Association study of candidate DNA-repair gene variants and acute graft versus host disease in pediatric patients receiving allogeneic hematopoietic stem-cell transplantation.

Acute Graft versus Host Disease (aGvHD) grades 2-4 occurs in 15-60% of pediatric patients undergoing allogeneic haematopoietic stem-cell transplantation (allo-HSCT). The collateral damage to normal tissue by conditioning regimens administered prior to allo-HSCT serve as an initial trigger for aGvHD. DNA-repair mechanisms may play an important role in mitigating this initial damage, and so the variants in corresponding DNA-repair protein-coding genes via affecting their quantity and/or function. We explored 51 variants within 17 DNA-repair genes for their association with aGvHD grades 2-4 in 60 pediatric patients. The cumulative incidence of aGvHD 2-4 was 12% (n = 7) in the exploratory cohort. MGMT rs10764881 (G>A) and EXO rs9350 (c.2270C>T) variants were associated with aGvHD 2-4 [Odds ratios = 14.8 (0 events out of 40 in rs10764881 GG group) and 11.5 (95% CI: 2.3-191.8), respectively, multiple testing corrected p ≤ 0.001]. Upon evaluation in an extended cohort (n = 182) with an incidence of aGvHD 2-4 of 22% (n = 40), only MGMT rs10764881 (G>A) remained significant (adjusted HR = 2.05 [95% CI: 1.06-3.94]; p = 0.03) in the presence of other clinical risk factors. Higher MGMT expression was seen in GG carriers for rs10764881 and was associated with higher IC50 of Busulfan in lymphoblastoid cells. MGMT rs10764881 carrier status could predict aGvHD occurrence in pediatric patients undergoing allo-HSCT.

Data for the measurement of serum vitamin D metabolites in childhood acute lymphoblastic leukemia survivors.

This article describes data related to a companion research paper entitled "Vitamin D nutritional status and bone turnover biomarkers in childhood acute lymphoblastic leukemia (cALL) survivors." (Delvin et al., submitted for publication) [1]. Various methods for the measurement of serum 25OHD3, the accepted biomarker for assessing vitamin D nutritional status, have been described (Le Goff et al., 2015; Jensen et al., 2016) [2], [3]. This article describes a novel mass spectrometry-QTOF method for the quantification of circulating 25OHD3, 3-epi-25OHD3 and 24,25(OH)2D3. It provides the description of the extraction, chromatography and mass spectrometry protocols, a sample of mass spectra obtained from standards and extracted serum, and a comparison with another HPLC-MS/MS (Jensen et al., 2016) [3] method for the measurement of serum concentrations of 25OHD3.

Data in support for the measurement of serum 25-hydroxyvitamin D (25OHD) by tandem mass spectrometry.

This article provides data and a method related to a research paper entitled "Assessing vitamin D nutritional status: is capillary blood adequate?" (Jensen et al., 2016) [1]. Circulating 25OHD, the accepted biomarker of the vitamin D nutritional status, is routinely measured by automated immunoassays, that although may be performed in hospital central laboratories, often suffer from a lack of specificity with regards to the different vitamin D metabolites, "Measurement of circulating 25-hydroxyvitamin D: a historical review" (Le Goff et al., 2015) [2]. Mass spectrometry offers this specificity. This article describes the performance of an in-house tandem mass spectrometry method for the individual measurement of 25OHD3, 25OHD2 and 3-épi-25OHD3.

Assessing vitamin D nutritional status: Is capillary blood adequate?

BACKGROUND: Venous blood is the usual sample for measuring various biomarkers, including 25-hydroxyvitamin D (25OHD). However, it can prove challenging in infants and young children. Hence the finger-prick capillary collection is an alternative, being a relatively simple procedure perceived to be less invasive. We elected to validate the use of capillary blood sampling for 25OHD quantification by liquid chromatography tandem-mass spectrometry (LC/MS-MS). METHODS: Venous and capillary blood samples were simultaneously collected from 15 preschool-aged children with asthma 10days after receiving 100,000IU of vitamin-D3 or placebo and 20 apparently healthy adult volunteers. 25OHD was measured by an in-house LC/MS-MS method. RESULTS: The venous 25OHD values varied between 23 and 255nmol/l. The venous and capillary blood total 25OHD concentrations highly correlated (r(2)=0.9963). The mean difference (bias) of capillary blood 25OHD compared to venous blood was 2.0 (95% CI: -7.5, 11.5) nmol/l. CONCLUSION: Our study demonstrates excellent agreement with no evidence of a clinically important bias between venous and capillary serum 25OHD concentrations measured by LC/MS-MS over a wide range of values. Under those conditions, capillary blood is therefore adequate for the measurement of 25OHD.

Thiopurine metabolite ratios for monitoring therapy in pediatric Crohn disease.

OBJECTIVES: Thiopurines (azathioprine, 6-mercaptopurine) are a mainstay of treatment in Crohn disease (CD). Monitoring intracellular metabolite (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine [6-MMP]) levels can help optimize therapeutic efficacy and minimize potential toxicity. Determination of 6-MMP/6-TGN ratios may provide additional useful information, such as the identification of individuals with excessive thiopurine methyltransferase activity and disadvantageous 6-MMP overproduction. These patients are at increased risk of therapeutic failure and hepatotoxicity. The aim of the study was to evaluate the correlation of 6-MMP/6-TGN ratios with therapeutic efficacy and risk of hepatotoxicity in CD. METHODS: The present study was a single-center cross-sectional study including pediatric patients with CD studied prospectively with clinical and laboratory assessments along with serial measurements of 6-MMP and 6-TGN. Clinical response was determined using established clinical indices. RESULTS: The study included 238 pediatric patients with CD with a total of 1648 evaluation points. The patients were in steroid-free remission at 59.1% of the evaluation points. 6-MMP/6-TGN ratios of 4 to 24 were protective against relapse (odds ratio [OR] 0.52, 95% confidence interval [CI] -0.39 to 0.69, P = 0.001). Hepatotoxicity was associated with high 6-MMP levels (>3919  pmol/8 × 10 red blood cell count: OR 7.65, 95% CI 3.7-15.9, P = 0.001) and high 6-MMP/6-TGN ratios (>24: OR 5.35, 95% CI -3.43 to 8.43, P = 0.001). CONCLUSIONS: We observed significant associations between 6-MMP/6-TGN ratios and clinical response, and risk of hepatotoxicity. Our results suggest that determination of thiopurine metabolite ratios is a valuable tool for identification of patients at increased risk of therapeutic failure and hepatotoxicity.

Glutathione S-transferase gene variations influence BU pharmacokinetics and outcome of hematopoietic SCT in pediatric patients.

BU is a key compound of conditioning regimens in children undergoing hematopoietic SCT (HSCT). Inter-individual differences in BU pharmacokinetics (PKs) might affect BU efficacy and toxicity. As BU is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship between GSTA1, GSTM1 and GSTP1 genotypes with first-dose BU PKs, and the relationship with HSCT outcomes in 69 children receiving myeloablative conditioning regimen. GSTM1 null genotype correlated with higher BU exposure and lower clearance in patients older than 4 years (P ≤ 0.04). In accordance with the suggested functional role, GSTA1*A2 haplotype was associated with lower drug levels and higher drug clearance (P ≤ 0.03). Gene-dosage effect was also observed (P ≤ 0.007). GSTA1 haplotypes were associated with HSCT outcomes. Patients with two copies of haplotype *A2 had better event free survival (P=0.03). In contrast, homozygous individuals for haplotypes *B and *B1 had higher occurrence of veno-occlusive disease (P=0.009). GSTM1 null individuals older than 4 years had more frequently graft versus host disease (P=0.03). In conclusion, we showed that GST gene variants influence BU PK and outcomes of HSCT in children. A model for the dosage adjustment with the inclusion of genetic and non-genetic factors should be evaluated in a future prospective validation cohort.

Influence of GST gene polymorphisms on busulfan pharmacokinetics in children.

Busulfan (BU) is a key compound in conditioning myeloablative regimens for children undergoing hematopoietic stem cell transplantation (HSCT). There are wide interindividual differences in BU pharmacokinetics, which increase the risk of veno-occlusive disease, graft rejection and disease relapse. As BU is mainly metabolized by glutathione S-transferase (GST), it is hypothesized that functional polymorphisms in GST genes may explain in part the variability in BU pharmacokinetics. We analyzed polymorphisms in GSTA1 (C-69T, A-513G, G-631T, C-1142G), GSTM1 (deletion) and GSTP1 (A1578G, C2293T) genes in 28 children undergoing HSCT. All patients had individualized dosing based on pharmacokinetics after the first dose of intravenous BU. GSTM1-null individuals had higher drug exposure (P(Cmax)=0.008; P(AUC)=0.003; P(Css)=0.02) and lower clearance (P(CL)=0.001). Multivariate regression models showed that, other than the drug dose and age, the GSTM1 genotype was the best predictor of first-dose pharmacokinetic variability. GSTM1-null patients also received lower cumulative BU doses (P=0.02). No association was found between BU exposure and major GSTA1 or GSTP1 gene variants. In children, GSTM1 polymorphism seems to modify BU pharmacokinetics after intravenous drug administration.

Intravenous busulfan for allogeneic hematopoietic stem cell transplantation in infants: clinical and pharmacokinetic results.

SUMMARY: High-dose busulfan is an important component of myeloablative regimens. Variable drug exposure may occur following oral administration. Therefore, the use of intravenous busulfan has been advocated. Previous work has suggested a cumulative dosage of 16 mg/kg for haematopoietic transplantation in children less than 3 years of age, but only limited data are available in infants. Pharmacokinetics of intravenous busulfan administered at the suggested dosage were studied in 14 infants (median age 4.7 months). Busulfan plasma concentrations were measured by either GC-MS or HPLC-UV. In seven patients, the dose was decreased to target an area- under- the- curve of 600-1300 micromol min. The median total dose given was 13.8 mg/kg. All patients engrafted. Severe veno-occlusive disease occurred in one patient. Our study demonstrates that a cumulative dosage of 16 mg/kg is associated with higher exposure than expected in infants. We suggest an initial dose of 0.8 mg/kg followed by pharmacokinetically guided dose adjustment.

Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease.

BACKGROUND & AIMS: The effects of 6-mercaptopurine (6-MP) are mediated via its intracellular conversion to 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) nucleotide metabolites, the latter genetically controlled by thiopurine methyltransferase (TPMT). We sought to determine optimal therapeutic 6-MP metabolite levels and their correlation with medication-induced toxicity and TPMT genotype. METHODS: Therapeutic response was determined in 92 pediatric patients with inflammatory bowel disease coincidentally with hematologic, pancreatic, and hepatic laboratory parameters, and compared with erythrocyte metabolite levels and TPMT genotype. RESULTS: Clinical response was highly correlated with 6-TG levels (P < 0.0001) but not with any other variable, including 6-MMP levels, drug dose, gender, and concurrent medications. The frequency of therapeutic response increased at 6-TG levels > 235 pmol/8 x 10(8) erythrocytes (P < 0.001). Hepatotoxicity correlated with elevated 6-MMP levels (>5700 pmol/8 x 10(8) erythrocytes; P < 0.05). Although leukopenia was associated with higher 6-TG levels (P < 0.03), it was observed in only 8% of responders. Patients heterozygous for TPMT (8/92) had higher 6-TG levels (P < 0.0001), and all responded to therapy. CONCLUSIONS: 6-MP metabolite levels and TPMT genotyping may assist clinicians in optimizing therapeutic response to 6-MP and identifying individuals at increased risk for drug-induced toxicity.

Cholinergic cell expression in the developing rat medial septal nucleus in vitro is differentially controlled by GABAA and GABAB receptors.

The early appearance and relative abundance of GABAergic neurons in basal forebrain cholinergic nuclei like the medial septum suggest that the maturation of the later developing cholinergic neurons in these nuclei may be controlled by GABA. To examine this possibility, the effects of both exogenous GABA and specific GABA receptor agonists, as well as that of endogenous GABA on the phenotypic expression and survival of the cholinergic neurons in primary cultures from the fetal rat medial septum, were studied. Treatment of these cultures for six days with GABA significantly decreased the enzymatic activity of choline acetyltransferase (EC 2.3.1.6) (ChAT) in a dose-dependent manner. This response to exogenous GABA was blocked by bicuculline, mimicked by muscimol and slightly potentiated by saclofen. Consistent with this latter observation, the GABAB receptor agonist, baclofen, dose-dependently increased septal ChAT activity. However, while the effect of baclofen on cholinergic expression was lost in the absence of glia, the suppressive effects of GABA or muscimol were more marked. Acetylcholinesterase (EC 3.1.1.7) (AChE) expression in mixed neuronal-glial cultures, was, like ChAT activity, increased or decreased in intensity with the inclusion of baclofen or muscimol, respectively. Although the number of AChE positive neurons in muscimol-treated cultures was significantly lower than that in controls, no changes in neither neuronal nor general cell viability were noted. Finally, as GABAA or GABAB receptor antagonists bicuculline and picrotoxin or saclofen, when applied alone to mixed cultures, increased or decreased ChAT activity, respectively, it appears that endogenous GABA, tonically released in the developing septum, may, via specific receptor types, differentially control the biochemical maturation of the cholinergic neurons.

Effects of basic fibroblast growth factor on the differentiation, growth, and viability of a new human medulloblastoma cell line (UM-MB1).

We presently report the effects of human recombinant basic fibroblast growth factor (bFGF) on a newly established medulloblastoma cell line, UM-MB1. This predominantly adherent cell line has a mean doubling time of 39.3 hours and was found, by karyotypic analysis, to be near triploid. UM-MB1 consists of undifferentiated cells expressing markers of neuronal lineage such as the three neurofilament subunits as well as neuron-specific enolase, synaptophysin, and microtubule-associated proteins 1 and 5. In contrast, no immunoreactivity for glial fibrillary acidic protein was evident. When exposed to nanomolar amounts of bFGF, UM-MB1 cells began to extend neurite-like processes with arborizations and growth-cone-like structures. In addition, UM-MB1 cells treated with bFGF exhibited ultrastructural alterations that reflect their enhanced differentiation as well as the increased expression of at least one of the neurofilament subunits as evidenced both immunocytochemically and on Western blots. Furthermore, bFGF significantly decreased UM-MB1 cell growth as well as induced their death. UM-MB1 cells treated with bFGF for several days displayed DNA cleavage, nuclear shrinkage, and chromatin condensation while retaining their cytoplasmic and mitochondrial membrane integrity, all early indices of apoptosis. After this, cell death was evident with the concomitant appearance of the classical apoptotic bodies. By flow cytometry, bFGF was found to increase the proportion of cells in G1 before inducing their death by apoptosis. In conclusion, UM-MB1 cells can, when appropriately stimulated, be induced to differentiate along their neuronal lineage pathway. Their differentiation induced by bFGF, although incomplete, appears to promote or inhibit the expression of apoptotic effectors or suppressors in these cells, respectively, so to induce their death by an apoptotic-like mechanism.

6-Mercaptopurine metabolism in Crohn's disease: correlation with efficacy and toxicity.

BACKGROUND: 6-Mercaptopurine (6-MP) has confirmed short and longterm efficacy in the treatment of IBD. However, the relation between its metabolism, efficacy, and side effects is not well understood. AIMS: To assay 6-MP metabolites and to correlate levels with drug compliance, disease activity, and adverse effects of treatment. PATIENTS: Heparinised blood was obtained prior to daily administration of 6-MP in 25 adolescent Crohn's disease patients (14 ileocolitis, 11 colitis) receiving 1.2 (range 0.4-1.6) mg/kg/day for a mean of 17 (range 4-65) months. METHODS: Erythrocyte free bases 6-thioguanine (6-TG) and 6-methyl-mercaptopurine (6-MMP) were measured (pmol/8 x 10(8) red blood cells) using reverse phase high performance liquid chromatography. RESULTS: Disease activity (modified Harvey-Bradshaw index) improved significantly with 6-MP (p = 0.001). Clinical remission was achieved in 72% of patients, who stopped taking prednisone, or were successfully weaned to a low alternate day dose (< 0.4 mg/kg/OD). Remission correlated well with erythrocyte 6-TG (p < 0.05), but not 6-MMP levels. Neutropenia was associated with 6-MP use (p < 0.005), but did not correlate with erythrocyte 6-MP metabolite levels. One patient refractory to 6-MP had 6-TG, but no measureable 6-MMP production, suggesting deficient thiopurine methyl-transferase activity or poor compliance. 6-MP induced complications (hepatitis, pancreatitis, and marrow suppression) were generally associated with increased 6-MMP levels. CONCLUSIONS: These results suggest that high performance liquid chromatography measurement of erythrocyte 6-MP metabolites may provide a quantitative assessment of patient responsiveness and compliance to treatment. The data support an immunosuppressive role for 6-TG, and potential cytotoxicity of raised 6-MMP levels.

Quantitation of 6-thioguanine in peripheral blood leukocyte DNA in Crohn's disease patients on maintenance 6-mercaptopurine therapy.

The effects of 6-mercaptopurine (6MP) in inflammatory bowel disease are believed to be primarily mediated by its metabolite 6-thioguanine (6TG). Our aim was to develop an assay for measuring leukocyte DNA 6TG levels in patients with Crohn's disease, and to correlate them with levels of 6TG in erythrocytes. Heparinized blood was obtained from 15 adolescent Crohn's disease patients receiving 6MP at an average dose of 1.3 mg.kg-1 day-1 (range 0.8-1.6 mg.kg-1 day-1) for a mean of 23.7 months (range 3-71 months). Leukocyte DNA and erythrocyte 6TG levels were measured by an HPLC assay. Leukocyte 6TG levels ranged from 100 to 2305 pmol/mg DNA, while erythrocyte 6TG levels ranged from 64 to 1038 pmol/8 x 10(8) red blood cells, demonstrating significant interpatient variability. Leukocyte DNA 6TG levels correlated directly with erythrocyte 6TG levels, as measured by the Spearman rank correlation coefficient (p < 0.05). The HPLC measurement of erythrocyte and leukocyte DNA 6TG levels can be useful clinically in monitoring compliance, as well as perhaps to tailor drug metabolite levels to achieve the desired clinical effect.

Safety, tolerability, and pharmacokinetic actions of diltiazem in pediatric liver transplant recipients on cyclosporine.

Thirty-two children who had undergone liver transplantation were paired according to their posttransplantation duration, renal function, and diagnoses when possible and randomized either to continue nifedipine (NIF group) or switch to diltiazem (DIL group), in addition to continuing their usual immunosuppressive medications. The cases were followed prospectively regarding diltiazem tolerance, cyclosporine dose requirements, effect on cyclosporine kinetics, diltiazem kinetics, as well as effect on renal function. Diltiazem was well tolerated at a dose of 3 mg to 6.4 mg/kg/day (max 180 mg/day) with infrequent self-limited mild side effects. Cyclosporine daily dose was reduced by a mean of 36.7% and 38.3% at 3 and 6 months, respectively, in the DIL group to achieve target trough cyclosporine levels without modifying liver function. No significant difference in renal function was observed after 3 to 6 months in either group based on blood urea nitrogen and creatinine levels and glomerular filtration rate by the DTPA method. Diltiazem appears to be well tolerated in children and allows for substantial dose reductions of CSA without apparent effects on liver graft function.

Simple, rapid and selective method using high-performance liquid chromatography for the determination of bretylium in plasma.

A high-performance liquid chromatographic method with ultraviolet detection has been developed for the determination of bretylium in plasma. Following a single-step solid-phase extraction procedure, bretylium is selectively isolated and well recovered from plasma. The assay sensitivity is 0.156 micrograms/ml from 250-microliters plasma samples and its linearity was assessed up to 40 micrograms/ml. The method is accurate (101.0 +/- 5.4%) and precise (maximum coefficient of variation of 8%). It provides a simple and time-saving alternative to existing methods and is particularly suitable for pharmacokinetic studies.

High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic procedure to determine spironolactone and its three major metabolites in biological specimens is described. The assay involves sequential extraction on C18 and CN solid phases, and subsequent separation on a reversed-phase column. In plasma samples, spironolactone and its metabolites were completely separated within 8 min using an isocratic mobile phase, while in urine samples a methanol gradient was necessary to achieve a good separation within 14 min. Recoveries for all analytes were greater than 80% in plasma and 72% in urine. Linear responses were observed for all compounds in the range 6.25-400 ng/ml for plasma and 31.25-2000 ng/ml for urine. The plasma and urine methods were precise (coefficient of variation from 0.8 to 12.5%) and accurate (-12.1% to 7.4% of the nominal values) for all compounds. The assay proved to be suitable for the pharmacokinetic study of spironolactone in healthy human subjects.

High-performance liquid chromatography-electrochemical detection of vecuronium and its metabolites in human plasma.

A high-performance liquid chromatographic assay coupled with electrochemical detection has been developed for the determination of vecuronium and its three putative deacetylated metabolites in human plasma. A novel solid-phase extraction procedure allowed good recovery of both vecuronium and its metabolites, together with ease and speed of execution. This method was sensitive, reproducible and accurate over the therapeutic range of concentrations of vecuronium and its metabolites, and was applied successfully to a study of the pharmacokinetics of vecuronium in anaesthetized patients.

Pharmacokinetics and pharmacodynamics of atracurium obtained with arterial and venous blood samples.

To determine the influence of sampling site on atracurium pharmacokinetic-pharmacodynamic relationships, blood was drawn simultaneously from the radial artery and peripheral vein during a 20-minute period after injection of atracurium, 0.2 mg/kg, in eight patients. Atracurium and laudanosine concentrations were measured by HPLC. Neuromuscular blockade was measured at the adductor pollicis, after stimulation of the ulnar nerve. Venous levels were lower than corresponding arterial values for up to 20 minutes, and this difference was marked for the early samples. Neuromuscular blockade was maximum after 5 to 7 minutes, much later than the peak venous concentration (1 to 3 minutes). Nonparametric analysis yielded (mean +/- SEM) a rate constant, concentration for 50% blockade, and slope of the effect-concentration relationship of 0.092 +/- 0.01 min-1, 379 +/- 27 ng/ml, and 7.3 +/- 1.67, respectively, when based on arterial samples. The values were statistically different (0.135 +/- 0.011 min-1, 235 +/- 42 ng/ml, and 3.41 +/- 0.37, respectively) when venous levels were used (p less than 0.05). It is concluded that forearm venous levels do not correspond to adductor pollicis neuromuscular blockade and the kinetics and kinetic-dynamic relationship for atracurium are heavily dependent on sampling site.

Pharmacokinetics and pharmacodynamics of atracurium with and without previous suxamethonium administration.

Suxamethonium increases neuromuscular block produced by non-depolarizing agents administered subsequently. To determine if this effect has a pharmacokinetic or pharmacodynamic origin, 18 ASA physical status I or II adults received atracurium 0.2 mg kg-1, with (n = 10) or without (n = 8) previous injection of suxamethonium 1 mg kg-1, during a thiopentone-nitrous oxide-isoflurane (0.5% end-tidal) anaesthetic. Arterial blood samples were obtained and plasma atracurium concentration measured by HPLC. Train-of-four stimulation was applied to the ulnar nerve and the force of contraction of the adductor pollicis muscle was recorded. Mean (SEM) volume of distribution was slightly greater with previous suxamethonium (143 (13) ml kg-1) than without (109 (5) ml kg-1) (P less than 0.04). Mean elimination half-life was unaffected (20.3 (0.8) min and 20.4 (1.6) min, respectively). Neuromuscular block was more intense and recovery was slower with previous administration of suxamethonium. Atracurium concentration at 50% block (Cpss50) was 305 (30) ng ml-1 with and 454 (25) ng ml-1 without previous suxamethonium (P less than 0.01). It is concluded that suxamethonium may be associated with a slight increase in the volume of distribution of atracurium, but this effect is more than compensated by a decrease in atracurium concentration required for a given effect.

Determination of atracurium and laudanosine in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method coupled with fluorometric detection has been developed for the determination of atracurium and its major end-product laudanosine in human plasma. The method enables good separation of atracurium from its metabolites after direct precipitation of plasma proteins. The assay is sensitive, reproducible and linear for atracurium concentrations ranging from 31.25 to 8000 ng/ml. In a clinical setting, drugs commonly administered during anesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.