Investigation Of Metabolism Research Articles

Synthesis and evaluation of a radiometal-labeled macrocyclic chelator-derivatised thymidine analog

Several radiolabeled thymidine analogs as metabolic probes of cell proliferation were developed specifically addressing DNA synthesis. Thymidine analogs containing carboranylalkyl groups for neutron capture therapy at the N-3 position were found to be good substrates for cytosolic thymidine kinase 1 (TK1). According to this approach, a DO3A macrocycle in N-3 position was attached to thymidine. The 3-DO3A thymidine analog was labeled with 68Ga and 111In. Different lipophilicities of the corresponding radiometal-thymidines were detected via RP-HPLC. [111In]DO3A-thymidine ([111In]D3T) was evaluated for cellular uptake in different cell lines (HL60 and DoHH2). Cellular uptake was low in both cell lines. Phosphorylation of the radioconjugates by TK1 was negligible. Although stable complexation of radiometals to thymidine was obtained, introduction of the macrocycle DO3A reduced the affinity to cytosolic TK1 drastically. Low cellular uptake can be ascribed to missing substrate specificity of [111In]DO3A-thymidine for TK1. The absence of substrate specificity may be due to the bulky macrocyclic chelator and partial charges remaining on the coordination sphere due to a more complex solution structure.

Foods and Food Components as Probes of Lipid Metabolism in a Challenge Meal

Chronic metabolic diseases are complex and difficult to diagnose/treat. The underlying metabolic disturbances which lead to the manifestation of disease symptoms are multiple and interconnected. New ways of assessing health status which can detect predispositions to disease as well as diagnose and monitor underlying metabolism are needed. Health is more accurately described as the ability of an organism to respond to a variety of challenges, including nutritional. Metabolomics measures a breadth of endpoints simultaneously as quantitative metabolite measurements. Metabolomic analyses also make it possible to use a mixture of nutritional challenges designed to test individual biological responses. In this study, challenge meals were used as probes of lipid metabolism in order to test the use of certain foods and food components to explore areas of lipid metabolism. Liquid meals were formulated and fed to volunteers. Blood was sampled before and at multiple time points after the meal, followed by lipoprotein fractionation, and quantitative lipomic analyses. Specialized bioinformatic tools developed for the analysis of the lipomic data revealed patterns in lipid metabolism that differed between individuals. This response to challenge approach holds promise as a new nutritional/health assessment that can be applied in clinical practice to improve the diagnosis and monitoring of health in individuals.

PIII-53Discordance between slow acetylator phenotype and genotype for N-acetyltransferase 2 (NAT-2) in a Hmong population

BACKGROUND Polymorphisms of NAT-2 acetylation contribute to drug toxicity, efficacy and cancer risk. Predominance of slow acetylation (SA) phenotype in ethnically distinct populations may have clinical implications for drug selection and cancer risk. The purpose of this study was to determine the genetic basis of SA phenotype predominance in Minnesota Hmong. METHODS Urine and DNA were obtained for phenotype and genotype analysis from unrelated healthy Hmong 18 and 65 years of age. Urinary molar ratios (MR) of caffeine metabolites (AFMU/1X) identified rapid acetylators (RA) phenotypes with a MR >= 0.6 and SA with MR < 0.6. Direct sequencing of the NAT-2 coding-region followed by cloning techniques for ambiguous genotypes identified individuals homozygous or heterozygous with a *4 and *13 allele as RA and variants as SA by genotype. RESULTS From 61 subjects (30±11 years, 27 male), analysis of 51 urine-DNA pairs identified 46 (90.2%) SA and 5 (9.8%) RA by phenotype. In contrast, genotypic analysis identified 5 (9.8%) SA and 46 (90.2%) RA. An 84% discordance between phenotype and genotype was observed. Direct sequencing did not reveal novel NAT-2 polymorphisms. CONCLUSIONS Genotypic analysis appears to demonstrate considerable discordance with the phenotype in Hmong. Genotyping alone, without a metabolic probe, would not have accurately predicted acetylation phenotype. Clinical Pharmacology & Therapeutics (2005) 79, P72–P72; doi: 10.1016/j.clpt.2005.12.261

Dexamethasone as an in vivo probe for CYP3A metabolism: application to docetaxel and vinorelbine

2036 Background: Since none endogenous marker is able to predict accurately clearance of metabolised drugs, two separate studies were conducted in order to assess the possibility to use dexamethasone as an in vivo probe for predicting docetaxel and/or vinorelbine clearance. Indeed, dexamethasone, vinorelbine and docetaxel are all mainly metabolised by cytochrome P450 3A4/5 and all are substrate of the ABCB1 transporter. Methods: Patients were receiving docetaxel (21 pts) or vinorelbine (20 pts) as part of established protocols. Dexamethasone (20 mg, iv) was given the day before chemotherapy. Plasma and urinary concentrations of both unchanged dexamethasone and its main metabolite, 6-beta-hydroxydexamethasone, were determined by HPLC Method: Genotyping of both CYP3A5 and ABCB1 have been also performed. The correlation between docetaxel or vinorelbine clearance and dexamethasone pharmacokinetic parameters was assessed using a population pharmacokinetic approach and NONMEM program. Results: A significant correlation between dexamethasone pharmacokinetic parameters and both docetaxel and vinorelbine clearance was found. Covariate models were obtained by considering dexamethasone plasma clearance (DPC) together with other covariates. For docetaxel, best covariate model was: CL (L/hr) = 356. fu. (1 - 0.17 . HPMT) . (1 + 0.126 . DPC) with fu for the unbound plasma fraction of docetaxel estimated from serum alpha 1 acid glycoprotein level, HPMT =1 or =0 if presence of hepatic metastasis or not, and DPC in L/hr. Interindividual variability in CL decreased from 38% (no covariate) to 15% by considering these three covariates. Mean total docetaxel AUC was not significantly larger in patients who experienced severe neutropenia, then the difference was significant (p=0.02) for AUCu (unbound docetaxel plasma exposure) between patients with or without severe toxicity. For vinorelbine, the best covariate model was: CL (L/hr) = 39.8. (DPC/13.2)0.524. (ALP/137)−0.198 with ALP for alkaline phosphatase level (IU). Interindividual variability in CL decreased from 29.7% (no covariate) to 14.7% by considering DPC and ALP. Conclusions: Dexamethasone may be used as a probe for individual dosing of docetaxel and vinorelbine. No significant financial relationships to disclose.

Biofilms Research—Implications to Biosafety and Public Health

Biofilms have been implicated in a variety of noso-comial infections associated with medical devices, hospital equipment, and other hard surfaces. In addition, household and workplace surfaces such as sinks, countertops, toilets, and cutting boards can act as reservoirs. This study's objective was to identify and evaluate literature reporting resistance to antimicrobial agents in biofilm populations. These review findings suggest that the research evaluating resistance in biofilms could be grouped into the following three mechanisms: (1) physicochemical barriers; (2) biological factors; and (3) phenotypic changes. Current research has identified potential mechanisms of antimicrobial resistance, but there is no clear evidence supporting any one mechanism. Moreover, no reported studies examine the potential impact of biofilms on biosafety practices and the public health risk of infectious diseases from biofilms in healthcare facilities and the workplace environment. Future research directions in biofilms are likely to focus on: (a) imaging of biofilms in situ, (b) in vitro and in vivo models of biofilms, (c) genetic, metabolic, and immunologic probes for real-time analysis, (d) antimicrobial resistance in multispecies biofilms, and (e) identification and characterization of phenotypic modifications. In our assessment, these studies will provide the basis to develop guidelines for biofilm-related biosafety and public health risk assessment.

Dexamethasone as a probe for vinorelbine clearance

To assess the value of using dexamethasone as an in vivo probe for predicting vinorelbine clearance (CL). A population approach (implemented with NONMEM) was used to analyse blood vinorelbine pharmacokinetic data from 20 patients who received a 20-min intravenous infusion of vinorelbine (from 20 to 30 mg m(-2)). Selected patient clinical data as well as known functional single CYP3A5 and ABCB1 genotype were also tested as covariates. The best covariate model (with +/- 95% confidence intervals) was based on dexamethasone plasma clearance (DPC) and alkaline phosphatase (ALP): vinorelbine blood CL (l h(-1)) = 39.8(+/- 4.0) x (DPC/13.2)(0.524(+/-0.322)) x (ALP/137)(-0.198(+/-0.158)). Interindividual variability in vinorelbine CL decreased from 29.7% (model without covariate) to 14.7% when including DPC and ALP. Vinorelbine CL was not correlated with body surface area (BSA) or associated with CYP3A5 and ABCB1 genotype. These results indicate that individualization of vinorelbine dose would be improved by using dexamethasone clearance rather than BSA. Dexamethasone merits further evaluation as a probe of CYP3A metabolism.

Association between CYP1A2 activity and riluzole clearance in patients with amyotrophic lateral sclerosis

Riluzole is used in a fixed dosing schedule of 50 mg twice daily to treat patients with amyotropic lateral sclerosis (ALS), one form of motor neurone disease. The large variability in the pharmacokinetics of riluzole may be a factor contributing to its limited therapeutic benefit. Riluzole is assumed to be mainly metabolized by the cytochrome P450 enzyme 1A2 (CYP1A2). The aim of the study was to investigate the relationship between CYP1A2 activity and riluzole clearance with a view to optimize drug treatment. A group of 30 ALS patients participated in the study. In each patient the CYP1A2 activity was determined using caffeine as a metabolic probe. Riluzole clearance was estimated from serum drug concentration measurements followed by Bayesian fitting. Riluzole clearance and the serum paraxanthine : caffeine (P/C) ratio showed a positive correlation (r = 0.693; P = 0.0002). Linear regression analysis identified the P/C ratio (beta: 1.16) and height (beta: 0.027) as independent predictors of riluzole clearance (adjusted r2 = 0.369). The P/C ratio, used as measure of CYP1A2 activity, significantly correlated with the riluzole clearance, although only 37% of the observed variability could be explained.

Contribution ???of ???Cytochrome??? P450??2D6??to??3,4-Methylenedioxymethamphetamine Disposition in Humans

3,4-Methylenedioxymethamphetamine (MDMA) is a synthetic amphetamine derivative typically used for recreational purposes. The participation of cytochrome P450 (CYP) 2D6 in the oxidative metabolism of MDMA may suggest an increased risk of acute toxicity in CYP2D6 poor metabolisers. This study was aimed at assessing the contribution of CYP2D6 to MDMA disposition in vivo using paroxetine as a metabolic probe inhibitor. Paroxetine, a CYP2D6 inhibitor, was repeatedly administered before MDMA administration. This was a randomised, double-blind, crossover, placebo-controlled trial conducted in seven healthy male volunteers who were CYP2D6 extensive metabolisers. Treatment conditions (paroxetine/MDMA and placebo/MDMA) were randomly assigned. Each volunteer participated in two 3-day sessions. On days 1, 2 and 3 subjects received a single oral dose of paroxetine or placebo 20 mg. On the third day, a single oral dose of MDMA 100 mg was administered in both paroxetine and placebo conditions. Plasma concentration-time profiles and urinary recoveries of MDMA and its metabolites were measured, as well as plasma concentrations of paroxetine, (3S,4R)-4-(4-fluorophenyl)-3-(3,4-methylenedioxyphenoxymethyl)-piperidine, and (3S,4R)-4-(4-fluorophenyl)-3-(3-methoxy-4-hydroxyphenoxymethyl)-piperidine (HM-paroxetine). Paroxetine given before MDMA resulted in significant increases of MDMA area under the plasma concentration-time curve from 0 to 27 hours (AUC(27)) [23%], AUC from zero to infinity (AUC(infinity)) [27%] and maximum plasma concentration (C(max)) [17%], without significant differences in MDMA time to reach C(max) (t(max)). MDMA elimination-related pharmacokinetic parameters showed a significant reduction of MDMA elimination rate constant (K(e)) [-14%] and plasmatic clearance (CL(P)) [-29%]. In the case of 3,4-dihydroxymethamphetamine (HHMA), a 21% decrease in C(max) with no significant differences in AUC(27), AUC(infinity), K(e) and elimination half-life) were found. 4-Hydroxy-3-methoxymethamphetamine (HMMA) showed a decrease in plasma concentrations with a reduction in AUC(27) (-28%), AUC(infinity) (-20%) and C(max) (-46%). In the case of 3,4-methylenedioxyamphetamine (MDA) an increase in C(max) (17%) and AUC(27) (16%) was found. Following paroxetine pretreatment, the urinary recovery (0-45 hours) of MDMA increased by 11%; HHMA and HMMA urinary recoveries were 27% and 16% lower, respectively compared with placebo. The ratio of C(max) values of paroxetine and its metabolite on days 1 and 3 showed a 3-fold reduction, with no differences in t(max). The contribution of CYP2D6 to MDMA metabolism in humans is not >30%, therefore other CYP isoenzymes may contribute to O-demethylenation of MDMA. Accordingly, the relevance of genetic polymorphism in CYP2D6 activity on MDMA effects and MDMA-induced acute toxicity should be examined as well as the interactions of other CYP2D6 substrates with MDMA, once the enzyme is inhibited. The pharmacokinetics of HM-paroxetine in humans after the administration of repeated doses is reported for the first time in this study.

Metabolism of 3′-deoxy-3′-[F-18]fluorothymidine in proliferating A549 cells: Validations for positron emission tomography

3′-Deoxy-3′-[F-18]fluorothymidine (FLT) is under clinical evaluation as a metabolic probe for imaging cell proliferation in vivo using positron emission tomography (PET). As part of our validation effort, we followed the short-term metabolism of FLT in exponentially growing tumor cells to demonstrate the enzyme activities within the DNA salvage pathway that influence retention of radioactivity. In A549 cells, thymidine kinase-1 ( TK1) activity produced FLTMP, which dominated the labeled nucleotide pool. Subsequent nucleotide phosphorylations by thymidylate kinase ( TMPK) and nucleotide diphosphate kinase ( NDPK) led to FLTTP. After 1h, the cellular metabolic pool contained ∼30% FLTTP. A putative deoxynucleotidase ( dNT), which degrades FLTMP to FLT, provided the primary mechanism for tracer efflux from cells. In contrast, FLTTP was resistant to degradation and highly retained. The uptake and retention characteristics of FLT were also compared to those of thymidine, FMAU (2′- arabino-fluoro-TdR) and FIAU (2′- arabino-fluoro-5-iodo-2′-dexoyuridine). Despite the fact that FLT lacks the 3′-hydroxy necessary for its incorporation into DNA it out performed both FMAU and FIAU in terms of uptake and retention.

Decrease in oral bioavailability of ciclosporin by intravenous pulse of methylprednisolone succinate in rats.

We examined the effects of high-dose methylprednisolone on the bioavailability of orally administered ciclosporin in rats. To emulate the clinical protocol of methylprednisolone pulse therapy, methylprednisolone sodium succinate (MPS), a prodrug of methylprednisolone, was intravenously administered as repeated doses (66.3 mg kg(-1)) for 3 days. The area under the blood ciclosporin concentration versus time curve after oral administration was significantly reduced by 60% by pulse treatment with MPS. Based on our previous finding that the total body clearance of ciclosporin was reduced by about 20% by the same methylprednisolone pulse protocol, the extent of reduction in the oral bioavailability of ciclosporin was estimated to be approximately 50%, indicating a drug interaction between high-dose methylprednisolone and orally administered ciclosporin, which affected the absorption process. In rats treated with MPS, an in-situ efflux experiment using rhodamine-123 demonstrated that the reverse transport function of P-glycoprotein (P-gp) in the small intestine was significantly enhanced, although there was no significant increase in the intestinal microsomal activity of triazolam alpha- and 4-hydroxylation, metabolic probes for CYP3A. In addition, a significant decrease was observed in the amount of secreted bile acids serving as an enhancer of gastrointestinal absorption of ciclosporin in MPS treatment. To directly estimate the absorptive capacity, an in-situ absorption test was conducted using a closed-loop of small intestine in control and MPS-treated rats. Intestinal absorption of ciclosporin was significantly decreased, not only in the absence of bile flow but also by treatment with MPS, which well reflected the change in the in-vivo pharmacokinetic behaviour of ciclosporin after methylprednisolone pulsing. These results demonstrate that bioavailability of ciclosporin is markedly reduced by MPS pulse treatment, and the mechanism of this interaction was confirmed to involve enhancement of small-intestinal P-gp function and decrease in bile secretion.

Short synthesis of labeled and unlabeled 6 Z,9 Z,12 Z,15-hexadecatetraenoic acid as metabolic probes for biosynthetic studies on diatoms

We describe a short synthesis of the unusual polyunsaturated 6 Z,9 Z,12 Z,15-hexadecatetraenoic acid found in marine and fresh water diatoms. Using a one pot reductive bis-Wittig-olefination, the homoconjugated tetraene backbone of the fatty acid can be generated from easy available precursors. Reductive olefination allows the non-statistical dissymmetrisation of a symmetrical bis-Wittig salt as key synthon. This short sequence was also applied to the generation of the corresponding 9,10-[ 2 H 2 ] labeled fatty acid. If administered to cell fragments of Thalassiosira rotula 9,10-[ 2 H 2 ]-6 Z,9 Z,12 Z,15-hexadecatetraenoic acid is transformed oxidatively to the aldehyde 1,2-[ 2 H 2 ]-2 E,4 E/ Z,7-octatrienal which is involved in the chemical defense of this alga. Using the synthetic standard it could be shown that the C16:4 ω1 fatty acid is released upon wounding of T. rotula cells. The synthesis with the labeled bis-Wittig salt is of general use and can also be applied for the fast generation of other internally labeled functionalized and non-functionalized polyunsaturated fatty acids. To our knowledge this represents the first synthesis of 6 Z,9 Z,12 Z,15-hexadecatetraenoic acid.

New stable isotope–mass spectrometric techniques for measuring fluxes through intact metabolic pathways in mammalian systems: introduction of moving pictures into functional genomics and biochemical phenotyping

The thesis of this review is that fully assembled metabolic pathways in living systems, rather than genes or proteins, are the true units of function in biology and biochemistry. A corollary is that measurement of metabolic fluxes (biochemical kinetics) is thereby required to understand biochemical control and gene function. Recent methodologic advances for improving observability of metabolic pathway fluxes in vivo are reviewed. Stable isotope–mass spectrometric techniques discussed here include mass isotopomer distribution analysis (combinatorial analysis), for measurement of polymerization biosynthesis; 2H 2O administration, for measuring synthesis of DNA (i.e., cell proliferation), RNA, proteins, lipids, glycolipids and other classes of molecules; non-invasive probes of intracellular metabolism, by sampling secreted metabolites in accessible body fluids, after isotopic labeling of the intracellular pathway; and measurement of multiple molecular fluxes concurrently, particularly through use of 2H 2O. Examples are given of pathway fluxes measured by each of these techniques, noting the often-surprising results. It is concluded that the introduction of “moving pictures” as tools for biochemical phenotyping could radically alter many signature areas of contemporary biology, including functional genomics, drug discovery and development, and disease research.

Vascular-Targeted Nanoparticles for Molecular Imaging and Therapy

This chapter describes the vascular-targeted nanoparticles for molecular imaging and therapy. Molecular imaging involves the noninvasive real-time observation of in vivo biologic events at the molecular level. In nearly all cases, molecular imaging will require the delivery of a probe to the tissue site of interest. The design of probes for molecular imaging target two basic classes of biologic events, including alteration in metabolic processes and changes in receptor expression. In the case of metabolic probes, small molecules are used that can perfuse most tissues and pathologic regions in the body. The integrins are one of the best characterized members of the adhesion molecule family that is upregulated in angiogenic endothelial cells found in tumors and certain inflammatory injuries. The integrins are transmembrane molecules that favor the anchorage of endothelial cells to a wide variety of extracellular matrix proteins with an exposed arginine, glycine, aspartate amino acid sequence. The in vivo imaging of angiogenic tumors using anti-avb3-targeted polymerized vesicles comprised of the murine antibody LM609 attached to PVs labeled with the MR contrast agent gadolinium in the V2 carcinoma model in rabbits is also elaborated.

Diversity of Actinoplanes and related genera isolated from an Italian soil.

Actinoplanes and related genera are good producers of bioactive secondary metabolites. However, many strains within these genera present similar morphological characteristics, and this prevents an effective discrimination of replicate strains during industrial isolation and screening programs. Using PCR-RFLP analysis of the 23S rDNA gene and of the 16S-23S intergenic spacer, we have analyzed 182 strains of Actinoplanes and related genera obtained through a selective isolation method from a single Italian soil. Combining the 23S and IGS data, 99 unique profiles were observed, and morphologically undistinguishable strains were discriminated. Further analyses on a restricted number of strains through 16S sequencing and hybridization to a probe for secondary metabolism established a good correlation between strain diversity seen by PCR-RFLP and that seen by the other methods. Overall, the data indicate the presence of a high diversity of Actinoplanes and related genera isolated from a single Italian soil.

Stereochemistry of a bifunctional dihydroceramide delta 4-desaturase/hydroxylase from Candida albicans; a key enzyme of sphingolipid metabolism.

The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.

Biosynthesis of sex pheromones in moths: stereochemistry of fatty alcohol oxidation in Manduca sexta

Six chiral deuterium labelled metabolic probes, ( R) and ( S)-enantiomers of [1,10,10- 2H 3]-hexadecan-1-ol, (11 E)-[1,10,10- 2H 3]-hexadec-11-en-1-ol and (11 Z)-[1,10,10- 2H 3]-hexadec-11-en-1-ol, were synthesized to examine the stereospecificity of the fatty alcohol oxidase from the female pheromone gland of the tobacco hawk moth ( Manduca sexta, Sphingidae). Both in vitro and in vivo oxidations were found to proceed by selective removal of the C1–H R hydrogen or deuterium atom ( Re-specificity) to yield the corresponding aldehydes. ( R) and ( S)-enantiomers of deuterium labelled salicyl alcohol and 2-thienyl-methanol, compounds entirely chemically diverse from the natural pheromone precursors, were also oxidised Re-specifically to salicylaldehyde and 2-thiophenecarb-aldehyde, respectively.

Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe.

The dismutation of chlorite into chloride and O(2) represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene cld was isolated and sequenced from the perchlorate-reducing bacterium Dechloromonas agitata strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence identical to that of the previously purified D. agitata chlorite dismutase enzyme. The predicted translation product of the D. agitata cld gene is a protein of 277 amino acids (aa), including a leader peptide of 26 aa. Primer extension analysis identified a single transcription start site directly downstream of an AT-rich region that could represent the -10 promoter region of the D. agitata cld gene. Northern blot analysis indicated that the cld gene was transcriptionally up-regulated when D. agitata cells were grown in perchlorate-reducing versus aerobic conditions. Slot blot hybridizations with a D. agitata cld probe demonstrated the conservation of the cld gene among perchlorate-reducing bacteria. This study represents the first description of a functional gene associated with microbial perchlorate reduction.

Plasma levels of TNF-α and IL-6 are inversely related to cytochrome P450-dependent drug metabolism in patients with congestive heart failure

Background: Cytochrome P450 (CYP) enzymes are important mediators of drug metabolism, and activity of these enzymes is a major determinant of the duration and intensity of drug effect. Circulating plasma concentrations of pro-inflammatory cytokines (e.g., tumor necrosis factor [TNF]-α and interleukin [IL]-6) are elevated in patients with heart failure and these cytokines have been shown to down-regulate CYP enzyme activity. The purpose of this study was to evaluate the relationship between plasma cytokine concentrations and CYP enzyme activities in patients with heart failure. Methods and Results: Sixteen patients with congestive heart failure (New York Heart Association classes II-IV) received a metabolic probe cocktail consisting of caffeine, mephenytoin, dextromethorphan, and chlorzoxazone to assess the activities of the CYP enzymes 1A2, 2C19, 2D6, and 2E1. Blood and urine samples were collected for drug and metabolite determinations by high-performance liquid chromatography (HPLC); cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). We found a striking inverse relationship between both TNF-α and IL-6 plasma concentrations and the activity of CYP2C19; metabolism of caffeine (CYP1A2) also had a negative association with IL-6 plasma concentrations. Conclusions: Cytokine-mediated decreases in drug metabolism may contribute to observed variability in drug response and augment the risk of adverse drug effects in CHF patients.

In vivo effect of 5- and 8-methoxypsoralens and cimetidine on R,S-warfarin metabolism in rat.

Several forms of cytochrome P-450 (CYP) metabolize R,S-warfarin in a regio- and enantioselective manner, therefore R,S-warfarin can be recognized as a metabolic probe for a number of CYP isoforms. We have applied a warfarin model in vivo in order to estimate the inhibitory properties of 5- and 8-methoxypsoralens on the activity of rat CYP isoforms. The area under the serum concentration versus time curve (AUC) values from time zero to 5 h for R- and S-warfarin and their metabolites were calculated. R,S-Warfarin kinetics measurements were made three times on each rat: a week before the 7-days inhibitor treatment, 3 h after the last dose of inhibitor and 3-7 days after the inhibitor was withdrawn. The inhibitory effect of cimetidine on CYP 2C11 and CYP 2C6 activities was confirmed in this approach and can be recognized as a positive control in validation of the in vivo experiment. Both 5- and 8-methoxypsoralen inhibited CYP 2C6 activity as the respective AUC for metabolite/warfarin enantiomer ratio decreased significantly. The activity of CYP 2C6 in 5- and 8-methoxypsoralen-treated rats increased over control values after the inhibitor was withdrawn. It was also observed that cimetidine additionally inhibits the absorption of R,S-warfarin and a decrease in the sum of AUC for R- and S-enantiomers became evident in spite of inhibition of the activity of both CYPs. 5-Methoxypsoralen modified the serum R-warfarin/S-warfarin ratio and a selective increase in AUC(S-warfarin) was observed, the most pronounced being after the inhibitor was withdrawn. This effect is not likely to be mediated by P-glycoprotein (P-gp) because quinidine--, a P-gp inhibitor at a dose of 15 mg kg(-1) body wt.--did not influence the AUC for either enantiomer.

Compartmentation of metabolism probed by [2-[formula omitted]]alanine: improved [formula omitted] NMR sensitivity using a CryoProbe detects evidence of a glial metabolon

The labelling of metabolites with the NMR active nucleus 13C allows not only metabolite enrichments to be monitored, but also the relative fluxes through competing pathways to be delineated. [2-13C, 15N]alanine was used as a metabolic probe to investigate compartmentation in superfused cerebral slices. Perchloric acid extracts of the tissue were investigated using 13C NMR spectroscopy. The spectra were obtained using a CryoProbe™ optimised for 13C detection (dual CryoProbe [13C, 1H]) in which the receiver and transmitter coils are cooled to ∼20K to reduce contributions to noise in the signal obtained. Compared with conventional inverse geometry probe, the signal-to-noise ratio (S/N) was increased by ∼17-fold using this device. A large proportion of alanine was initially metabolised over the first 20min by glial cells, as indicated by the relative importance of the glial, only enzyme pyruvate carboxylase to the labelling pattern of glutamate, with the ratio of pyruvate carboxylase to pyruvate dehydrogenase derived glutamate being 0.25, and exported [2-13C, 15N]aspartate.Using the increased sensitivity of the CryoProbe™, [2-13C, 15N]aspartate was also detected in the extracts of cerebral tissue. This metabolite could only have been derived via the pyruvate carboxylase pathway, and given the large export of the metabolite into the superfusion buffer suggests the occurrence of a ‘metabolon’ arrangement of enzymes within glial cells.