma roxithromycin concentration over the dosing interval was 2.6-fold higher than that for erythromycin, which was a smaller excess than would have been predicted from the single dose data. The results suggest that roxithromycin exerts less inducing and inhibiting effects on human cytochrome P450 than erythromycin. Roxithromycin has a favorable pharmacokinetic profile suitable for twice daily dosing and may have a lower potential than erythromycin for cytochrome P450-mediated drug interactions.

A simple high-performance liquid chromatographic method for the routine measurement of flecainide in plasma.

A simple and sensitive high-performance liquid chromatographic method for the analysis of flecainide in plasma has been developed. Alkalinized plasma was extracted with 1-chlorobutane and then back extracted into dilute phosphoric acid. An aliquot of the aqueous acid layer was injected onto the HPLC column and the eluent monitored at 297 nm. A phenyl reversed-phase column was used for the separation. The mobile phase was 42% acetonitrile and 58% 20mM sodium acetate solution, with the pH of the mobile phase adjusted to 6.5. The recovery of flecainide was 88.5%, and the coefficient of variation of two quality control (0.2 mg/L and 1.0 mg/L) samples analyzed on a within-day basis (N = 10) was 3.0% in both cases. The coefficient of variation for the two quality control samples analyzed on a between-day basis (N = 10) was less than 10% in both cases. The limit of sensitivity for the assay was 0.033 mg/L. A variety of other drugs likely to be used in patients with cardiovascular disease were screened and found not to interfere with the assay.

Trial of an intervention to reduce passive smoking in infancy.

We tested a health education intervention program to reduce passive smoking in infancy. The aim was to develop an instrument for study of tobacco smoke exposure and childhood respiratory illness. One hundred and eighty-four women who had smoked during pregnancy were allocated by month of delivery to an intervention group, to a minimal contact group, or to a follow-up only comparison group. Exposure to smoke was assessed 3 months later by questionnaire and by measurement of cotinine in samples of maternal and infant urine. There was a reduction in maternal smoking associated with contact with research staff, but this was not statistically significant. There were no differences between the groups in the exposure of infants to tobacco smoke. Reasons for this finding may include the timing of the intervention, the heterogeneity of the target group, and the manner in which information was presented on health risks caused by parental smoking.

Breast feeding and smoking hygiene: major influences on cotinine in urine of smokers' infants.

The determinants of urine cotinine levels were studied in a group of 101 infants aged 3 months, including 79 infants whose mothers were current smokers. At a pre-arranged home visit the infants' mothers completed an interviewer-administered questionnaire, and samples of maternal urine and breast milk and infants' urine were collected. Cotinine and nicotine levels were determined by gas-liquid chromatography. Infant urine cotinine levels ranged from 0 to 140 micrograms/l (0-1120 ng cotinine/mg creatinine). A linear dose response relation between mother's smoking rate and infant urine cotinine level was observed among breast-fed infants (r = 0.79, p less than 0.001). The relation was weaker among infants fed by both breast and bottle (r = 0.56, p = 0.01) and was not apparent among bottle-fed infants (r = 0.15, p = 0.16). In addition to mode of feeding and mother's smoking rate, mother's smoking "hygiene" (assessed by the reported frequency of smoking while feeding and with infant in same room) was independently associated with infant urine cotinine level. Father's smoking pattern and exposure to smoke outside the household did not relate significantly to infant cotinine levels. We conclude that when mothers smoke, breast feeding is the principal determinant of cotinine in infants' urine. It is likely that most of this cotinine comes from cotinine in mothers' breast milk, but further research is needed to establish how much nicotine is ingested by breast-fed infants of mothers who smoke, and to investigate possible health effects.

Influence of gender and oral contraceptive steroids on the metabolism of salicylic acid and acetylsalicylic acid.

Salicylic acid and acetylsalicylic acid (aspirin) disposition after an oral dose of aspirin, 900 mg (equivalent to 689.7 mg of salicylic acid) was studied in eight males, eight females and eight females receiving oral contraceptive steroids (OCS). Salicylic acid clearance was 61% higher in males compared to the control female group, an effect due largely to enhanced activity of the glycine conjugation pathway (salicyluric acid formation) in males. Salicylic acid clearance was 41% higher in OCS-users compared to the control female group due to increases in both the glycine and glucuronic acid conjugation pathways in the pill users. There was no difference in any salicylic acid disposition parameter between males and OCS-users. Area under the plasma concentration-time curve (AUC) and elimination half-life of aspirin was significantly greater and aspirin plasma hydrolysis rate was significantly lower in both female groups compared to males. There was no difference between OCS-users and the control female group in any of these parameters. Aspirin AUC and elimination half-life were significantly correlated with aspirin plasma hydrolysis rate. These data confirm the importance of hormonal factors in the regulation of drug conjugation reactions in humans and suggest that sex-related differences in salicylic acid and aspirin disposition may be of clinical importance.

PIP: To characterize the influence of gender and oral contraceptives (OCs) on drugs metabolized by conjugation reactions, salicylic metabolism was studied in 8 males, 8 females, and 8 additional females taking OCs. In addition, differences in acetylsalicylic acid (aspirin) disposition were studied in these 3 groups of subjects. Salicylic acid clearance was 61% higher in males than in female controls due to enhanced activity of the glycine conjugation pathway (salicyluric acid formation) in males. Salicylic acid clearance was 41% higher in OC users than in female controls due to increases in both the glycine and glucuronic acid conjugation pathways in the OC users. There were no differences in salicylic acid disposition parameters between males and OC users. The area under the plasma concentration time curve and the elimination half-life of aspirin were significantly lower in both female groups compared to males, as was the aspirin plasma hydrolysis rate. Overall, these data confirm the importance of hormonal factors in the regulation of drug conjugation reactions in humans and suggest that sex-related differences in salicylic acid and aspirin disposition may be of clinical significance.

Ranitidine disposition in patients with renal impairment.

Ranitidine disposition has been studied in 12 patients with renal impairment following 50 mg given intravenously and 150 mg given by mouth on separate occasions. The clearance of ranitidine from plasma (y) was correlated with creatinine clearance (x): y = 10.47 + 0.289x,r2 = 0.751, but there was no significant correlation of creatinine clearance with distribution volume or bioavailability. The mean (s.e. mean) distribution volume was 1.62 (0.08) 1/kg and the mean bioavailability 0.81 (0.05). These data suggest that in order to obtain similar ranitidine plasma concentrations in anephric patients and patients with normal renal function, the maintenance dose in the anephric patients should be 25-30% of that for patients with normal renal function.

Bioavailability and diurnal variation in absorption of sustained release theophylline in asthmatic children.

The absolute oral bioavailability of a sustained release theophylline tablet (Nuelin-SR250), given 12 hourly was determined in 14 asthmatic children aged 5 to 13 years. In 4 of the patients, mean bioavailability of the fourth dose was 38.9 +/- 8.4% and that of the sixth dose was 67.9 +/- 25.9% (p less than 0.05) in the other ten patients. This suggests steady-state had not been achieved after four doses. In the initial study with 9 patients, a significant diurnal variation in predose plasma theophylline concentrations was observed, as the mean morning predose concentrations were 2.9 fold greater than the mean evening predose concentrations (p less than 0.005). Dual peak plasma concentrations occurred in 5 out of the 9 patients. The mechanism of this diurnal variation was investigated in a further 5 asthmatic children (10.8 years +/- 1.6). Morning and night steady-state plasma theophylline concentrations during a continuous intravenous infusion of aminophylline were not different (14.9 +/- 5.3 mg/l vs. 15.6 +/- 5.9 mg/l), demonstrating that there was no diurnal variation in the plasma clearance of theophylline. The diurnal variation in predose concentrations with Neulin-SR250 was confirmed with the morning concentrations again being 2.6 fold greater than those in the evening. However, bioavailability was not significantly different for day (09.00-21.00) and night (21.00-09.00) dosing intervals after doses 6 and 7 respectively of Nuelin-SR250. The plasma concentration versus time profiles suggested that the diurnal variation in predose concentrations was due to slower absorption of the evening dose.

A colourimetric procedure for the determination of thiocyanate in plasma.

A colourimetric method is described for the determination of thiocyanate using a modification of the König reaction. The reagents used are stable, relatively non-toxic, and produce a colour with lambda max 543 nm which is stable for at least 90 minutes after formation. Although the method requires only 0.1--0.5 mL of plasma, 0.25 mg/L of thiocyanate may be determined using this procedure. The calibration curve is linear to at least 60 mg/L and is highly reproducible. Plasma samples analysed on a within-day (10.0 mg/L) and between-day (4.9 mg/L) basis, had coefficients of variation (CV) of 2.39% and 3.09%, respectively. Recovery of thiocyanate added to plasma is quantitative.