A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots.

An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r≥0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1 µg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were <20% at the LLOQs and <15% at all other concentrations tested. This method was successfully applied to the analysis of the AEDs in DBS samples taken from children with epilepsy for the assessment of their adherence to prescribed treatments.

Public awareness and views on unlicensed use of medicines in children.

AIMS: To explore awareness and views of the general public on unlicensed use of medicines in children and on the participation of children in clinical trials. METHODS: Members of the public completed a questionnaire survey administered by face-to-face interview in public areas in N. Ireland. The main outcome measures were the views on unlicensed use of medicines in children and on clinical trials in children. RESULTS: One thousand participants (59.2% female) took part; 610 were parents. Most participants (86%) had no previous knowledge about unlicensed use of medicines in children. Being a parent did not influence this nor did being a parent of a child who suffered from a health problem (P > 0.05). Most participants (92%) felt that parents should be told about unlicensed use of medicines, with the doctor most frequently selected as the person who should inform parents. At the outset, only 1.8% of participants felt that the use of medicines in children was unsafe. However, having been informed about unlicensed use of medicines, this proportion increased dramatically (62.4%; P < 0.001). Views on whether participants would enter a child of their own into a clinical trial varied according to the health status of the child (P < 0.05) i.e. a child in good health (3.9%) vs a child with a life-threatening condition (41.9%). CONCLUSIONS: There is limited public knowledge of unlicensed use of medicines in children and a general reluctance to involve children in clinical trials unless the child to be involved has a life-threatening condition.

Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples.

An HPLC method has been developed and validated for the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 microl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30-1000 ng/ml for spironolactone and 25-1000 ng/ml for 7 alpha-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7 alpha-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.

Intravenous indometacin in preterm infants with symptomatic patent ductus arteriosus. A population pharmacokinetic study.

AIMS: To characterize the population pharmacokinetics of indometacin in preterm infants with symptomatic patent ductus arteriosus and to investigate the influence of various factors on the response to treatment. METHODS: Data were collected from 35 infants (gestational age 25-34 weeks; postnatal age 1-77 days) in neonatal units in Belfast and Copenhagen. Infants received an initial course of up to three doses of intravenous indometacin (0.1-0.2 mg kg(-1)) as considered appropriate by the treating physician. For those infants who did not respond to therapy or in whom the ductus reopened, a second course was sometimes given. Population analysis of the 185 plasma concentrations obtained was conducted using NONMEM and pharmacokinetic and demographic differences between responders and nonresponders were compared. RESULTS: The concentration-time course of indometacin was best described by a one-compartment model. The final population parameter estimates of clearance (CL) and volume of distribution (V) (standardized to the median weight of 1.17 kg) were 0.00711 l h(-1) and 0.266 l, respectively. CL increased from birth by approximately 3.38% per day and V by approximately 1.47% per day. Concomitant digoxin therapy resulted in a 30% decrease in V. Interindividual variability in CL and V was 41% and 21%, respectively. Interoccasion variability for CL was 43%. Residual variability corresponded to a standard deviation of 0.148 mg l(-1). Closure occurred in 75% of infants with a plasma concentration > or = 0.4 mg l(-1) 24 h after the last dose. CONCLUSIONS: Dosing regimens for indometacin should take into account the weight and postnatal age of the infant and any concomitant digoxin therapy. The population estimates can be used to determine typical values of CL and V allowing the prediction of individualized doses of indometacin that should increase the probability of achieving a 24 h plasma concentration > or = 0.4 mg l(-1). Although the pharmacokinetic estimates will be affected by both interindividual and within-individual variation, it is anticipated that this approach will decrease the variability of exposure and optimize treatment outcome.

Liquid chromatographic determination including simultaneous "on-cartridge" separation of ranitidine cisapride drug combinations from paediatric plasma samples using an automated solid-phase extraction procedure.

HPLC methodology was investigated for the simultaneous determination of cisapride and ranitidine in small volume paediatric plasma samples. Such a simultaneous determination proved difficult due to the small sample volumes, the low concentrations of the drugs and the different log P values of the two compounds. The two drugs and their respective internal standards were separated "on-cartridge" using HLB Solid Phase Extraction cartridges and the samples quantified by individual HPLC methodologies. The technique has been applied successfully to 60 paediatric plasma samples containing both cisapride and ranitidine.

The pharmacokinetics of ethosuximide enantiomers in the rat.

A chiral gas chromatographic assay previously developed for quantitative analysis of ethosuximide and its major metabolites in rat urine has been adapted for the analysis of the drug in plasma. Ethosuximide, both as a racemic mixture and as the individual enantiomers, was administered to conscious rats by the intravenous (i.v.) and intraperitoneal (i.p.) routes. Pharmacokinetic parameters were estimated using standard non-compartmental methods. Comparison of the pharmacokinetic parameters of (S)-ethosuximide and (R)-ethosuximide showed that total body clearance of (R)-ethosuximide was significantly larger than that of (S)-ethosuximide and that elimination half-life was significantly shorter following administration of both 40 mg i.v. and i.p. doses, indicating that there is stereoselective elimination of ethosuximide. However, no significant differences were found between apparent volumes of distribution. In addition, no significant differences were found for either enantiomer between the estimates of the pharmacokinetic parameters obtained following administration as the individual enantiomer and as a constituent of the racemic mixture. This indicates that, at the doses studied, the preferential faster elimination of (R)-ethosuximide is not dependent upon the presence of the (S)-enantiomer. Also, for each enantiomer, the lack of any significant difference between estimates of clearance when administered as part of a racemic mixture and when administered separately indicates that neither enantiomer affects the clearance of the other.

Quantification of urinary excretion of ethosuximide enantiomers and their major metabolites in the rat.

A chiral gas chromatographic assay has been developed for quantitative analysis of ethosuximide and its major metabolites in rat urine. The extraction procedure was found to be precise and reproducible. Recovery was in the range of 94-98%, intraday CV(%) was 0.92% for (S)-ethosuximide (50 microg/ml) and 0.51% for (R)-ethosuximide (50 microg/ml). Interday CV(%) was 1.12% for (S)-ethosuximide and 0.72% for (R)-ethosuximide. The limit of detection was determined to be around 0.01 microg/ml for each enantiomer. Following administration of rac-ethosuximide by i.v., i.p. and oral routes, unchanged ethosuximide was detected in urine up to 72 h after drug administration. The appearance of all detected metabolites occurred within 24 h of drug administration. Significantly more (S)-ethosuximide was excreted unchanged than (R)-ethosuximide with all three routes studied. A substantial amount of the drug was eliminated as the 2-(1-hydroxyethyl)-2-methylsuccinimide (2 pairs of diastereoisomers). Much less drug was eliminated as the 2-ethyl-3-hydroxy-2-methylsuccinimide with only one diastereoisomer observed. Examination of the one pair of diastereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide that was resolved showed preferential excretion of one isomer. Comparison of both pairs of diastereoisomers showed that one pair was formed in preference to the other with a ratio of approximately 0.8:1. It is concluded that stereoselective metabolism of ethosuximide occurs.

The use of polymeric solid phase extraction and HPLC analysis for the determination of ranitidine in routine plasma samples obtained from paediatric patients.

A sensitive HPLC method for the determination of ranitidine in small-volume (0.5 mL) paediatric plasma samples is described. Plasma samples were extracted using a simple, rapid solid phase extraction (SPE) technique developed using disposable copolymer packed SPE cartridges. Chromatographic separation was achieved by reverse-phase HPLC with isocratic elution using a microBondapak C18 column and a phosphate buffer (10 mM, pH 3.75)-acetonitrile (87:13 v/v) mobile phase with UV detection at 313 nm. The HPLC system exhibited linearity in the range 8-800 ng mL(-1). Intraday % CV and % bias values were in the range 1.28-8.09% (% bias -4.33 to -0.87) and interday % CV and % bias values were in the range 0.73-15.28% (% bias -1.80 to + 1.65). The limits of detection and quantitation obtained were 2 ng mL(-1) and 8 ng mL(-1), respectively, and ranitidine extraction recoveries from plasma ranged from 92.30 to 103.88%. In this study, the developed HPLC and SPE methodologies have been successfully applied to the determination of ranitidine concentrations in 68 paediatric plasma samples. The sampled population was drawn from patients already receiving the study drug therapeutically. Patients recruited had received ranitidine by two main routes - oral and intravenous. The plasma concentrations of ranitidine encountered in paediatric samples following oral or intravenous administration of a range of prescribed doses are presented graphically. These profiles are based on analysis of the first 68 plasma samples obtained from the first 35 patients recruited to the study receiving ranitidine by the oral or intravenous route.

The use of hydrophilic lipophilic balanced (HLB) copolymer SPE cartridges for the extraction of diclofenac from small volume paediatric plasma samples.

The extraction of diclofenac from spiked aqueous and plasma samples by liquid-liquid extraction (LLE) and solid phase extraction (SPE) methods is compared. The SPE methodology utilised a hydrophilic lipophilic balanced (HLB) copolymer as the extraction phase. Using a literature HPLC method, a calibration curve for diclofenac was constructed in the range 1.0-50.0 microg/ml. Diclofenac spiked samples (aqueous and plasma) were extracted by LLE and SPE methodologies. The SPE resulted in higher extraction efficiencies (mean 94.9%) than the LLE (mean 78.9%) with %R.S.D.s similar in both methods (3.2 vs. 2.1%, respectively). The SPE method was suitable for the extraction of diclofenac from small volume paediatric plasma samples.

Topical administration of racemic ibuprofen.

In vitro experiments to investigate possible stereoselective aspects of the topical administration of ibuprofen have been conducted. Incubation of ibuprofen with rat skin homogenates in the presence of coenzyme A, ATP, and magnesium provided no evidence for the formation of ibuprofenyl coenzyme A (the initial intermediate in the metabolic inversion of [R]- to [S]-ibuprofen). Similar incubation studies gave no indication of a change in the enantiomeric ratios of ibuprofen over the time course of the experiments. Percutaneous penetration studies of ibuprofen gel through porcine skin indicated that the ibuprofen enantiomer levels in the reservoir solutions were consistent with racemic ibuprofen having traversed the skin with no metabolic inversion. These results suggest that, in the models studied, skin metabolism does not result in the chiral inversion of (R)- to (S)-ibuprofen and that the topical administration of ibuprofen will result in the delivery of 50% "isomeric ballast."

Metabolism of paracetamol in children with chronic liver disease.

OBJECTIVE: To study the metabolism of single doses of paracetamol in paediatric patients with chronic liver disease admitted to a hospital liver disease clinic. RESULTS: Thirteen paediatric patients, aged 7 months to 12 years, with chronic liver disease of varying severity were studied. In these children, paracetamol elimination half-life was negatively correlated with serum albumin and positively with prothrombin time, as previously reported in adults with liver disease. The rate constant of glucuronide formation was higher in the children with liver disease compared to the value reported in healthy children of similar ages. The rate constant of the formation of paracetamol sulphate was no different from that in normal children. The 36 h urinary paracetamol glucuronide to sulphate ratio was 1.4 (95% CI 0.8 to 1.7). This mean ratio was higher than in healthy children (0.81 and 0.75) but not significantly so, probably because of a Type 1 error due to the inevitable small sample size arising from the nature of the population being studied. CONCLUSION: The present study provides reassuring additional data to indicate that, at least for single doses, there is no cause for concern in the use of paracetamol in children with chronic liver disease.

The effect of three H2 receptor antagonists on the disposition of cyclosporin A in the in situ perfused rat liver model.

The in situ perfused rat liver model was used to investigate the effect of three H2 receptor antagonists on the disposition of cyclosporin A (CyA) and the major human metabolite, AM1. Perfusion experiments, using standard techniques, were carried out on four groups (one control and three H2-receptor antagonist-treated groups) of male Sprague-Dawley rats (300-350 g). All animals received CyA, 2.5 mg; the three treated groups received cimetidine (8 mg), ranitidine (3 mg), or famotidine (0.4 mg). Perfusate and bile samples were collected and assayed for CyA, AM1, and the H2 receptor antagonists by HPLC. Results indicated that CyA perfusate concentrations in the controls and cimetidine and ranitidine-treated groups were not significantly different, although levels in the famotidine group were significantly higher at all times (p < 0.05), except 30 min, compared to the controls. However, examination of the AM1 perfusate and bile data and the apparent metabolic clearance data indicated that CyA metabolism was still occurring, despite the presence of the H2 receptor antagonist. It is suggested that the absence of a interaction may be attributed to a lack of specificity of the H2 receptor antagonists for CYP3A, the isoenzyme responsible for CyA metabolism.

Chiral aspects of the metabolism of ethosuximide.

Ethosuximide is a chiral drug substance primarily indicated for the treatment of absence seizures. This drug is used clinically as the racemate. The urinary metabolites of ethosuximide (following i.p. administration of the racemate or individual enantiomers to rats) have been studied using chiral gas chromatography (GC) and gas chromatography-mass spectroscopy (GCMS). The metabolites identified were unchanged ethosuximide enantiomers, all four stereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide, and a single stereoisomer of 2-ethyl-3-hydroxy-2-methylsuccinimide [derived from (R)-ethosuximide]. Preliminary quantitative studies indicate a degree of stereoselectivity in the fate of ethosuximide since the ratio of (R)- to (S)-ethosuximide in the urine was found to be 0.77:1 (0-24 h sample), 0.64:1 (24-48 h sample), and 0.83:1 (48-72 h sample). This would suggest that the (R)-isomer is preferentially metabolised. Results obtained following the administration of individual enantiomers of ethosuximide indicate that the 2-(1-hydroxyethyl)-2-methylsuccinimide diastereoisomers derived from (R)-ethosuximide are produced in approximately equal proportions [ratio 1.05:1 (0-24 h sample), 1.10:1 (24-48 h sample)], whilst those from (S)-ethosuximide are produced in unequal proportions [ratio 1.65:1 (0-24 h sample), 1.74:1 (24-48 h sample)].

The effect of three H2-receptor antagonists on the disposition of midazolam in the rat in-situ perfused liver model.

The rat in-situ perfused liver model was used to investigate the effect of three H2-receptor antagonists on the pharmacokinetic disposition of the short-acting benzodiazepine, midazolam. Perfusion experiments, using standard techniques, were carried out on four groups (one control and three H2-receptor antagonist-treated groups) of male Sprague-Dawley rats (300-350 g). All animals received midazolam 1 mg; the three treated groups received cimetidine (8 mg), ranitidine (3 mg) or famotidine (0.4 mg). Perfusate and bile samples were collected and assayed for midazolam using gas chromatography. The perfusate data indicated that midazolam disposition was impaired at 10, 50 and 60 min of the experimental period following the addition of cimetidine, whereas ranitidine and famotidine produced an effect at 10 min only; midazolam levels in bile were not affected by the presence of an H2-receptor antagonist. It was concluded that the limited inhibitory effect of cimetidine may be attributed to its lack of specificity for CYP3A, the isoenzyme responsible for the metabolism of midazolam.

Cyclosporin A and erythromycin: a study of a drug interaction in the in situ perfused rat liver model.

Using the in situ perfused rat liver model, the effect of erythromycin (Ery) on the disposition of cyclosporin A (CyA) and the major human metabolite, AM1, was investigated. Prior to perfusion experiments, oral dosing was carried out for three days on three groups of male Sprague-Dawley rats (300-350 g), involving pretreatment with water (control and H2O/Ery groups) or erythromycin (Ery oral group). On the fourth day, perfusion experiments took place using standard techniques, with the addition of 20 mg Ery to the H2O/Ery and Ery oral groups, and 2.5 mg CyA to all groups. Perfusate and bile samples were collected and assayed for CyA and AM1 by HPLC. Results indicated that inhibition of CyA metabolism had occurred as the CyA concentration in perfusate was significantly higher in both Ery groups at all times compared to the control group, and the levels of AM1 in both perfusate and bile were significantly lower than in the control group. There was also a marked reduction in the apparent metabolic clearance of CyA in the Ery groups. It was concluded that AM1 production had been inhibited by Ery, the most likely mechanism being inhibition of the isoenzyme CYP3A with which Ery forms a stable complex.

The metabolism of ethosuximide.

The metabolism of the antiepileptic drug ethosuximide (3-ethyl-3-methylpyrollidine-2,5-dione) (I) in animals and humans is reviewed. Chiral aspects of the metabolism of ethosuximide are discussed. Clarification of the precise nature of the hydroxymetabolites of ethosuximide is presented.

Factors affecting quantitative assessment of Pseudomonas aeruginosa adherence to buccal epithelial cells.

The influence of experimental protocol on estimation of adherence of P. aeruginosa to buccal epithelial cells is highlighted. Use of membrane filtration to remove non-adherent bacteria was significantly affected by membrane pore size and rinse volume, but was not affected by inoculum size or test isolate. Even with optimum filtration conditions, over 58% of the non-adherent bacterial population were retained on the filters. Separation of non-adherent bacteria was greatly improved when density centrifugation was employed, with less than 30% of non-adherent bacteria present in the buccal epithelial suspension. Adherence values measured by a radiological and fluorometric method were significantly higher than those determined by microscopic counting. Radiological assays had the lowest variance. Gradient centrifugation was used to prepare a standard suspension of buccal epithelial cells. However, cells collected on different days showed significantly different adherence measurements. Adherence of bacteria to trypsinized buccal epithelial cells and to buccal epithelial cells from cystic fibrosis patients was similar, whereas adherence to normal buccal epithelial cells was significantly lower.

The influence of particle size on the bioavailability of inhaled temazepam.

1. Temazepam was administered by aerosol using a standard protocol to healthy volunteers. Two studies are reported in which different dosage formulations were used: a) 30 mg of the 5 mu diameter particle (n = 6); b) 10 mg of the 2 mu diameter particle (n = 6). 2. An open crossover design was followed in each study. On one occasion in both studies subjects used a gargling procedure to remove drug which had been deposited in the mouth and oropharynx. 3. Serial venous blood samples were drawn for a period of 24 h. The mean total AUC of the 5 mu preparation was significantly reduced by gargling (3153 ng ml-1 h to 1066 ng ml-1 h) (F = 0.32). Gargling also had a significant effect on the mean AUC(0-1 h). 4. In contrast gargling had no significant effect on the mean AUC associated with the smaller diameter particle preparation (630 ng ml-1 h) vs 397 ng ml-1 h (F = 0.74). 5. These findings also indicate that temazepam deposition in the pulmonary tree is enhanced by the use of a 2 mu rather than a 5 mu diameter particle. However, the plasma drug concentrations achieved are unlikely to produce a sufficiently marked sedative effect for endoscopic investigations such as gastroscopy.

Prediction of netilmicin disposition in neonates.

Multiple regression analyses of data from 33 neonates who received netilmicin therapy showed that concurrent treatment with other drugs (Drg), creatinine clearance (CLcr), gestational age (GA), and an apgar score of less than 6 at 1 min (Agl') were significant determinants of netilmicin clearance. Apparent volume of distribution was significantly affected by postnatal age (PNA), gender, the presence of ascites and/or oedema (A/O), and whether or not the neonate was small for gestational age (SGA). The following formulae were obtained: [formula: see text] The regression formulae were shown to predict netilmicin plasma concentrations with good precision and a non-significant bias in a further 15 neonates studied prospectively.

Development of resistance to ciprofloxacin in nutrient-rich and nutrient-limited growth conditions in vitro by Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

Five serial exposures of mucoid Pseudomonas aeruginosa from patients with cystic fibrosis to subinhibitory concentrations of ciprofloxacin resulted in stepwise increases in the MIC, with a mean proportional increase of 10. MICs were significantly lower in an iron-limited chemically defined medium than in Iso-Sensitest broth. The mucoid phenotype was maintained in chemically defined medium. Acquired resistance was retained either partially or completely in 85% of the isolates following 10 transfers in drug-free media. In cases in which susceptibility was regained, an increase in the MIC was observed on one further exposure to ciprofloxacin.