High-performance liquid chromatographic assays for a second-generation novel oral iron chelator (APCP363) and their application to pharmacokinetic studies in rats.

Sensitive and specific HPLC assays for APCP363 in biological matrices (rat plasma, urine and feces) were developed. The recovery of APCP363 ranged from 81.2 to 99.9% in plasma, from 82.1 to 92.8% in urine, and from 65 to 68% in feces. Standard deviations were below 10% for all analyses. The limits of quantitation were 0.1, 10 and 30 microg/ml in plasma, urine and feces, respectively. The HPLC assays, which are the first reports for APCP363 analysis in biological matrices, have been successfully applied to preliminary pharmacokinetic studies in rats. The stool assay is the first non-radiolabeled method for hydroxypyridinones in feces.

Pregnancy-induced changes in drug metabolism in epileptic women.

The pharmacokinetics of antiepileptic drugs may be altered during pregnancy, resulting in decline of serum concentrations and subsequent suboptimal control of seizures. We investigated changes which may occur during pregnancy in hepatic drug handling by comparing metabolic ratios of 15 pregnant epileptic women to 15 nonpregnant epileptic women, as well as 10 pregnant nonepileptic and 10 nonpregnant nonepileptic controls. We used the caffeine test to describe several enzyme activities: P450 1A2, xanthine oxidase, n-acetyltransferase and hydroxylation. For this end, ratios were calculated among a number of metabolites of the main demethylation pathway of caffeine. In addition, we measured D-glucaric acid excretion for specific characterization of antiepileptic drug metabolism. Paired comparison of epileptic women in late pregnancy and six to eight weeks post partum revealed statistically significant decreases in P450 1A2, xanthine oxidase and n-acetyltransferase activities, and a significantly increased hydroxylation activity during pregnancy. Twenty-one of the 30 epileptic women (70%) were found to be fast acetylators, whereas the normal distribution in the nonepileptic control groups was 50%. Excretion of D-glucaric acid was significantly increased in all epileptic patient groups as compared to the matched nonepileptic control groups. Importantly, it was also significantly increased in the pregnant nonepileptic control group as compared to the nonpregnant nonepileptic women. Overall, our results suggest that enzymatic pathways involved in antiepileptic drug metabolism tend to be increased during pregnancy as a potential cause for observed lower serum concentrations of these drugs.

Peroxidase-dependent oxidation of sulfonamides by monocytes and neutrophils from humans and dogs.

The hydroxylamine and nitroso metabolites formed by N4-oxidation of sulfonamides are thought to be involved in the pathogenesis of idiosyncratic reactions to this class of drugs. Idiosyncratic reactions to sulfonamides are characterized by multisystemic toxicity, including hepatitis, nephritis, dermatitis, and blood dyscrasias (aplastic anemia, agranulocytosis). We have previously shown that cytochrome P-450 in the liver metabolizes sulfamethoxazole to its hydroxylamine metabolite. In this paper we report the N4-oxidation of sulfamethoxazole by activated monocytes and neutrophils (human and canine) to form sulfamethoxazole hydroxylamine and nitrosulfamethoxazole. The presumed nitroso intermediate was not detected. Purified myeloperoxidase and prostaglandin H synthase were also capable of mediating the oxidation of sulfamethoxazole. The present studies suggest that myeloperoxidase is responsible for the observed oxidation by phagocytic cells. Oxidation by neutrophils may play a role in agranulocytosis, and oxidation by monocytes may facilitate antigen presentation. Extrahepatic bioactivation of sulfonamides by peroxidases in phagocytic cells and other tissues may be important in determining the range of adverse reactions to sulfonamides that occur.

Comparison of deferoxamine pharmacokinetics between asymptomatic thalassemic children and those exhibiting severe neurotoxicity.

The use of deferoxamine for iron chelation in transfusion-dependent thalassemia major is limited by serious neurotoxicity (hearing and vision loss). We assessed whether interpatient variability in handling deferoxamine and resultant accumulation of the drug may account for the neurotoxicity. We studied steady-state deferoxamine pharmacokinetics during intravenous infusion in two groups of patients--one group exhibited severe manifestations of auditory and visual loss and one group was asymptomatic. The groups were matched for age, sex distribution, weight, treatment period, ferritin levels, and hemoglobin levels. Similarly, doses of deferoxamine at the time of the study were not different. Clearance rates were not different between the symptomatic and asymptomatic patients (39.83 +/- 4.54 versus 30.66 +/- 4.39 ml/min.kg). However, patients who exhibited toxicity received significantly higher daily doses of subcutaneous deferoxamine at the time of diagnosis of neurotoxicity (9.03 +/- 0.96 and 5.58 +/- 0.61 mg/kg.hr, respectively; p less than 0.005). These data suggest that deferoxamine induced neurotoxicity is dose-dependent and cannot be attributed to accumulation of the drug caused by slower clearance rates.

A high-performance liquid chromatographic method for the measurement of deferoxamine in body fluids.

A high-performance liquid chromatography method for the analysis of deferoxamine (DFO) in 100 microliters of serum or plasma is described. The procedure involves the addition of the internal standard ciprofloxacin to the sample, followed by ultrafiltration to remove protein. The ultrafiltrate is then directly injected into the chromatography system. Separation is achieved using a reverse-phase mu Bondapak C18 column and a ternary solvent system (sodium phosphate:acetonitrile:methanol) running at 2.0 ml/min. Assay time is 10 min, and chromatograms show no interference from coadministered drugs during this period of time. Coefficients of variation were found to be less than 5%, and analytical recovery of DFO was 85%. Validation experiments in an experimental dog model and in patients with iron overload demonstrate that the method is appropriate for studying the pharmacokinetics of DFO in thalassemic patients receiving drug for the treatment of chronic iron overload.

Temporal variation in the disposition of theophylline and its metabolites.

The temporal aspects of theophylline disposition are of interest, as there are predictable time-dependent fluctuations in the pulmonary function of patients with asthma and theophylline serum concentrations may vary throughout a 24-hour period. We studied the extent to which there are significant temporal changes in theophylline kinetics and the relative contribution of distribution, metabolism, and excretion to this phenomenon. Eight healthy men received an intravenous dose (6 mg/kg) of theophylline at 8 AM and 8 PM at 1-week intervals. Serum and urine were analyzed for theophylline and its three major metabolites by HPLC. Distribution volumes and total body and nonrenal clearances showed no differences between morning and evening dosing. The elimination rate was 12% greater after morning dosing. Renal clearance was 24% greater after morning dosing and was accompanied by an increased excretion fraction of unchanged theophylline. Based on total urinary metabolite excretion and the metabolite serum AUCs, there was no evidence of time-dependent variation in theophylline biotransformation. Although theophylline renal clearance is greater after morning dosing, it is only a small fraction of the overall drug elimination and does not change the total body clearance after morning or evening dosing.

Theophylline pharmacokinetics in adolescent females following coadministration of oral contraceptives.

The influence of 3-9 months of combined low dose oral contraceptives on theophylline pharmacokinetics was studied in 10 adolescent females (age 15-18 years, mean +/- SD 17 +/- 1) and compared to 10 age-matched control subjects (age 13.8-19 years, mean +/- SD 16.5 +/- 1.6). The distribution volume (0.44 +/- 0.06 L/kg in control vs. 0.44 +/- 0.09 L/kg oral contraceptive group), total body clearance (0.78 +/- 0.13 ml/kg/min vs 0.78 +/- 0.18 ml/kg/min) and elimination T 1/2 (402 +/- 78 min vs. 409 +/- 126 min) were identical in the two groups. It appears that during the first 3-9 months of low dose oral contraceptive treatment, these steroids do not alter the pharmacokinetic behaviour of theophylline in adolescent females.

Cloxacillin absorption and disposition in cystic fibrosis.

Because of reports of lowered antibiotic serum concentrations in patients with cystic fibrosis (CF), a bioavailability and pharmacokinetic study of cloxacillin was conducted in 12 control and 16 patients with CF after intravenously and orally administered doses of cloxacillin 25 mg/kg. The patients had mild to moderate CF and were in stable condition. Significantly lower serum concentrations in CF were a result of a 78% increase in total body clearance (P less than 0.005) and a 38% increase in the apparent volume of distribution (P less than 0.025). The bioavailability in CF (0.50) was not significantly different than in controls (0.38), but more variability was seen in the group with CF. After the intravenously given dose the fraction of cloxacillin excreted in the urine unchanged was similar in controls (0.644) and patients with CF (0.547). Compared with that in the control subjects, the mean renal clearance in patients with CF was 30% greater (P less than 0.10) and the nonrenal clearance was 144% greater (P less than 0.07). Enhanced nonrenal clearance explains most of the demonstrated difference between serum concentrations in controls and patients with CF after identical weight-adjusted doses. The data suggest enhanced cloxacillin biotransformation in CF.

Ceftazidime disposition in acute and stable cystic fibrosis.

Ceftazidime disposition after an intravenous dose of 50 mg/kg infused over 20 min was followed in 10 subjects with cystic fibrosis (CF) hospitalized with acute pulmonary exacerbations and in 10 healthy subjects. Serum ceftazidime elimination t 1/2 decreased from 105.3 +/- 12.4 min (mean +/- SD) in controls to 90.0 +/- 11.1 min in subjects with CF. Calculated distribution volumes were both larger in subjects with CF. When normalized for body surface area, total body clearance (Cl) was 41.9% greater in the CF group (142.4 +/- 16.9 and 100.5 +/- 10.3 ml/min/1.73 m2). Normalization for body weight revealed 64.8% greater Cl in subjects with CF. Fraction of dose recovered in urine was of the same order for each group, while renal clearance (ClR) was 40.9% greater in the subjects with CF (130.1 +/- 11.4 and 92.7 +/- 11.6 ml/min/1.73 m2). Five subjects with CF were restudied while infection-free 119 to 219 days after the original study day. With the exception of a 10% increase in the volume of distribution at steady state while infection-free, kinetic parameters were much the same. No changes in Cl or ClR were evident from one study day to the next. Acute pulmonary infection does not appear to alter ceftazidime clearance in CF. The mechanism underlying increased ceftazidime Cl and ClR in CF is not apparent from the present data.

High-pressure liquid chromatographic analysis of ceftazidime in serum and urine.

A rapid and sensitive high-pressure liquid chromatographic procedure was developed for quantitative analysis of a new semisynthetic cephalosporin, ceftazidime, in serum and urine. A good linear relationship was established between peak height and the amount of ceftazidime injected over a concentration range of 1.9 to 30 micrograms/ml. Recovery was approximately 90% at concentrations of 3, 15, and 30 micrograms/ml. The method is specific for ceftazidime, with no interference noted from 11 other beta-lactam antibiotics tested. The assay is accurate, reproducible, and useful for pharmacokinetic studies in humans as demonstrated in two subjects.

A rapid high performance liquid chromatographic procedure for the analysis of cloxacillin and/or nafcillin in serum.

A rapid, reliable procedure for the analysis of cloxacillin and/or nafcillin in 100 microliter of serum or plasma is described. Percentage analytical recovery of cloxacillin, nafcillin, and internal standard (5-(p-hydroxyphenyl)-5-phenylhydantoin) were 94, 91, and 85%, respectively. The between-day precision of the method at cloxacillin concentrations of 2, 8, and 20 mg/liter was 14.7, 11.6, and 10.3%, respectively. At identical serum concentrations, the values obtained for nafcillin were 15.3, 12.4, and 12.2%, respectively. The method will be used to study the pharmacokinetics of both drugs in patients with cystic fibrosis who are on long-term therapy. Preliminary data providing the i.v. half-life, clearance, and volume of distribution are presented for two normal individuals.