Adherence to antiepileptic medicines in children: a multiple-methods assessment involving dried blood spot sampling.

PURPOSE: To evaluate adherence to prescribed antiepileptic drugs (AEDs) in children with epilepsy using a combination of adherence-assessment methods. METHODS: A total of 100 children with epilepsy (≤17 years old) were recruited. Medication adherence was determined via parental and child self-reporting (≥9 years old), medication refill data from general practitioner (GP) prescribing records, and via AED concentrations in dried blood spot (DBS) samples obtained from children at the clinic and via self- or parental-led sampling in children's own homes. The latter were assessed using population pharmacokinetic modeling. Patients were deemed nonadherent if any of these measures were indicative of nonadherence with the prescribed treatment. In addition, beliefs about medicines, parental confidence in seizure management, and the presence of depressed mood in parents were evaluated to examine their association with nonadherence in the participating children. KEY FINDINGS: The overall rate of nonadherence in children with epilepsy was 33%. Logistic regression analysis indicated that children with generalized epilepsy (vs. focal epilepsy) were more likely (odds ratio [OR] 4.7, 95% confidence interval [CI] 1.37-15.81) to be classified as nonadherent as were children whose parents have depressed mood (OR 3.6, 95% CI 1.16-11.41). SIGNIFICANCE: This is the first study to apply the novel methodology of determining adherence via AED concentrations in clinic and home DBS samples. The present findings show that the latter, with further development, could be a useful approach to adherence assessment when combined with other measures including parent and child self-reporting. Seizure type and parental depressed mood were strongly predictive of nonadherence.

Microassay of drugs and modern measurement techniques.

Details of the development of conventional analytical methods for the determination of drugs in pediatric plasma/serum samples via microassays are presented. Examples of the development of small-volume sampling and the use of the newer detection systems such as LC/MS/MS for enhanced detection are presented. Dried blood spot sampling has conventionally been used for the study of inborn errors of metabolism using Guthrie cards. Limited applications in the area of drug-level determination, for example, in therapeutic drug monitoring had been reported but the methodology had not been widely used up until relatively recently. In the last few years, there has been a resurgence of interest in this methodology for drug-level determinations, and examples of drug analysis in pediatric and neonatal patients where the small-volume samples are particularly useful are presented. The application of the methodology in pharmacokinetic/pharmacodynamic studies is discussed. The utilization of solid-phase microextraction and stir bar sorptive extraction in drug analysis is presented. Clinical applications of these methodologies are reported including the development of in vivo solid-phase microextraction.

Influence of ABCB1 polymorphisms and haplotypes on tacrolimus nephrotoxicity and dosage requirements in children with liver transplant.

AIMS: The aim of this study was to investigate the influence of genetic polymorphisms in ABCB1 on the incidence of nephrotoxicity and tacrolimus dosage-requirements in paediatric patients following liver transplantation. METHODS: Fifty-one paediatric liver transplant recipients receiving tacrolimus were genotyped for ABCB1 C1236>T, G2677>T and C3435>T polymorphisms. Dose-adjusted tacrolimus trough concentrations and estimated glomerular filtration rates (EGFR) indicative of renal toxicity were determined and correlated with the corresponding genotypes. RESULTS: The present study revealed a higher incidence of the ABCB1 variant-alleles examined among patients with renal dysfunction (> or =30% reduction in EGFR) at 6 months post-transplantation (1236T allele: 63.3% vs 37.5% in controls, P= 0.019; 2677T allele: 63.3% vs. 35.9%, p = 0.012; 3435T allele: 60% vs. 39.1%, P= 0.057). Carriers of the G2677->T variant allele also had a significant reduction (%) in EGFR at 12 months post-transplant (mean difference = 22.6%; P= 0.031). Haplotype analysis showed a significant association between T-T-T haplotypes and an increased incidence of nephrotoxicity at 6 months post-transplantation (haplotype-frequency = 52.9% in nephrotoxic patients vs 29.4% in controls; P= 0.029). Furthermore, G2677->T and C3435->T polymorphisms and T-T-T haplotypes were significantly correlated with higher tacrolimus dose-adjusted pre-dose concentrations at various time points examined long after drug initiation. CONCLUSIONS: These findings suggest that ABCB1 polymorphisms in the native intestine significantly influence tacrolimus dosage-requirement in the stable phase after transplantation. In addition, ABCB1 polymorphisms in paediatric liver transplant recipients may predispose them to nephrotoxicity over the first year post-transplantation. Genotyping future transplant recipients for ABCB1 polymorphisms, therefore, could have the potential to individualize better tacrolimus immunosuppressive therapy and enhance drug safety.

The development of an objective methodology to measure medication adherence to oral thiopurines in paediatric patients with acute lymphoblastic leukaemia--an exploratory study.

AIMS: To develop a method that prospectively assesses adherence rates in paediatric patients with acute lymphoblastic leukaemia (ALL) who are receiving the oral thiopurine treatment 6-mercaptopurine (6-MP). METHODS: A total of 19 paediatric patients with ALL who were receiving 6-MP therapy were enrolled in this study. A new objective tool (hierarchical cluster analysis of drug metabolite concentrations) was explored as a novel approach to assess non-adherence to oral thiopurines, in combination with other objective measures (the pattern of variability in 6-thioguanine nucleotide erythrocyte concentrations and 6-thiouric acid plasma levels) and the subjective measure of self-reported adherence questionnaire. RESULTS: Parents of five ALL patients (26.3%) reported at least one aspect of non-adherence, with the majority (80%) citing "carelessness at times about taking medication" as the primary reason for non-adherence followed by "forgetting to take the medication" (60%). Of these patients, three (15.8%) were considered non-adherent to medication according to the self-reported adherence questionnaire (scored > or = 2). Four ALL patients (21.1%) had metabolite profiles indicative of non-adherence (persistently low levels of metabolites and/or metabolite levels clustered variably with time). Out of these four patients, two (50%) admitted non-adherence to therapy. Overall, when both methods were combined, five patients (26.3%) were considered non-adherent to medication, with higher age representing a risk factor for non-adherence (P < 0.05). CONCLUSIONS: The present study explored various ways to assess adherence rates to thiopurine medication in ALL patients and highlighted the importance of combining both objective and subjective measures as a better way to assess adherence to oral thiopurines.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells.

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

Population pharmacokinetic and pharmacogenetic analysis of 6-mercaptopurine in paediatric patients with acute lymphoblastic leukaemia.

AIMS: To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites. METHODS: Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m(-2) day(-1)). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites. RESULTS: The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P < 0.005) when incorporated as a covariate affecting the fractional metabolic transformation of 6-MP into 6-TGNs. The other genetic covariates tested were not statistically significant and therefore were not included in the final model. CONCLUSIONS: The developed pharmacokinetic model (if successful at external validation) would offer a more rational dosing approach for 6-MP than the traditional empirical method since it combines the current practice of using body surface area in 6-MP dosing with a pharmacogenetically guided dosing based on TPMT genotype.

Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine.

AIMS: To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the influence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or inflammatory bowel disease (IBD). METHODS: Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94-->A and IVS2+21A-->C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined. RESULTS: Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients, P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A-->C variants among ALL and IBD patients had significantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients, P = 0.047 in IBD patients). The study confirmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a significant association between inosine triphosphatase IVS2+21A-->C variants with thrombocytopenia (P = 0.012). CONCLUSIONS; Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose individualization.

Diastereotopic analysis of mesoridazine besylate (Serentil).

NMR studies were conducted with the aim of determining the diastereoisomeric ratio of a commercially supplied sample of mesoridazine (MES) and to compare the results with a freshly synthesised sample of MES. The results indicated that the commercially supplied MES consisted almost entirely of one diastereoisomeric pair, which was in agreement with previous findings reported by Eap et al. The synthesised sample of MES was analysed by NMR in two stages: 1) as the initial product isolated as the free base from the direct synthesis, and 2) as the free base isolated from the crystallised besylate salt of the synthetic product. The NMR results show that the initial synthetic product consisted of two equal pairs of diastereoisomers. The diastereoisomeric pairs were further separated by the addition of the chiral shift reagent (R)-(-)-N-(3,5 dinitrobenzoyl)-alpha-benzylamine to reveal equal quantities of all four enantiomers, clearly observed at the methyl sulfoxide proton peak of the NMR scan. The sample obtained from the crystallisation of MES besylate, however, indicated a significant difference, with a diastereoisomeric ratio of 75:25. The results suggest that MES besylate undergoes preferential crystallisation of one pair of diastereoisomers, with the other pair remaining in solution.

A sensitive gas chromatographic/mass spectrometric method for the resolution and quantification of ethosuximide enantiomers in biological fluids.

A modified specific, sensitive and reproducible chiral gas chromatographic (GC) method for the resolution and quantification of ethosuximide enantiomers in urine and plasma was developed. The samples were extracted by liquid-liquid extraction, using diethylether and the enantiomers were separated and quantified on a chiral gas chromatographic column (25QC2 / CYDEX- beta 0.25). The method involved the use of GC/MS instrumentation for the acquisition of data in the electron impact selective-ion monitoring mode, collecting ions characteristic of both ethosuximide and alpha, alpha - dimethyl - beta - methylsuccinimide, the internal standard and of mass-to-charge ratio (m/z) exactly equal to 55 and 70 units. The limit of quantitation of the method was 2.5 microg/ml for both urine and plasma with both enantiomers. The method proved to be linear, precise and reproducible in the 5-300 microg/ml concentration range for urine samples and in the 10-250 microg/ml concentration range for plasma samples. Future research work envisaged the application of this method in pharmacokinetic and pharmacodynamic studies.