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.

Development and validation of a dried blood spot LC-MS/MS assay to quantify ranitidine in paediatric samples.

A novel approach has been developed to determine ranitidine in paediatric samples using dried blood spots (DBS) on Guthrie cards (Whatman 903). A selective and sensitive HPLC-MS/MS assay has been developed and validated using small volumes of blood (30 µl). A 6 mm disc was punched from each DBS and extracted with methanolic solution of the internal standard (IS) nizatidine. This was further subjected to solid phase extraction (SPE), followed by reversed phase HPLC separation, using a XBridge™ C18 column and mobile phase 10 mM ammonium acetate/methanol (98:2 v/v) with a flow rate of 0.3 mL/min. This was combined with multiple reaction monitoring (MRM) mass detection using electrospray ionisation (ESI). The calibration curve for ranitidine was found linear over the range 10-500 ng/mL (r=0.996). The limit of quantification (LOQ) of the method was validated at 10 ng/mL. Accuracy and precision values for within and between days were <20% at the LOQ and <15% at all other concentrations. The validated DBS method was successfully applied to a clinical study employing 81 samples from 36 paediatric patients.

Determination of diclofenac from paediatric urine samples by stir bar sorptive extraction (SBSE)-HPLC-UV technique.

A novel stir bar sorptive extraction (SBSE) method coupled with high performance liquid chromatography (HPLC) and UV detection for the extraction of diclofenac (DIC) from paediatric urine samples has been developed and validated. Selectivity and sensitivity being the prime objectives of the bioanalytical method for clinical samples, an optimised SBSE protocol was developed that selectively extracted DIC from various concurrently administered drugs. The validated assay was found to be linear (r=0.9999) over a concentration range of 100-2000 ng mL(-1). SBSE showed consistent recoveries (∼ 70%) of DIC across the validated linearity range. Overall, the method exhibited excellent accuracy and precision across all QC concentrations, tested over three days. Calculated LOD and LOQ were found to be 12.03 ng mL(-1) and 36.37 ng mL(-1), respectively, however, for the experimental purposes, 100 ng mL(-1) was considered as the validated LOQ (accuracy and precision at this LQC was <20%). Further, studies on various attributes of the stir bar/SBSE, showed no significant inter- and intra-stir bar variability for DIC extraction. There was no carryover effect with re-use of conditioned stir bars and for the first time, a systematic investigation on the effect of ageing of stir bars on their extraction efficiency was carried out. Results showed that, for the present study, stir bars which were used 150 times were still functional based on in-house acceptance criteria and extraction efficiency. The validated method was successfully applied to the analysis of DIC in paediatric clinical trial samples.

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.

Stir bar sorptive extraction of diclofenac from liquid formulations: a proof of concept study.

A new stir bar sorptive extraction (SBSE) technique coupled with HPLC-UV method for quantification of diclofenac in pharmaceutical formulations has been developed and validated as a proof of concept study. Commercially available polydimethylsiloxane stir bars (Twister™) were used for method development and SBSE extraction (pH, phase ratio, stirring speed, temperature, ionic strength and time) and liquid desorption (solvents, desorption method, stirring time etc) procedures were optimised. The method was validated as per ICH guidelines and was successfully applied for the estimation of diclofenac from three liquid formulations viz. Voltarol(®) Optha single dose eye drops, Voltarol(®) Ophtha multidose eye drops and Voltarol(®) ampoules. The developed method was found to be linear (r=0.9999) over 100-2000ng/ml concentration range with acceptable accuracy and precision (tested over three QC concentrations). The SBSE extraction recovery of the diclofenac was found to be 70% and the LOD and LOQ of the validated method were found to be 16.06 and 48.68ng/ml, respectively. Furthermore, a forced degradation study of a diclofenac formulation leading to the formation of structurally similar cyclic impurity (indolinone) was carried out. The developed extraction method showed comparable results to that of the reference method, i.e. method was capable of selectively extracting the indolinone and diclofenac from the liquid matrix. Data on inter and intra stir bar accuracy and precision further confirmed robustness of the method, supporting the multiple re-use of the stir bars.

Development and validation of a dried blood spot-HPLC assay for the determination of metronidazole in neonatal whole blood samples.

A selective and sensitive high-performance liquid chromatography method with UV detection for the determination of metronidazole in dried blood spots (DBS) has been developed and validated. DBS samples [spiked or patient samples] were prepared by applying blood (30 microL) to Guthrie cards. Discs (6 mm diameter) were punched from the cards and extracted using water containing the internal standard, tinidazole. The extracted sample was chromatographed without further treatment using a reversed phase system involving a Symmetry(R) C18 (5 microm, 3.9 x 150 mm) preceded by a Symmetry(R) guard column of matching chemistry and a detection wavelength of 317 nm. The mobile phase comprised acetonitrile/0.01 M phosphate solution (KH(2)PO(4)), pH 4.7, 15:85, v/v, with a flow rate of 1 mL/min. The calibration was linear over the range 2.5-50 mg/mL. The limits of detection and quantification were 0.6 and 1.8 microg/mL, respectively. The method has been applied to the determination of 203 DBS samples from neonatal patients for a phamacokinetic/pharmacodynamic study.

Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples.

A selective and sensitive liquid chromatography (LC)-atmospheric pressure chemical ionisation (APCI)-mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 microl of spiked whole blood onto Guthrie cards. A 6mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17alpha-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC-APCI-MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25-1000 ng/ml (r>0.994). Accuracy (% RE) and precision (% CV) values for within and between day were <20% at the lower limit of quantification (LLQC) and <15% at all other concentrations tested. The LLOQ of the method was validated at 25 ng/ml. Clinical validation of the method was achieved by employing the validated method for analysis of 160 DBS samples from 37 neonatal and paediatric patients.

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.