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

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT center dot The cytotoxic effects of 6-mercaptopurine (6-MP) were found to be due to drug-derived intracellular metabolites (mainly 6-thioguanine nucleotides and to some extent 6-methylmercaptopurine nucleotides) rather than the drug itself. center dot Current empirical dosing methods for oral 6-MP result in highly variable drug and metabolite concentrations and hence variability in treatment outcome. WHAT THIS STUDY ADDS center dot The first population pharmacokinetic model has been developed for 6-MP active metabolites in paediatric patients with acute lymphoblastic leukaemia and the potential demographic and genetically controlled factors that could lead to interpatient pharmacokinetic variability among this population have been assessed. center dot The model shows a large reduction in interindividual variability of pharmacokinetic parameters when body surface area and thiopurine methyltransferase polymorphism are incorporated into the model as covariates. center dot The developed model offers a more rational dosing approach for 6-MP than the traditional empirical method (based on body surface area) through combining it with pharmacogenetically guided dosing based on thiopurine methyltransferase genotype. 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. 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. 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

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 in?uence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or in?ammatory 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 signi?cantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients,P = 0.047 in IBD patients). The study con?rmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a signi?cant 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.

Application of NMR based metabolomics for mapping metabolite variation in European wheat

In this study, we report on the use of NMR-based metabolomics to access variation in low molecular weight polar metabolites between the European wheat cultivars Apache, Charger, Claire and Orvantis. Previous unassigned resonances in the published NMR spectra of wheat extracts were identified using C NMR and two dimensional proton-carbon NMR. These included a peak for trans-aconitate (d3.43) and resonances corresponding to fructose in the crowded carbohydrate region of the spectra. Large metabolite differences were observed between two different growth stages, namely the coleoptile and two week old leaf tissue extracts which were consistent across cultivars. Two week old leaf tissue extracts had higher abundances of glutamine, glutamate, sucrose and trans-aconitate and less glucose and fructose than were observed in the coleoptile extracts. Across both growth stages the cultivars Apache and Charger showed the greatest differences in metabolite profiles. Charger had higher abundances of betaine, the single most influential metabolite in the principal component analysis, in addition to fructose and sucrose. However, Charger had lower levels of aspartate, choline and glucose than Apache. These findings demonstrate the potential for a biochemical mapping approach using NMR, across European wheat germplasm, for metabolites of known importance to functional characteristics. © Springer Science+Business Media, LLC 2009.

Regioselectivity and stereoselectivity of dioxygenase catalysed cis-dihydroxylation of mono- and tri-cyclic azaarene substrates

cis-Dihydrodiol metabolites were obtained from dioxygenase-catalysed asymmetric dihydroxylations of. five monocyclic (azabiphenyl) and four tricyclic (azaphenanthrene) azaarene substrates. Enantiopurity values and absolute configuration assignments were determined using a combination of stereochemical correlation, X-ray crystallography and spectroscopy methods. The degree of regioselectivity found during cis-dihydroxylation of monocyclic azaarenes (2,3 bond >> 3,4 bond) and of tricyclic azaarenes (bay region > non-bay region bonds) was dependent on the type of dioxygenase used. The cis-dihydrodiol metabolite from an azaarene (3-phenylpyridine) was utilised in the chemoenzymatic synthesis of the corresponding trans-dihydrodiol.

Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness?

Bailey DM, Taudorf S, Berg RMG, Lundby C, McEneny J, Young IS, Evans KA, James PE, Shore A, Hullin DA, McCord JM, Pedersen BK, Moller K. Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness? Am J Physiol Regul Integr Comp Physiol 297: R1283-R1292, 2009. First published September 2, 2009; doi: 10.1152/ajpregu.00366.2009.-This study examined whether hypoxia causes free radical-mediated disruption of the blood-brain barrier (BBB) and impaired cerebral oxidative metabolism and whether this has any bearing on neurological symptoms ascribed to acute mountain sickness (AMS). Ten men provided internal jugular vein and radial artery blood samples during normoxia and 9-h passive exposure to hypoxia (12.9% O-2). Cerebral blood flow was determined by the Kety-Schmidt technique with net exchange calculated by the Fick principle. AMS and headache were determined with clinically validated questionnaires. Electron paramagnetic resonance spectroscopy and ozone-based chemiluminescence were employed for direct detection of spin-trapped free radicals and nitric oxide metabolites. Neuron-specific enolase (NSE), S100 beta, and 3-nitrotyrosine (3-NT) were determined by ELISA. Hypoxia increased the arterio-jugular venous concentration difference (a-v(D)) and net cerebral output of lipid-derived alkoxyl-alkyl free radicals and lipid hydroperoxides (P

The Yin and Yang of vitamin D receptor (VDR) signaling in neoplastic progression: Operational networks and tissue-specific growth control

Substantive evidence implicates vitamin D receptor (VDR) or its natural ligand 1a,25-(OH)2 D3 in modulation of tumor growth. However, both human and animal studies indicate tissue-specificity of effect. Epidemiological studies show both inverse and direct relationships between serum 25(OH)D levels and common solid cancers. VDR ablation affects carcinogen-induced tumorigenesis in a tissue-specific manner in model systems. Better understanding of the tissue-specificity of vitamin D-dependent molecular networks may provide insight into selective growth control by the seco-steroid, 1a,25-(OH)2 D3. This commentary considers complex factors that may influence the cell- or tissue-specificity of 1a,25-(OH)2 D3/VDR growth effects, including local synthesis, metabolism and transport of vitamin D and its metabolites, vitamin D receptor (VDR) expression and ligand-interactions, 1a,25-(OH)2 D3 genomic and non-genomic actions, Ca2+ flux, kinase activation, VDR interactions with activating and inhibitory vitamin D responsive elements (VDREs) within target gene promoters, VDR coregulator recruitment and differential effects on key downstream growth regulatory genes. We highlight some differences of VDR growth control relevant to colonic, esophageal, prostate, pancreatic and other cancers and assess the potential for development of selective prevention or treatment strategies.

Chemoenzymatic synthesis of chiral 2,2 '-bipyridine ligands and their N-oxide derivatives: applications in the asymmetric aminolysis of epoxides and asymmetric allylation of aldehydes

A series of enantiopure 2,2'-bipyridines have been synthesised from the corresponding cis-dihydrodiol metabolites of 2-chloroquinolines. Several of the resulting hydroxylated 2,2'-bipyridines were found to be useful chiral ligands for the asymmetric aminolysis of meso-epoxides leading to the formation of enantioenriched amino alcohols (-> 84%ee). N-oxide and N,N'-dioxide derivatives of these 2,2'-bipyridines, including separable atropisomers, have been synthesised and used as enantioselective organocatalysts in the asymmetric allylation of aldehydes to give allylic alcohols (-> 86%ee).

Solutes determine the temperature windows for microbial survival and growth

Microbial cells, and ultimately the Earth's biosphere, function within a narrow range of physicochemical conditions. For the majority of ecosystems, productivity is cold-limited, and it is microbes that represent the failure point. This study was carried out to determine if naturally occurring solutes can extend the temperature windows for activity of microorganisms. We found that substances known to disorder cellular macromolecules (chaotropes) did expand microbial growth windows, fungi preferentially accumulated chaotropic metabolites at low temperature, and chemical activities of solutes determined microbial survival at extremes of temperature as well as pressure. This information can enhance the precision of models used to predict if extraterrestrial and other hostile environments are able to support life; furthermore, chaotropes may be used to extend the growth windows for key microbes, such as saprotrophs, in cold ecosystems and manmade biomes.

Binding of serum albumin to the anthelmintic drugs albendazole, triclabendazole and their sulphoxides

The binding of drugs to plasma proteins – especially serum albumin – is an important factor in controlling the availability and distribution of these drugs. In this study we have investigated the binding of two drugs commonly used to treat liver fluke infections, albendazole (ABZ) and triclabendazole (TCBZ), and their sulphoxide metabolites to bovine serum albumin (BSA). Both ABZ and TCBZ caused shifts in the mobility of BSA in native gel electrophoresis. No such changes were observed with the sulphoxides under identical conditions. The drugs, and their sulphoxides, caused quenching of the intrinsic tryptophan fluorescence of BSA, indicating association between the drugs and this protein. Quantification of this quenching suggested a 5–10-fold reduction in affinity of the sulphoxides compared to the parent compounds. These results are discussed in respect to previous work on the pharmacodynamics of these fasciolicides and will inform the design of novel anthelmintics.

Synthesis and Reactions of Enantiopure Substituted Benzene cis-Hexahydro-1,2-diols

Enantiopure cis-dihydro-1,2-diol metabolites, obtained from toluene dioxygenase-catalysed cis-dihydroxylation of six monosubstituted benzene substrates, have been converted to their corresponding cis-hexahydro-12-diol derivatives by catalytic hydrogenation via their cis-tetrahydro-1,2-diol intermediates. Optimal reaction conditions for total catalytic hydrogenation of the cis-dihydro-1,2-diols have been established using six heterogeneous catalysts. The relative and absolute configurations of the resulting benzene cis-hexahydro-1,2-diol products have been unequivocally established by X-ray crystallography and NMR spectroscopy. Methods have been developed to obtain enantiopure cis-hexahydro-1,2-diol diastereoisomers, to desymmetrise a meso-cis-hexahydro-1,2-diol and to synthesise 2-substituted cyclohexanols. The potential of these enantiopure cyclohexanols as chiral reagents was briefly evaluated through their application in the synthesis of two enantiomerically enriched phosphine oxides from the corresponding racemic phosphine precursors.

Metabolic profile changes in the testes of mice with streptozotocin-induced type 1 diabetes mellitus

Contrary to the traditional view, recent studies suggest that diabetes mellitus has an adverse influence on male reproductive function. Our aim was to determine the effect of diabetes on the testicular environment by identifying and then assessing perturbations in small molecule metabolites. Testes were obtained from control and streptozotocin-induced diabetic C57BL/6 mice, 2, 4 and 8 weeks post-treatment. Diabetic status was confirmed by glycated haemoglobin, non-fasting blood glucose, physiological condition and body weight. A novel extraction procedure was utilized to obtain protein free, low-molecular weight, water soluble extracts which were then assessed using H-1 nuclear magnetic resonance spectroscopy. Principal component analysis of the derived profiles was used to classify any variations, and specific metabolites were identified based on their spectral pattern. Characteristic metabolite profiles were identified for control and type 1 diabetic animals with the most distinctive being from mice with the largest physical deterioration and loss of body weight. Eight streptozotocin-treated animals did not develop diabetes and displayed profiles similar to controls. Diabetic mice had decreases in creatine, choline and carnitine and increases in lactate, alanine and myo-inositol. Betaine levels were found to be increased in the majority of diabetic mice but decreased in a few animals with severe loss of body weight and physical condition. The association between perturbations in a number of small molecule metabolites known to be influential in sperm function, with diabetic status and physiological condition, adds further impetus to the proposal that diabetes influences important spermatogenic pathways and mechanisms in a subtle and previously unrecognized manner.

Short-term phytoestrogen supplementation alters insulin-like growth factor profile but not lipid or antioxidant status

Phytoestrogens are plant compounds that have been proposed to have a variety of health benefits. The aim of this study was to assess the effects of these compounds on a number of physiological endpoints. Subjects were given a single intake of a phytoestrogen-rich (80 mg total phytoestrogens) supplement containing soy, rye and linseed (Phase 1), followed by a week-long intervention using the same supplement (Phase 2) (80 mg total phytoestrogens daily). A number of biochemical endpoints were assessed including urinary phytoestrogen metabolites, lipids, antioxidant status, DNA damage and insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 (IGFBP-1) and -3 (IGFBP-3). Ten healthy female subjects took part in the study. Excretion of the isoflavones genistein, daidzein and equol in urine increased in both phases of the study. No other endpoint was altered in Phase 1. However, in Phase 2, concentrations of IGF-1 and IGFBP-3 were increased by phytoestrogen supplementation [IGF-1, median (IQ range), baseline 155 (123, 258), postweek 265 (228, 360) ng/ml, P

An introduction to biological nuclear magnetic resonance spectroscopy

ABSTRACT Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of 1H NMR spectroscopy in mixture analysis and metabolomics, the use of 13C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways (‘fluxomics’) and the use of 31P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances— such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation—are opening new avenues for today’s biological NMR spectroscopists.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Prokaryotes on the tentacles of deep-sea holothurians: A novel form of dietary supplementation

Bathyal and abyssal epibenthic holothurians have a layer of bacteria lying over the tentacular epidermis and below the cuticle. Thus the tentacles of deep-sea holothurians may provide ideal conditions for subcuticular bacteria. These bacteria appear to be regulated by phagocytosis, which, together with pinocytosis would facilitate transfer of bacterial metabolites to the holothurian. Their abundance suggests a previously unknown pathway for energy transformation and assimilation of particular significance in an environment where food is limiting.