Global DNA methylation profiling reveals silencing of a secreted form of Epha7 in mouse and human germinal center B-cell lymphomas.

Most human lymphomas originate from transformed germinal center (GC) B lymphocytes. While activating mutations and translocations of MYC, BCL2 and BCL6 promote specific GC lymphoma subtypes, other genetic and epigenetic modifications that contribute to malignant progression in the GC remain poorly defined. Recently, aberrant expression of the TCL1 proto-oncogene was identified in major GC lymphoma subtypes. TCL1 transgenic mice offer unique models of both aggressive GC and marginal zone B-cell lymphomas, further supporting a role for TCL1 in B-cell transformation. Here, restriction landmark genomic scanning was employed to discover tumor-associated epigenetic alterations in malignant GC and marginal zone B-cells in TCL1 transgenic mice. Multiple genes were identified that underwent DNA hypermethylation and decreased expression in TCL1 transgenic tumors. Further, we identified a secreted isoform of EPHA7, a member of the Eph family of receptor tyrosine kinases that are able to influence tumor invasiveness, metastasis and neovascularization. EPHA7 was hypermethylated and repressed in both mouse and human GC B-cell non-Hodgkin lymphomas, with the potential to influence tumor progression and spread. These data provide the first set of hypermethylated genes with the potential to complement TCL1-mediated GC B-cell transformation and spread.

NMR and QSAR studies on the transacylation reactivity of model 1beta-O-acyl glucuronides. I: design, synthesis and degradation rate measurement.

1. The products arising from intramolecular acyl migration reactions of drug ester glucuronides are reactive towards cellular proteins and can potentially cause toxic side-effects. The relationship between molecular structure and the degradation rates (kd) of 1beta-O-acyl glucuronides were investigated systematically using a series of model compounds based on 4-substituted benzoic acids. 2. A rational method for selecting suitable compounds for inclusion was used and 10 glucuronide esters, predicted to produce a wide range of transacylation rates, were synthesized via a simple "one-pot" method using an imidazolide intermediate. The 10 substituents, where X = NO2, CN, I, Br, F, H, nPr, Et, OMe, O-nPr, had degradation rate half-lives (t1/2 = loge(2)/kd) ranging from 0.9 to 106.6 h. The reactions resulted in mixtures, which predominantly consisted of the desired 1beta-O-acyl glucuronides. 3. It was demonstrated that further purification was unnecessary for determination of kd of the synthetic 1beta-O-acyl glucuronides. Degradation rates (kd) were calculated by following the disappearance of the 1H-NMR signal from the 1beta-anomeric proton of the glucuronic acid moiety as the reaction progressed in pH 7.4 buffer inside an nuclear magnetic resonance tube. Each measured degradation rate represents a pseudo-first-order rate constant, which is a combination of the transacylation rate (1beta to 2beta isomer) and the hydrolysis rate. 4. Degradation rates show a clear relationship with substituent properties, with half-life increasing as the substituent becomes more electron-donating, e.g. 4-nitro t1/2 = 0.9 h and 4-propoxy t1/2 = 106.6 h.

Nuclear magnetic resonance (NMR) and quantitative structure-activity relationship (QSAR) studies on the transacylation reactivity of model 1beta-O-acyl glucuronides. II: QSAR modelling of the reaction using both computational and experimental NMR parameters.

In a previously reported study, a number of 4-substituted benzoic acid acyl glucuronides were synthesized and their degradation rates determined using nuclear magnetic resonance (NMR) spectroscopy. It was shown that this reaction was strongly influenced by the nature of the substituent at the 4-position of the benzoyl moiety. The overall degradation reaction rates for this series of compounds have been modelled successfully using Hammett substituent constants, computational chemistry-derived partial atomic charges and the experimentally determined carbonyl carbon 13C-NMR chemical shifts of the benzoic acids and their ethyl and glucuronide esters. The primary contribution to reactivity is the scale of the electron-donating or -withdrawing effect of the substituent; however, additional contributions such as steric parameters must also be considered when modelling reactions outside a single chemical series. The derived property-reactivity relationships should find utility in medicinal chemistry efforts for optimizing chemical series in pharmaceutical discovery programmes.

High-resolution diffusion and relaxation-edited magic angle spinning 1H NMR spectroscopy of intact liver tissue.

High-resolution magic angle spinning (HRMAS) (1)H NMR spectroscopy is ideal for monitoring the metabolic environment within tissues, particularly when spectra are weighted by physical properties such as T(1) and T(2) relaxation times and apparent diffusion coefficients (ADCs). In this study, spectral-editing using T(1) and T(2) relaxation times and ADCs at variable diffusion times was used in conjunction with HRMAS (1)H NMR spectroscopy at 14.1 T in liver tissue. To enhance the sensitivity of ADC measurements to low molecular weight metabolites a T(2) spin echo was included in a standard stimulated gradient spin-echo sequence. Fatty liver induced in rats by chronic orotic acid feeding was investigated using this modified sequence. An increase in the combined ADC for the co-resonant peaks glucose, betaine, and TMAO during fatty liver disease was detected (ADCs = 0.60 +/- 0.11 and 0.35 +/- 0.1 * 10(-9) m(2)s(-1) (n = 3) for rats fed with and without orotic acid), indicative of a reduction in glucose and betaine and an increase in TMAO.

HPLC/1H NMR spectroscopic studies of the reactive alpha-1-O-acyl isomer formed during acyl migration of S-naproxen beta-1-O-acyl glucuronide.

A widely held view in drug metabolism and pharmacokinetic studies is that the initial 1-isomer to 2-isomer step in the intramolecular acyl migration of drug ester glucuronides is irreversible, and that alpha-1-O-acyl isomers do not occur under physiological conditions. We investigated this hypothesis using high-performance liquid chromatography directly coupled to proton nuclear magnetic resonance spectroscopy (HPLC/1H NMR) and mass spectrometry (LC/MS) to probe the migration reactions of S-naproxen beta-1-O-acyl glucuronide, in phosphate buffer at pH 7.4, 37 degrees C. We report the first direct observation of the alpha-1-O-acyl isomer of a drug ester glucuronide (S-naproxen) formed in a biosystem via the facile acyl migration of the corresponding pure beta-1-O-acyl glucuronide. The unequivocal identification of the reactive product was achieved using stopped-flow one-dimensional HPLC/1H NMR and two-dimensional 1H-1H total correlation spectroscopy (1H-1H TOCSY). Parallel LC/ion-trap mass spectrometry yielded the confirmatory glucuronide masses. Moreover, "dynamic" stopped-flow HPLC/1H NMR experiments revealed transacylation of the isolated alpha-1-O-acyl isomer to a mixture of alpha/beta-2-O-acyl isomers; the reverse reaction from the isolated alpha/beta-2-O-acyl isomers to the alpha-1-O-acyl isomer was also clearly demonstrated. This application of "dynamic" stopped-flow HPLC/1H NMR allows key kinetic data to be obtained on a reactive metabolite that would otherwise be difficult to follow by conventional HPLC and NMR methods where sample preparation and off-line separations are necessary. These data challenge the widely held view that the alpha-1-O-acyl isomers of drug ester glucuronides do not occur under physiological conditions. Furthermore, the similar formation of alpha-1-O-acyl isomers from zomepirac and diflunisal beta-1-O-acyl glucuronides has recently been confirmed (Corcoran et al., unpublished results). Such reactions are also likely to be widespread for other drugs that form ester glucuronides in biological systems. Ultimately, the presence of significant quantities of the kinetically labile alpha-1-O-acyl glucuronide isomer may also have toxicological implications in terms of reactivity toward cellular proteins.

LC-1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems.

The reactive metabolite S-naproxen-beta-1-O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC-1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-beta-1-O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase 1 (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4-O-acyl isomers, beta-1-O-acyl glucuronide, 3-O-acyl isomers, 2-O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: alpha/beta-4-O-acyl isomers, S-naproxen aglycone, beta-1-O-acyl glucuronide, 3-O-acyl isomers, and alpha/beta-2-O-acyl isomers. In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-1H NMR (U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113-122]. The alpha-1-O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-beta-1-O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37 degrees C. In both HPLC systems it eluted just before the beta-1-O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a alpha-1-O-acyl isomer when studying glucuronide reactivity and degradation.

The biochemical profile of rat testicular tissue as measured by magic angle spinning 1H NMR spectroscopy.

The testis is the principal organ of male fertility, responsible for the production of spermatozoa and their maturation into sperm. However, the underlying biochemistry of the testis is relatively understudied. The fluidic and homogeneous nature of the testis makes it an ideal organ for high resolution magic angle spinning (MAS) 1H NMR spectroscopy. In this study we have catalogued the low molecular weight metabolites. The testis contains large amounts of creatine, of which a substantial proportion was shown to be extracellular using bipolar gradients to measure apparent diffusion coefficients. The tissue also contained relatively high amounts of uridine.

The initial pathogenesis of cadmium induced renal toxicity.

The novel application of magic angle spinning 1H NMR spectroscopy, coupled with pattern recognition techniques, has identified biochemical changes in lipid and glutamate metabolism that precede classical nephrotoxicity. These changes occurred in the bank vole (Clethrionomys glareolus) after chronic dosing, at a low level of exposure and at a renal Cd(2+) concentration (8.4 microgram/g dry wt) that was nearly two orders of magnitude below the WHO critical organ concentration (200 microg/g wet wt). These early stage effects of Cd(2+) on the biochemistry of renal tissue may reflect adaptation mechanisms to the toxic insult or the preliminary stages of the toxicological cascade.

Degradation of 4-fluorobiphenyl by mycorrhizal fungi as determined by (19)F nuclear magnetic resonance spectroscopy and (14)C radiolabelling analysis.

The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus Tylospora fibrilosa and several other mycorrhizal fungi were investigated by using (19)F nuclear magnetic resonance (NMR) spectroscopy in combination with (14)C radioisotope-detected high-performance liquid chromatography ((14)C-HPLC). Under the conditions used in this study T. fibrillosa and some other species degraded 4FBP. (14)C-HPLC profiles indicated that there were four major biotransformation products, whereas (19)F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4'-ol and 4-fluorobiphen-3'-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely, meta cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.

Degradation of 4-fluorobiphenyl in soil investigated by 19F NMR spectroscopy and 14C radiolabelling analysis.

The incubation of the model pollutant [U-14C]'-4-fluorobiphenyl (4FBP) in soil, in the presence and absence of biphenyl (a co-substrate), was carried out in order to study the qualitative disposition and fate of the compound using 14C-HPLC and 19F NMR spectroscopy. Components accounted for using the radiolabel were volatilization, CO2 evolution, organic solvent extractable and bound residue. Quantitative analysis of these data gave a complete mass balance. After sample preparation. 14C-HPLC was used to establish the number of 4FBP related components present in the organic solvent extract. 19F NMR was also used to quantify the organic extracts and to identify the components of the extract. Both approaches showed that the composition of the solvent extractable fractions comprised only parent compound with no metabolites present. As the 14C radiolabel was found to be incorporated into the soil organic matter this indicates that metabolites were being generated, but were highly transitory as incorporation into the SOM was rapid. The inclusion of the co-substrate biphenyl was to increase the overall rate of degradation of 4FBP in soil. The kinetics of disappearance of parent from the soil using the data obtained were investigated from both techniques. This is the first report describing the degradation of a fluorinated biphenyl in soil.

Direct characterization of drug glucuronide isomers in human urine by HPLC-NMR spectroscopy: application to the positional isomers of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid glucuronide.

In this work, 400 and 600 MHz 1H HPLC-NMR spectroscopic methods were developed and applied to separate and identify the positional glucuronide isomers and anomers of the model nonsteroidal antiinflammatory drug, 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid, in whole human urine. The HPLC methods utilized either an isocratic system, comprising 30% acetonitrile in water at pH 2.5, or a gradient elution system increasing from 30% to 60% acetonitrile, in order to achieve improved separation of the 2-, 3-, and 4-O-acylglucuronide isomers from the faster eluting endogenous urinary metabolites. Directly coupled stop-flow 1H HPLC-NMR spectroscopic measurements were made at the retention times indicated by the UV-monitored chromatographic peaks. The glucuronide isomers were identified from the 1H NMR spectra on the basis of their chemical shifts and spin-spin coupling patterns. The elution order was 4-O-acyl-, 3-O-acyl-, and finally 2-O-acylglucuronide, with tR values of 10.04, 11.68, and 12.64 min, respectively. Although the alpha- and beta-anomers of each of the positional isomers could not be separated in these solvent systems, they could be identified in the individual 1H NMR spectra. This work shows for the first time that directly coupled HPLC-NMR spectroscopy can be used directly to isolate and characterize acyl-migrated isomers of drug glucuronides in whole urine. This approach will be of value in the study of glucuronide acyl migration reactions of nonsteroidal antiinflammatory drugs and other xenobiotic ester glucuronides in whole biofluids.

750 MHz HPLC-NMR spectroscopic studies on the separation and characterization of the positional isomers of the glucuronides of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid.

Ester glucuronides (beta-1-O-acyl-D-glucopyranuronates) of many drugs can undergo a series of acyl migration reactions, resulting in positional isomers and anomers which can react with serum proteins with possible toxicological consequences. We have investigated the acyl migration of the ester glucuronides of the model drug 6,-11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid in pH 7.4 buffer using directly coupled 750 MHz stopped-flow HPLC-NMR spectroscopy. Using a reversed phase isocratic HPLC method with 21% acetonitrile and 79% D2O in the mobile phase, it was possible to separate and hence identify the individual positional isomers of the model drug glucuronide by 750 MHz HPLC-NMR. The order of elution of the isomers from the C18 column was 4alpha-, 4beta-, aglycon, 1beta-, 3beta-, 3alpha-, 2alpha-, 2beta- (alpha- and beta- referring to the anomerization state at C1 on the glucuronide ring and the numbers referring to the carbon number on the glucuronide ring to which the drug moiety has migrated). It is shown that directly coupled ultra-high-field HPLC-NMR spectroscopy offers a unique analytical advantage for obtaining structural information of interconverting compounds in equilibrium mixtures, and this method will be of value in the study of reactive drug glucuronides of toxicological importance.

Disposition of [14C]velnacrine maleate in rats, dogs, and humans.

This study describes the disposition of [14C]velnacrine maleate in rats, dogs, and humans, and the isolation and identification of metabolites in dog urine. Following oral administration of [14C]velnacrine maleate, drug-related material was well absorbed in all three species, with the majority of the dose recovered in the urine. Fecal elimination of radioactivity accounted for the remainder of the dose. The majority of the radioactivity was eliminated within 24 hr. Pharmacokinetic parameters for the elimination of radioactivity from the plasma of rats and dogs were similar after oral dosing compared with intravenous dosing. In humans, the plasma and urinary levels of velnacrine maleate were substantially lower, and the elimination half-life shorter than for total radioactivity, indicating the presence of one or more metabolites with a longer half-life than the parent compound. Preliminary TLC analysis of urine, plasma, and feces showed that metabolism appeared to be similar in the three species investigated. Velnacrine maleate was extensively metabolized with only approximately 10%, 19%, and 33% of the dose appearing in the urine as unchanged drug in humans, dogs, and rats, respectively. Isolation and identification of dog urinary metabolites was conducted. The identity of the isolated metabolites was determined by GC/MS and proton NMR. One of the main metabolic routes was found to be via hydroxylation of the tetrahydroaminoacridine ring with other minor hydroxylated and dihydroxylated metabolites being detected. In addition two dihydrodiol metabolites were also identified. Phase II metabolism did not appear to be a significant route.

Studies on the biochemical effects of the aldose reductase inhibitor 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione (Al 1576, HOE 843). Detection of D-glucaric and D-glucuronic acid excretion by high resolution 1H and 13C NMR spectroscopy.

The effects of two aldose reductase inhibitors on the biochemical composition of rat urine were investigated using high resolution 1H and 13C NMR spectroscopy. We report the elevated excretion of D-glucaric acid (DGA) and D-glucuronic acid (GCA) following treatment with 2,7-difluorospirofluorene-9,5'-imidazolidine-2'4'-dione (Imirestat, IM, Al 1576, HOE 843) at 50 mg/kg/day for 1 month, but not with 3-4-bromo-2-fluorobenzyl-4-oxo-3-phthalazine-1-ylacetic acid (Ponalrestat, Statil), dosed at 50 mg/kg/day for 2 weeks. Sugar aciduria was also detected following treatment with the cytochrome P450 inducer phenobarbitone (PB) at 45 mg/kg/day for 1 month, although the qualitative and quantitative pattern of excretion of sugar acids differed greatly between the IM and PB treatment groups. The levels of GCA excreted are elevated 11-fold by IM treatment from 19.0 to 210.0 mumol/24 hr, but only 2.5-fold by PB, from 9.7 to 23.9 mumol/24 hr. DGA was not detectable in control urine, although levels did increase by 30% during the study from 7.5 to 10.9 mumol/24 hr, between day 8 and day 29, with IM treatment, and by 60% from 1.7 to 4.9 mumol/24 hr following PB administration for the same time period. This predominant elevation of DGA and GCA caused by IM treatment far exceeds previous records. In contrast, PB treatment resulted in an increase in intensity of a number of partially resolved sugar resonances, but at a much lower level than resulted from IM treatment. A raised level of DGA and GCA is usually associated with hepatic P450 induction; however, we report here profound DGA and GCA uria as a result of the inhibition of the aldehyde reductase, hexonate dehydrogenase (EC 1.1.1.19, EC 1.1.1.20). This mechanism is not closely linked to P450 induction, corroborating the current view that elevated excretion of DGA is not a reliable indicator of hepatic enzyme induction. This study further demonstrates the use of high resolution NMR spectroscopy in the detection of a novel biochemical effect which may go unnoticed during routine clinical chemistry tests.

Application of 19F-n.m.r. spectroscopy to the identification of dog urinary metabolites of imirestat, a spirohydantoin aldose reductase inhibitor.

1. Urine from a dog dosed orally at 20 mg/kg with 14C-imirestat, a spirohydantoin aldose reductase inhibitor, contained 17.7 and 12.5% of the administered radioactivity at 0-48 and 48-72 h respectively. 2. Radio-h.p.l.c. of the 0-48 h urine revealed a complex mixture of metabolites and a small proportion of parent drug (1.6% of dose). Direct 19F-n.m.r. spectroscopy of this urine showed the fluoride ion, numerous metabolites which were predominantly glucuronide conjugates and, as a minor component, the parent drug. 3. After incubation with beta-glucuronidase the 0-48 h urine gave a 19F-n.m.r. spectrum showing fewer signals. This finding is consistent with aromatic ring hydroxylation followed by glucuronidation being the major metabolite pathways. 4. Deconjugated urine was analysed by proton-coupled 19F-n.m.r. and two-dimensional 19F-19F correlated spectroscopy. Results indicate that major components included three monohydroxy metabolites, a diphenol with both phenolic functions in the same ring, and a phenolic metabolite containing only one fluorine atom. 5. Semi-preparative h.p.l.c. of 0-48 h dog urine gave individual glucuronides isolated as mixtures of C-9 epimers. These fractions were hydrolysed and purified a second time by h.p.l.c. to give aglycones which were analysed by multi-nuclear n.m.r. and g.l.c.-mass spectrometry. The 3- and 4-hydroxy derivatives of imirestat were identified, as was the 2-hydroxy product obtained during or following defluorination. The other major aglycone was postulated to be the 3-fluoro-2-hydroxy metabolite. This represents a novel 'NIH-shift' type pathway for the metabolism of fluorobenzenes.

19F and 1H magnetic resonance strategies for metabolic studies on fluorinated xenobiotics: application to flurbiprofen [2-(2-fluoro-4-biphenylyl)propionic acid].

Strategies for the use of 1H and 19F nuclear magnetic resonance (NMR) spectroscopy as an aid to the study of the metabolic fate of fluorinated drugs are discussed with reference to the application of these methods to flurbiprofen metabolism in man. 1H and 19F NMR analysis of untreated urine enabled the detection of two major and eight minor metabolites of the drug. The two major metabolites were identified using a combination of NMR spectroscopy, solid-phase extraction chromatography with 19F and 1H NMR detection and chemical hydrolysis to a flurbiprofen glucuronide and the glucuronide of the 4-hydroxy metabolite. 1H-19F 2D shift correlated spectroscopy and spin-echo difference experiments are discussed in relation to their use in the structural identification of drug metabolites.

The metabolism of [14C]N-ethoxycarbonyl-3-morpholinosydnonimine (molsidomine) in laboratory animals.

[14C]N-Ethoxycarbonyl-3-morpholinosydnonimine (molsidomine, Corvaton) was found to be extensively metabolized following oral dosing to rat and dog and intravenous dosing to rabbit. The majority of the radiolabel was rapidly excreted in the urine with the main radiolabelled components being characterized as acidic metabolites resulting from oxidative metabolism of the morpholine ring. A new metabolite, (N-cyanomethylenamino-2-aminoethoxy)-acetic acid, was identified and shown to be a major component of the 14C-labelled urinary metabolites in all three species. However, the previously identified metabolite, N-cyanomethylenaminomorpholine-2-one (compound D) was not detected and may therefore have been formed artefactually in the earlier studies. The long terminal half-life for plasma radioactivity observed in previous studies was shown to be the result of the production of small amounts of 14C-thiocyanate from the nitrile-containing metabolites of molsidomine.