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The 2-phenylindole system has proved to be a versatile structure for the design of potent antiestrogens, especially when functional groups have been introduced into the alkyl side chain in position 1. In analogy to steroidal structures such as ICI 164,384 a number of 2-phenylindoles with carbamoylalkyl and aminoalkyl side chains were synthesized. They bind to the calf uterine estrogen receptor with relative binding affinities between 2.1 and 21 (estradiol = 100). The antiestrogenic effect of these compounds was demonstrated by the inhibition of transcriptional activity which was measured in a new luciferase assay with the EREwtc luc as reporter plasmid. The derivative with a methyl-n-propyldodecanamide side chain (4h) antagonized the effect of estradiol (10(-9) M) completely at concentrations of 10(-7) M and higher. As a sensitive model for quantification of estrogenic and antiestrogenic effects in vitro we used HeLa-cells cotransfected both with the reporter plasmid and estrogen receptor expression vectors HEG0 and HE0. In cells transfected with these vectors transcriptional activity was strongly dependent on side chain structure. With mutated receptors we were able to show that this activity was mainly due to TAF-1 whereas TAF-2 remained silent. When we studied the effect of some of the new compounds in vivo using the mouse uterine weight assay, we observed a correlation between transcriptional activity in transfected HeLa cells and estrogenic effects in mice. Two of the 1-carbamoylalkyl-2-phenylindoles (4f, 4h) proved to be "pure" antiestrogens both in vitro and in vivo. In estrogen-sensitive MCF-7 breast cancer cells, they strongly inhibit cellular growth. Some of the IC50-values were close to 10(-8) M.

The male rabbit liver microsomal cytochrome P450 metabolism of 1-nitropyrene and 3-nitrofluoranthene was investigated. In this study, we used inhibitory antibodies specific for rabbit P450 2C3 and determined that, in untreated male rabbit liver microsomes, the antibody inhibited approximately 75% of the cytochrome P450-mediated C-oxidation of both 1-nitropyrene and 3-nitrofluoranthene. These results verify our previous prediction that mainly one cytochrome P450 is responsible for microsomal metabolism of 1-nitropyrene in the untreated rabbit liver.

Respiratory depression is the main obstacle for the safe administration of morphine for acute pain after injury. Due to this complication, new delivery methods are needed to insure that safe and effective doses of opioid analgesics are administered during emergencies. A depot formulation containing a naloxone pro-drug was designed to release the antidote when morphine causes dangerous hypoxic conditions in the blood. The aim of this work was to test the naloxone release in vivo in response to a severe overdose of morphine in the Sprague-Dawley rat model. Non-invasive two-chamber plethysmography was used to monitor and record respiration and to test the capability of the naloxone pro-drug to respond to and rescue morphine-induced respiratory depression in the animal. We show that the pro-drug formulation can both prevent and reverse severe morphine induced respiratory depression. The animal model demonstrates that co-administration of the naloxone pro-drug reliably antagonizes profound respiratory depressive effects of morphine.

Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or “degrons”) contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.

The cytochromes P450 (P450) involved in the epoxidation of the rat carcinogen acrylonitrile (ACN) to the mutagen 2-cyanoethylene oxide (CEO) have been investigated in hepatic microsomes from F-344 rats and humans. Induction of P450 2E1 by acetone treatment increased the Vmax for rat microsomal CEO formation 5-fold, while ACN treatment had little effect. Treatment with beta-naphthoflavone, dexamethasone, and phenobarbital had little effect upon Vmax but increased the KM 3- to 5-fold. The P450 ligand 1-phenylimidazole and substrate ethanol were potent inhibitors of ACN epoxidation after all treatments. 2-(Diethylamino)ethyl 2,2-diphenylvalerate (SKF 525A; 0.1 mM) was not an effective inhibitor with microsomes from untreated or acetone-treated rats, but inhibited approximately 50% following dexamethasone or phenobarbital treatment. Antibodies to P450 2E1 inhibited > 85% of the ACN epoxidation activity in microsomes from untreated or beta-naphthoflavone- or acetone-treated rats, but only produced 40% and 60% inhibition following dexamethasone or phenobarbital treatments, respectively. These results indicate that P450 2E1 is the major catalyst of ACN epoxidation in untreated rats and that other forms of P450 can also epoxidize ACN. Diethyldithiocarbamate (0.1 mM) was a potent irreversible inhibitor of ACN epoxidation after all of the induction treatments, indicating that it is not specific for P450 2E1. Chlorzoxazone (2 mM) produced 75-90% inhibition after all of the induction treatments, indicating that it interacts with several rodent P450 isoforms in addition to 2E1. Human hepatic microsomes (n = 6) epoxidized ACN with Vmax'S ranging from 129 to 315 pmol of CEO formed/(min.mg of protein) and KM's from 12 to 18 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

When reconstituted with cytochrome b5 and NADPH cytochrome P450 oxidoreductase, cytochrome P450 2E1 metabolized lauric, stearic, oleic, linoleic, linolenic, and arachidonic acid to multiple metabolites. Two major metabolites, accounting for 78% of the total metabolism, were produced with arachidonic acid. The Vmax for total metabolite formation from arachidonic acid was 5 nmol/min/nmol P450 with an apparent Km of 62 microM. Gas chromatography-mass spectrometry analysis identified the two major metabolites as monohydroxylated eicosatetraenoic acids (HETEs). The major HETE was 19-hydroxyeicosatetraenoic acid (19-HETE) and comprised 46% of the total metabolite produced. The second metabolite was the omega-2 hydroxylated metabolite (18-HETE) and comprised 32% of the total product formed. Chiral analysis demonstrated that 19-HETE was 70% 19(S)-HETE and 30% 19(R)-HETE. In contrast, 18-HETE was essentially 100% R isomer. Approximately 18% of the total metabolite produced from arachidonic acid coeluted with epoxyeicosatrienoic acid (EET) standards. The EET metabolites were 56.4% 14,15-EET and 43.6% as a mixture of 11,12-EET and 8,9-EET. 5,6-EET was not detected. Anti-P450 2E1 IgG inhibited arachidonic acid metabolism by renal and hepatic microsomes prepared from acetone-treated rabbits. With renal cortex microsomes, the formation of 18-HETE and 19-HETE was inhibited 67 and 25%, respectively, by the antibody. Liver microsomal formation of 18-HETE was inhibited by 87% and 19-HETE by 70%. Thus, under conditions where cytochrome P450 2E1 is induced, the enzyme could contribute significantly to the formation of the omega-1 and omega-2 hydroxylated metabolites of arachidonic acid.

Our prior work on tricyclic acridones combined with a desire to minimize the tricyclic system led to an interest in antimalarial quinolones and a reexamination of endochin, an experimental antimalarial from the 1940's. In the present article, we show that endochin is unstable in the presence of murine, rat, and human microsomes which may explain its relatively poor antimalarial activity in mammalian systems. We also profile the structure-activity relationships of ≈ 30 endochin-like quinolone (ELQ) analogs and highlight features that are associated with enhanced metabolic stability, potent antiplasmodial activity against multidrug resistant strains of Plasmodium falciparum, and equal activity against an atovaquone-resistant clinical isolate. Our work also features an ELQ construct containing a polyethylene glycol carbonate pro-moiety that is highly efficacious by oral administration in a murine malaria model. These findings provide compelling evidence that development of ELQ therapeutics is feasible.

Lipoic acid is a natural anti-oxidant available as an oral supplement from a number of different manufacturers. Lipoic acid administered subcutaneously is an effective therapy for murine experimental autoimmune encephalomyelitis, a model of multiple sclerosis. The aim of this study was to compare serum lipoic acid levels with oral dosing in patients with multiple sclerosis with serum levels in mice receiving subcutaneous doses of lipoic acid. We performed serum pharmacokinetic studies in patients with multiple sclerosis after a single oral dose of 1200 mg lipoic acid. Patients received one of the three different racemic formulations randomly: tablet (Formulation A) and capsules (Formulations B and C). Mice pharmacokinetic studies were performed with three different subcutaneous doses (20, 50 and 100 mg/kg racemic lipoic acid). The pharmacokinetic parameters included Maximum Serum Concentrations (Cmax in μg/ml) and area under the curve 0—infinity (AUC 0—infinity in μg*min/ml). We found mean Cmax and AUC 0—infinity in patients with multiple sclerosis as follows: group A ( N = 7) 3.8 ± 2.6 and 443.1 ± 283.9; group B ( N = 8) 9.9 ± 4.5 and 745.2 ± 308.7 and group C ( N = 8) 10.3 ± 3.8 and 848.8 ± 360.5, respectively. Mean Cmax and AUC 0—infinity in the mice were: 100 mg/kg lipoic acid: 30.9 ± 2.9 and 998 ± 245; 50 mg/kg lipoic acid: 7.6 ± 1.4 and 223 ± 20; 20 mg/kg lipoic acid: 2.7 ± 0.7 and 119 ± 33. We conclude that patients taking 1200 mg of lipoic acid from two of the three oral formulations achieved serum Cmax and AUC levels comparable to that observed in mice receiving 50 mg/kg subcutaneous dose of lipoic acid, which is a highly therapeutic dose in experimental autoimmune encephalomyelitis. A dose of 1200 mg oral lipoic acid can achieve therapeutic serum levels in patients with multiple sclerosis.

A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified from renal cortex. A small amount of the unresolved omega/omega-1 hydroxylated eicosatetraenoic acid products were also produced. The enzyme had a specific content of 8.4 nmol of P450/mg of protein and exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after silver staining. Sequencing revealed a single NH2-terminal amino acid sequence with the first 20 residues identical to rabbit cytochrome P450 2C2. We suggest this enzyme be termed P450 2CAA (for arachidonic acid) until the complete sequence and substrate selectivity are established. Purified P450 2CAA was in the low spin state as evidenced by an absorption maximum at 415 nm; the reduced-carbonyl complex exhibited a maximum at 451 nm. The specific activity for metabolism of 7 microM arachidonic acid was 1.1 nmol of product formed/min/nmol of P450. About 75% of the metabolites were two of the four possible epoxyeicosatrienoic acids identified as the 11,12- and 14,15-epoxyeicosatrienoic acids by coelution with synthetic and commercial standards on reversed and normal-phase high pressure liquid chromatographic separations. The ratio of the 11,12- to 14,15-epoxyeicosatrienoic acids was 1.5:1. The purified enzyme exhibited no significant activity toward 7-ethoxyresorufin or progesterone, but demethylated aminopyrine and benzphetamine. Other fatty acids were also substrates for the enzyme. Oleic, linoleic, and lauric acids, all at about 10 microM, were metabolized at rates of 0.32, 0.72, and 0.73 nmol/min/nmol of P450, respectively. Monoclonal antibody that cross-reacts with P450 2C2 inhibited 63% of the microsomal epoxidation activity from renal cortex microsomes from phenobarbital-treated rabbits. The production of the epoxide metabolites of arachidonic acid suggests that P450 2CAA may have a significant role in arachidonic acid-mediated intra- and intercellular signalling pathways.

Introduction Chemical sphincterotomy, the use of pharmacological agents to reduce anal sphincter resting pressure, has become more and more popular in the treatment of chronic anal fissures (CAFs). It offers the possibility to avoid a lateral internal sphincterotomy and its associated risk of incontinence. In our hospital, patient with a chronic anal fissure are consecutively treated with isosorbide dinitrate 1% ointment, applied 6 times a day for 8 weeks, followed by diltiazem 2% ointment, applied 2 times a day for 8 weeks and Botulin Toxin A injections (Dysport®; Ipsen, Hoofddorp, the Netherlands) in the internal anal sphincter. In a previous study (1), we describe high healing rates with this regime. Objective The objective of this study is to evaluate the effect of the combination of fissurectomy and Botulin Toxin A in the treatment of CAFs. Methods Twenty-one patients (10 male patients, median age 48 years) with persistent symptoms of chronic anal fissures after following the above mentioned treatment, were enrolled in this study. Fissurectomy was combined with Botulinum Toxin A (80 U of Dysport®) under regional anaesthesia in day care. Results After 12 weeks 19/21 CAFs (90%) had healed. Median follow-up was 16 (9-30) months. No recurrences were seen. Conclusion Fissurectomy in combination with Botulinum Toxin A injection in the internal anal sphincter is an effective treatment for medically resistant CAFs.

At different European institutes software has been developed for evaluation of the gravitational potential of the Earth using high degree spherical harmonic expansions. In this report the results of a comparison of a number of these software packages are presented. We compared the results for the second order derivatives (gravity gradients). It appeared that one of the most critical points in these computations is the definition of the coordinates, which should be as accurate as possible. Machine dependency and algorithm setup were of less importance, the former being only reflected in CPU timing results.

An investigation was carried out to demonstrate the possibility of recovering spherical harmonic potential coefficients up to high degree and order from gradiometric measurements. Observations were taken from one month simulated GRM gradient data computed at the Center of Space Research at the University of Texas. For least squares adjustment a linear observation model was chosen from which the radial orbit uncertainties were eliminated. A solution up to degree and order 180 proved to be very promising, whereas a 360 solution showed degraded results due to the too short mission and the non-homogeneous data coverage.

The volatile anesthetic isoflurane poses a number of experimental challenges in the laboratory. Due to its rapid evaporation, the open conditions of most in vitro electrophysiological recording systems make the determination of actual isoflurane concentrations a challenge. Since the absolute anesthetic concentration in solution is directly related to efficacy, concentration measurements are important to allow comparisons between laboratory and clinical studies. In this study we quantify the sources of isoflurane loss during experimentation and describe a method for the measurement of isoflurane concentrations using gas chromatography and mass spectrometry simultaneous to in vitro electrophysiological measurements. Serial samples of perfused bath solution allowed correlation of isoflurane concentrations with ongoing biological effects. Saturated physiological solutions contained 13.4 +/- 0.2 mM isoflurane and were diluted to desired "nominal" concentrations for experiments. The perfusion system established stable isoflurane concentrations within the bath by 2 minutes. However, bath isoflurane concentrations varied substantially and unpredictably between experiments. The magnitudes of such discrepancies in isoflurane concentrations spanned clinically important levels. Our studies suggest that, despite countermeasures, solution handling significantly impacted the isoflurane content in the tissue bath. The magnitude of these discrepancies appears to necessitate systematic direct measurement of bath isoflurane concentrations during most in vitro conditions.

Single nucleotide polymorphisms (SNPs) in the humanEPHX2gene have recently been implicated in susceptibility to cardiovascular disease, including stroke.EPHX2encodes for soluble epoxide hydrolase (sEH), an important enzyme in the metabolic breakdown of arachidonic acid-derived eicosanoids referred to as epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs are protective against ischemic cell death in culture. Therefore, we tested the hypothesis that polymorphisms in the humanEPHX2gene alter sEH enzyme activity and affect neuronal survival after ischemic injuryin vitro. HumanEPHX2mutants were recreated by site-directed mutagenesis and fused downstream of TAT protein transduction domain. Western blot analysis and immunocytochemistry staining revealed high-transduction efficiency of human TAT-sEH variants in rat primary cultured cortical neurons, associated with increased metabolism of 14,15-EET to corresponding 14,15-dihydroxyeicosatrienoic acid. A human variant of sEH with Arg103Cys amino acid substitution, previously demonstrated to increase sEH enzymatic activity, was associated with increased cell death induced in cortical neurons by oxygen-glucose deprivation (OGD) and reoxygenation. In contrast, the Arg287Gln mutation was associated with reduced sEH activity and protection from OGD-induced neuronal cell death. We conclude that sequence variations in the humanEPHX2gene alter susceptibility to ischemic injury and neuronal survival in a manner linked to changes in the hydrolase activity of the enzyme. The findings suggest that humanEPHX2mutations may in part explain the genetic variability in sensitivity to ischemic brain injury and stroke outcome.

Axonal regeneration is important for functional recovery following nerve damage. Centella asiatica Urban herb, also known as Hydrocotyle asiatica L., has been used in Ayurvedic medicine for centuries as a nerve tonic. Here, we show that Centella asiatica ethanolic extract (100 μg mL−1) elicits a marked increase in neurite outgrowth in human SH-SY5Y cells in the presence of nerve growth factor (NGF). However, a water extract of Centella was ineffective at 100 μg mL−1. Sub-fractions of Centella ethanolic extract, obtained through silica-gel chromatography, were tested (100 μg mL−1) for neurite elongation in the presence of NGF. Greatest activity was found with a non-polar fraction (GKF4). Relatively polar fractions (GKF10 to GKF13) also showed activity, albeit less than GKF4. Thus, Centella contains more than one active component. Asiatic acid (AA), a triterpenoid compound found in Centella ethanolic extract and GKF4, showed marked activity at 1 μm (0.5 μg mL−1). AA was not present in GKF10 to GKF13, further indicating that other active components must be present. Neurite elongation by AA was completely blocked by the extracellular-signal-regulated kinase (ERK) pathway inhibitor PD 098059 (10 μm). Male Sprague-Dawley rats given Centella ethanolic extract in their drinking water (300–330 mg kg−1 daily) demonstrated more rapid functional recovery and increased axonal regeneration (larger calibre axons and greater numbers of myelinated axons) compared with controls, indicating that the axons grew at a faster rate. Taken together, our findings indicate that components in Centella ethanolic extract may be useful for accelerating repair of damaged neurons.

Background and aims: A herbal prescription, Yan-gan-wan (YGW), has been known to offer hepatoprotective effects in Asian countries for years. This study investigated its mechanisms of action. Methods: The effects of YGW on CCl4 induced liver damage were tested in mice and cultured hepatocytes. Microarray analysis screened genes affected by YGW. YGWs effects on the expression of cytochrome P450 (CYP) 2E1 and other isozymes were determined. YGWs effects on TNFα expression and NF-κB activation in Kupffer cells (KC), and TNFα promoter activity in RAW264.7 cells, were also assessed. Results: Administration of YGW reduced the plasma ALT, centrilobular necrosis, neutrophilic infiltration, and TNFα mRNA in the livers of mice acutely given CCl4. The in vivo herb treatment reduced ALT release and necrosis of isolated hepatocytes directly exposed to CCl4. Microarray analysis demonstrated marked reductions in CYP4A10 and 4A14 by YGW but no changes in other CYP isozymes as confirmed by immunoblot analysis. The herb treatment suppressed LPS-stimulated TNFα release in vivo and by cultured KC. Direct addition of the aqueous herb extract suppressed NF-κB activation by KC and TNFα promoter activity in RAW cells under LPS stimulation. This activity to suppress TNFα expression was largely separated into gel filtration fractions with the molecular size of 102–107 Da. YGW also attenuated liver fibrosis induced by chronic treatment of CCl4 or porcine serum. Conclusions: The protective effects of YGW on CCl4 hepatotoxicity are due in part to inhibition of KC NF-κB activation and TNFα expression by small water soluble molecules, and may also be related to suppressed hepatic expression of CYP4A10 and 4A14 that are considered as alternative prooxidant cytochromes.

The occurrence of malignant tumors of the upper gastrointestinal tract and liver is, based largely on epidemiological evidence, causally related to the consumption of ethanol. It is widely recognized that oxidants play a key role in alcohol-induced liver injury; however, it is unclear how oxidants may be involved in DNA damage. We asked whether nicotinamide adenine dinucleotide phosphate oxidase, cytochrome P450 CYP2E1, or both are responsible for the production of DNA damage. The rodent Tsukamoto-French model of intragastric ethanol infusion was used. Wistar rats, Cyp2e1-, p47(phox)-null, and hCyp2e1 transgenic mice were used. The abundance of oxidative DNA adducts, mutagenic apurinic/apyrimidinic sites, and expression of base excision DNA repair genes was determined. In rats and wild-type mice, ethanol treatment for 4 weeks led to an increase in oxidative DNA damage and induction of expression of the base excision DNA repair genes that are known to remove oxidative DNA lesions. No increase in either of the endpoints was observed in ethanol-treated Cyp2e1-null mice, whereas the magnitude of response in p47(phox)-null mice and transgenic hCyp2e1 was identical to that in wild types. The increase in expression of DNA repair genes was completely abolished by treatment with the P450 inhibitor 1-aminobenzotriazole. In conclusion, the data support the hypothesis that oxidative stress to DNA is induced in liver by ethanol. Furthermore, although it was shown that nicotinamide adenine dinucleotide phosphate oxidase-derived oxidants are critical for the development of ethanol-induced liver injury, CYP2E1 is required for the induction of oxidative stress to DNA, and thus may play a key role in ethanol-associated hepatocarcinogenesis.

It is the aim of the GOCE mission to determine a model of the Earth’s gravity field with high accuracy and resolution. For this purpose, gravity gradients will be measured in combination with high–low satellite-to-satellite tracking. The gravity gradients are derived from pair-wise differenced accelerations as determined by the six three-axes accelerometers that form the GOCE gradiometer. Since the measured accelerations suffer from errors of a random and systematic nature, the gravity gradients may suffer from random and systematic errors as well. Systematic errors are, for example, a scale factor and a bias. The common accelerations of the paired accelerometers also are contaminated with such errors. The common accelerations are used in the drag-free control of the satellite and are important for the separation of the gravitational and non-gravitational forces in the gravity field determination. The checking of the gravity gradients and the common accelerations against independent data (i.e. external to the GOCE satellite) in order to free the observations as well as is possible from systematic errors is called external calibration. The possibilities and limitations of using terrestrial gravity data and global gravity models for external calibration of the gravity gradients are reviewed. It turns out that the determination of a gravity gradient scale factor and bias using just the accurate knowledge of the central term and the flattening (J2) of the Earth’s gravity field is not good enough. When global gravity field models are used for the calibration, higher degrees and orders should be taken into account as well. With today’s existing global models it seems to be possible to remove the greater part of the systematic errors of the GOCE gradients. A gravity gradient bias can accurately be recovered using terrestrial gravity data in a regional approach with least squares collocation. However, since regional data are used it may not be possible to determine calibration parameters valid for the whole (global) gravity gradient data set. Nevertheless, regional terrestrial gravity data could be used to validate the measured and calibrated gravity gradients. In addition, a possible use of GOCE high–low satellite-to-satellite tracking data to calibrate the common accelerations is explored; it is shown that this approach fails. If more accurate gravity field information becomes available then such a calibration may become feasible.

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.

CYP2E1 is an ethanol- and drug-metabolizing enzyme that can also activate procarcinogens and hepatotoxicants and generate reactive oxygen species; it has been implicated in the pathogenesis of liver diseases and cancer. Cigarette smoke increases CYP2E1 activity in rodents and in humans and we have shown that nicotine (0.1-1.0 mg/kg s.c. × 7 days) increases CYP2E1 protein and activity in the rat liver. In the current study, we have shown that the induction peaks at 4 h postnicotine (1 mg/kg s.c. × 7 days) treatment and recovers within 24 h. No induction was observed after a single injection, and 18 days of treatment did not increase the levels beyond that found at 7 days. We found that CYP2E1 is induced by very low doses of chronic (× 7 days) nicotine with an ED50 value of 0.01 mg/kg s.c.; 0.01 mg/kg in a rat model results in peak cotinine levels (nicotine metabolite) similar to those found in people exposed to environmental tobacco smoke (passive smokers; 2-7 ng/ml). Previously, we have shown no change in CYP2E1 mRNA, and our current mechanistic study indicates that nicotine does not regulate CYP2E1 expression by protein stabilization. We postulated that a nicotine metabolite could be causing the induction but found that cotinine (1 mg/kg × 7 days) did not increase CYP2E1. Our findings indicate that nicotine increases CYP2E1 at very low doses and may enhance CYP2E1-related toxicity in smokers, passive smokers, and people treated with nicotine (e.g., smokers, patients with Alzheimer's disease, ulcerative colitis or Parkinson's disease).

GOCE will be the first satellite gravity mission measuring gravity gradients in space using a dedicated instrument called a gradiometer. High resolution gravity field recovery will be possible from these gradients. Such a recovery requires a proper description of the gravity gradient errors, where the a priori error model is for example based on end-to-end instrument simulations. One way to test the error model against real data, i.e. to see if the a priori model really describes the actual error, is to compare along track interpolated gradients with the measured gradients. The difference between the interpolated and measured gravity gradients is caused by, among others, the interpolation error and the measurement errors. The idea is that if the interpolation error is small enough, then the differences should be predicted reasonably well by the error model. This paper discusses a simulation study where the gravity gradient errors are generated with an end-to-end instrument simulator. The measurement error will be compared with the interpolation error and we will assess the latter as a function of the sampling interval.

Administration of bacterial lipopolysaccharide (LPS) to laboratory animals and cultured macrophages induces tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine. Pretreatment with Ginkgo biloba extract (EGb 761) inhibited the in vivo production of TNF-alpha (measured by ELISA) after challenge with LPS. To begin to understand the mechanism of this inhibition, we evaluated the in vitro effects of EGb 761 and its flavonoid component, quercetin, on LPS-treated RAW 264.7 macrophages. Pretreatment with EGb 761 or quercetin concentration-dependently inhibited TNF-alpha release, as measured by the L929 fibroblast assay. Northern blotting demonstrated that quercetin inhibited LPS-induced TNF-alpha mRNA, but did not alter its half-life. Activation of mitogen-activated protein kinases (MAPKs) and the redox-sensitive transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1), are key events in the signal transduction pathways mediating TNF-alpha induction. Phosphorylation of extracellular signal-related kinases 1 and 2 (ERK 1/2), p38 MAPK, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), members of the MAPK family, was analyzed by western blotting. Our results suggest that quercetin is unique in its ability to inhibit TNF-alpha transcription by inhibiting the phosphorylation and activation of JNK/SAPK and, therefore, suppressing AP-1-DNA binding [assessed by electrophoretic mobility shift analysis (EMSA)]. Results from western analysis, EMSA, and transient transfections suggest that EGb 761 diminishes LPS-induced NF-kappaB but has no effect on LPS-induced TNF-alpha transcription. Both EGb 761 and quercetin inhibited ERK1/2 phosphorylation and p38 MAPK activity, which are important in the post-transcriptional regulation of TNF-alpha mRNA.

Administration of bacterial lipopolysaccharide (LPS) to laboratory animals and cultured macrophages is known to induce the production of nitric oxide (NO) from inducible nitric oxide synthase (iNOS). Here we show that pre-treatment with Ginkgo biloba extract (EGb 761) suppresses the in vivo production of NO (measured by the Griess reaction) after challenge with LPS. In order to begin to understand the mechanism of this inhibition, we evaluated in vitro effects of EGb 761 and its flavonoid component, quercetin, on LPS-treated RAW 264.7 macrophages. Pre-treatment with EGb 761 or quercetin dose-dependently inhibited NO release. Both substances scavenged NO generated from the decomposition of sodium nitroprusside. Western analysis showed that EGb 761 and quercetin inhibited LPS-induced levels of iNOS protein. Northern blotting demonstrated that EGb 761 and quercetin decreased LPS-induced iNOS mRNA levels without altering the half-life. Activation of mitogen activated protein kinases (MAPKs) and the redox-sensitive transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1) are key events in the signal transduction pathways mediating iNOS induction. In our studies, both EGb 761 and quercetin inhibited p38 MAPK activity, which is necessary for iNOS expression in LPS-stimulated RAW 264.7 macrophages. However, differences in the response of NF-kappaB, AP-1, and Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) and its downstream substrates to EGb 761 and quercetin suggest that quercetin is not the sole component responsible for the in vivo inhibition of LPS-induced iNOS activation by EGb 761.

The role of Kupffer cells in CCl4-induced fibrosis was investigated in vivo. Male Wistar rats were treated with phenobarbital and CCl4for 9 wk, and a group of rats were injected with the Kupffer cell toxicant gadolinium chloride (GdCl3) or were fed glycine, which inactivates Kupffer cells. After CCl4alone, the fibrosis score was 3.0 ± 0.1 and collagen protein and mRNA expression were elevated, but GdCl3or glycine blunted these parameters. Glycine did not alter cytochrome P-450 2E1, making it unlikely that glycine affects CCl4metabolism. Treatment with GdCl3or glycine prevented CCl4-induced increases in transforming growth factor (TGF)-β1 protein levels and expression. CCl4treatment increased α-smooth muscle actin staining (score 3.0 ± 0.2), whereas treatment with GdCl3and glycine during CCl4exposure blocked this effect (1.2 ± 0.5); there was no staining with glycine treatment. These results support previous in vitro data and demonstrate that treatment of rats with the selective Kupffer cell toxicant GdCl3prevents stellate cell activation and the development of fibrosis.

The expected high resolution and precision of a global gravity field model derived from satellite gradiometric observations is unprecedented compared to nowadays satellite-only models. However, a dedicated gravity field mission will most certainly fly in a non-polar (sun-synchronous) orbit, such that small polar regions will not be covered with observations. The resulting inhomogeneous global data coverage, together with the downward continuation problem, leads to unstable global solutions and regularization is necessary. Regularization gives rise to a bias in the solution, mainly in the polar areas although in other regions as well.

The Gravity Field and Steady-State Ocean Circulation Explorer (GOCE) mission has been selected by the Earth Sciences Advisory Committee (ESAC) of the European Space Agency (ESA) as the first of two core missions of ESA’s Earth Explorer Programme to be launched in 2004. The main objective of the GOCE mission is to provide a high-accuracy high-resolution global model of the Earth’s static gravity field and the geoid from a combination of spaceborne gravity gradiometry (SGG) and high-low satellite-to-satellite tracking (hl-SST). Of the order of a hundred thousand gravity field parameters have to be estimated from tens of millions measured gravity gradients, which makes a brute force approach in terms of a least-squares solution of the observation equations an enormous numerical task. This huge number of observations and gravity field parameters does not allow to set-up the design matrix and the normal matrix explicitly. In turn this implies that existing software libraries for the platform of choice cannot be used directly. Therefore, we discuss a (parallellized) data processing approach for SGG data on the CRAY T3E supercomputer at Delft University of Technology, which only requires the application of the design matrix or its transposed to some vectors. Moreover, we discuss the performance of the algorithm in terms of degree of parallelism and scalability properties based on simulations.

GOCE (Gravity Field and Steady-State Ocean Circulation Explorer) is one of the four selected ESA Earth Explorer Missions. The main objective of GOCE is the determination of the Earth’s gravity field with high spatial resolution and with high homogeneous accuracy. For this purpose, two observation concepts will be realised. Satellite-to-Satellite Tracking (SST) in high-low mode will be used for the orbit determination and for the retrieval of the long-wavelength part of the gravity field. Satellite Gravity Gradiometry (SGG) will be employed for the derivation of the medium/short-wavelength parts of the gravity field..

We investigate the time-wise approach to the data anlaysis for the GOCE mission. The number of observations collected during the mission, the number of potential coefficients to be estimated, and the complexity of the mathematical model for the time-wise approach require a special strategy, which has to reduce the CPU-time and storage requirements considerably. Our approach is based on (1) the iterative solution of the normal equations using a Richardson-iteration scheme and (2) the approximation of the design matrix in order to assemble the right-hand side in each iteration step efficiently. We demonstrate the performance of the approach for white noise and coloured noise observations along a simulated GOCE orbit up to degree and order 180. We provide error estimates anal show that the solution is unbiased. We also prove that the method does not converge to the solution of the normal equations. However, the approximation error can be neglected in our simulations.

A tetracycline (Tc)-controlled gene expression system that quantitatively controls gene expression in eukaryotic cells () was used to express cytochrome P-450 2E1 (CYP2E1) in HeLa cells in culture. The rabbit CYP2E1 cDNA was subcloned into the Tc-controlled expression vector (pUHD10-3) and transfected into a HeLa cell line constitutively expressing the Tc-controlled transactivator, a positive regulator of expression in the absence of Tc. The expression of CYP2E1 was tightly regulated. There was a time-dependent induction of CYP2E1 after removal of Tc. In the absence of Tc, the enzyme was induced more than 100-fold and expressed about 18 pmol of CYP2E1/mg microsomal protein. At maximal levels of expression the enzyme catalyzed the formation of 158 pmol 6-hydroxychlorzoxazone/min/mg total cellular protein. In addition, the level of the enzyme could be modulated by the concentration of Tc in the media. In the absence of Tc, exposure of cells to N-nitrosodimethylamine caused a significant dose-dependent decrease in cell viability. In contrast, menadione, a redox cycling toxicant, was less toxic to the cells after induction of CYP2E1 when compared with noninduced cells. Pulse-chase studies conducted 72 h after removal of Tc indicated a rapid turnover of CYP2E1 with a half-life of 3.9 h. Addition of the ligand, 4-methylpyrazole, and the suicide substrate, 1-aminobenzotrizole, decreased the degradation of CYP2E1. This cell line offers a useful system to examine the role of CYP2E1 in the cytotoxicity of xenobiotics and to investigate post-translational regulation of the enzyme.

The beneficial effects of moderate red wine consumption have been attributed, in part, to the presence of antioxidant components. Oxidant stress is an activating stimulus for the NF (nuclear factor)-KB/Rel family of transcription factors, which have binding sites in the promoter regions of many genes involved in inflammatory and immune responses. The effect of lipopolysaccharide (LPS)-stimulated activation of NF-KB and the subsequent production of tumor necrosis factor alpha (TNF-alpha) and NO was determined in the macrophage cell line RAW 264.7. Unexpectedly, the wine polyphenolics quercetin and resveratrol and the antioxidant N-acetylcysteine (NAC) did not inhibit LPS-induced activation of the NF-KB complex p50/65, as determined by mobility shift. Quercetin inhibited LPS-induced p50/50. Northern blot analysis indicated that quercetin (0.1 and 0.2 mM) inhibited LPS-dependent production of inducible nitric oxide synthase (iNOS) mRNA and decreased NO release, as measured by the Griess reaction. This flavonoid had no effect on LPS-induced TNF-alpha mRNA, but decreased LPS-stimulated TNF-alpha release, as measured by ELISA. Resveratrol (0.05 and 0.1 mM) posttranscriptionally decreased LPS-induced nitrite release. It increased basal levels of TNF-alpha mRNA and protein and enhanced LPS-induced TNF-alpha mRNA and cytokine release. Our results do not support the view that wine antioxidants inhibit LPS-induced NF-KB activation but instead that they have a more selective action on genes activated by LPS.

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.

Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its omega-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 +/- 0.3 nmol/min/nmol P450; 0.25 +/- 0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparent Km of 74.8 microM with a VMAX of 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp --> 12Gly) over the first 17 residues with the human CYP4F2 cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 omega-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 +/- 5%; n = 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1 but was unaffected by lauric acid, palmitic acid, and PGF2alpha. Finally, a strong correlation (r = 0.86; P < 0.002; n = 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 omega-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid.

Cyclosporine suppresses rat hepatic cytochrome P450 in a time-dependent manner. Background Cyclosporine is a potent immunosuppressant known to selectively suppress specific cytochrome P450 (P450) isoforms following chronic therapy in the rat. Cyclosporine undergoes significant hepatic metabolism in the rat, primarily due to P450 3A isoforms. Hence, alterations in hepatic metabolism of cyclosporine may lead to changes in drug pharmacokinetics or pharmacodynamics. The purpose of this study was to examine the temporal effect of chronic cyclosporine dosing on P450 protein expression and metabolic activity in a rat model of chronic cyclosporine nephropathy. Methods Adult male rats were administered cyclosporine 15mg/kg/day or vehicle 1 ml/kg/day by subcutaneous injection for up to 28days. To examine whether or not metabolic activity recovered following drug removal, additional rats were administered cyclosporine for 28days followed by vehicle for up to an additional 15days. Hepatic P450 protein expression and microsomal metabolic activity were measured by Western blot analysis and in vitro steroid hydroxylation, respectively. Results Cyclosporine trough levels progressively increased over the 28days period and were still measurable for up to 15days after discontinuation. Immunoblot analysis indicated that chronic cyclosporine treatment suppressed P450 3A2 expression and in vitro steroid hydroxylation in a time-dependent manner. Fifteen days following discontinuation of cyclosporine dosing, hepatic metabolic activity and microsomal P450 3A2 levels returned to near pre-dosing levels. Conclusions We conclude that the time-dependent P450 suppression by cyclosporine may at least partially explain the variability in cyclosporine pharmacokinetics. These studies support the hypothesis that hepatic isoforms other than P450 3A2 may be responsible for cyclosporine metabolism during chronic treatment in the rat.

Cytochrome P450-catalyzed desaturation reactions have been reported infrequently in the literature. Previously, we documented the formation of the terminal olefinic metabolite of valproic acid by various members of the CYP2B and CYP4B sub-families. However, despite the extensive use of fatty acid substrates in drug metabolism studies, other examples of terminal desaturation at non-activated carbon centers are lacking. The goals of the present studies were to determine whether the archetypal P450 substrate, lauric acid (dodecanoic acid; DDA), also undergoes desaturation reactions, identify specific rabbit P450 isoforms which catalyze this reaction and examine its mechanism. A highly sensitive, capillary GC/MS assay was developed to separate and quantitate the trimethylsilyl derivatives of 11-ene-DDA, cis- and trans-10-ene-DDA and cis- and trans-9-ene-DDA. Among all of these potential olefinic metabolites, only 11-ene-DDA was formed at a significant rate by rabbit liver microsomes. The formation of 11-ene-DDA was NADPH-dependent, and was induced markedly by acetone pre-treatment, but not by phenobarbital, rifampin or Arochlor 1254. Studies with seven purified, reconstituted rabbit P450 isoforms showed that the most rapid rates of desaturation were obtained with CYP2E1, CYP4A5/7 and CYP4B1. Non-competitive, intermolecular isotope effect experiments, conducted with [12,12,12-2H3]DDA and [11,11-2H2]DDA, demonstrated further that CYP4B1-mediated terminal desaturation of DDA is initiated by removal of a hydrogen atom from the ω-1 rather than the ω position.

5017 Background: Diets high in cruciferous vegetables are strongly associated with lower prostate cancer risk. Sulforaphane is a constituent of these foods postulated to harbor the anti-neoplastic activity based on pre-clinical evidence in multiple tumor models. Our own work demonstrates that sulforaphane inhibits HDAC function and suppresses AR signaling in prostate cancer cells (Gibbs, et al PNAS 2009). However, the anti-tumor efficacy and safety of sulforaphane in men with prostate cancer was unknown. Methods: In this single arm study, we treated patients with biochemical (PSA)-recurrence of prostate cancer with 200 µmol of sulforaphane extracts for up to 20 weeks. The primary endpoint was PSA response rate (>50% decline in PSA). Other efficacy endpoints included: maximal PSA decline and percent change in PSA from baseline to end of study. We also analyzed PSA doubling time changes using a mixed effects model. Genotyping for GSTM1 that contributes to sulforaphane metabolism, sulforaphane pharmacokinetics (PK), and pharmacodynamic (PD) measurements of HDAC inhibition in mononuclear cells (MCs) were also performed. Results: Twenty patients were enrolled, and 16/20 (80%) completed the pre-planned 20 weeks of treatment. One patient experienced a PSA decline >50%. Thirty-five percent of patients had lesser PSA declines (3% to 20%), and 15% of patients had a final PSA lower than baseline. There was a significant reduction in PSA doubling time (6 months pre-study vs. 9.4 months on-study, p=.013). Of note, testosterone levels remained non-castrate in all subjects. PK analysis demonstrated that GSTM1 null genotype correlated with longer sulforaphane T1/2 (half-life) (2.6 hours for GSTM1 null vs. 2.1 hours for GSTM1 intact, p=0.04). Sulforaphane treatment also increased histone acetylation in PD assays in MCs. Finally, no grade three adverse events were seen, and only one patient discontinued study treatment for toxicity (grade one GI discomfort). Conclusions: Treatment with 200 µmol per day of sulforaphane is feasible, safe, and inhibits HDAC function. This combined with the preliminary observation of PSA modulation, which may indicate biologic activity, provides the basis for dose escalation studies of sulforaphane in men with prostate cancer. Clinical trial information: NCT01228084.

The regioselective epoxidation of oleic, linoleic, alpha-linolenic, and gamma-linolenic acid by cytochromes P4502CAA and P4502C2 was characterized. Epoxide metabolites for all fatty acids were resolved by normal phase HPLC and identified by gas chromatography and mass spectrometry. Both isoforms epoxidized the single double bond in oleic acid and both double bonds in linoleic acid. The ratio of the two epoxides produced with linoleic acid (1.6:1 for the 12,13- and 9,10-epoxides) was similar for both enzymes. When alpha-linolenic acid was the substrate, all three epoxides were produced in about equal ratios with both enzymes. In contrast for the omega-6 fatty acid, gamma-linolenic acid, both enzymes produced only the 9,10- and 12,13-epoxides. Furthermore, the ratio of the metabolites produced by each enzyme was significantly different. The ratios of 12,13-epoxide to 9,10-epoxide for gamma-linolenic were 11.0 +/- 0.19 and 5.8 +/- 1.2 for P4502CAA and P4502C2, respectively. These results suggest that there may be subtle differences in the structure of 2C2 and 2CAA and also indicate that P450 may be important in the generation of potentially active epoxide metabolites of unsaturated fatty acids other than arachidonic acid.

Cyclosporine is an immunosuppressant that undergoes extensive hepatic biotransformation to hydroxylated and demethylated metabolites. At present, the CYP3A gene family is thought to be the primary enzyme system responsible for cyclosporine metabolism. The effect of chronic cyclosporine therapy on the suppression of drug metabolism was studied in male and female rats maintained on a low-salt diet. After 28 days of subcutaneous cyclosporine dosing 15 mg/kg, cyclosporine-treated rats had significant renal dysfunction as compared with gender-matched control rats. Creatinine clearance in male cyclosporine-treated rats was reduced by 47% (P.01) as compared with male controls. Female rats demonstrated a 38% (P.01) decrease in creatinine clearance as a result of chronic cyclosporine therapy. Despite similar nephrotoxicity, female rats had whole blood cyclosporine levels 48% (P.01) less than male rats. Immunoblot analysis of hepatic microsomal proteins indicated that chronic cyclosporine treatment decreased the protein levels of P450 3A2 in male rats. This loss was paralleled by reduced production of 6 beta-hydroxytestosterone, the primary product of P450 3A activity, by hepatic microsomes from cyclosporine-treated male rats by 76% (P.001). In addition, cyclosporine treatment of male rats also reduced the formation of 2 alpha-hydroxytestosterone and 16 alpha-hydroxytestosterone by 81% (P.01) and 84% (P.001), respectively. At the end of the study period, steroid 5 alpha-reductase activity in control male rats was only 4% (P.001) of female counter-parts; however, cyclosporine treatment increased steroid 5 alpha-reductase activity in male rats to 79% (P.001) of female values. These alterations in testosterone metabolism are consistent with the suppression of the predominately male-associated P450 3A2, P450 2C11 and P450 2C13 isoforms. Levels of 6 alpha-hydroxytestosterone and 7 alpha-hydroxytestosterone were not statistically different between rat groups. Taken together, the steady-state blood levels and metabolism studies suggest that, after chronic cyclosporine treatment, isoforms other than those from the CYP3A family or unidentified members of the CYP3A family are likely responsible for cyclosporine metabolism.

Rabbit P450 2E1 was stably expressed in Chinese hamster ovary cells after cotransfection with pRC/CMV-2E1 and pFR400 which expresses murine dihydrofolate reductase with a single arginine to leucine substitution at position 22. This mutation permits amplification of expression with increasing methotrexate concentrations in CHO-K1 cells that are not dihydrofolate reductase deficient. After amplification with 1 μM methotrexate, a representative clone expressed about 15 pmol of P450 2E1/mg microsomal protein. Cells from a single 35-mm plate catalyzed the formation of 1.02 nmol 6-hydroxychlorzoxazone/10 6 cells/h or about 127 pmol/mg total cell protein/min. The enzyme was rapidly labeled when pulsed with [ 35 S]-methionine. Initial pulse-chase experiments indicate that the expressed protein has a half-life of 4.8 h.

For the analysis of the yield of a satellite gradiometry mission, different methods can be employed, see e.g. (Koop, 1993) or (Rummel et al., 1993). Most studies carried out so far rely on covariance propagation. In the present study full simulations are used to evaluate future missions like step. This gives the opportunity to investigate several effects in more detail than before.

Objective: Assess the safety of Polyphenon E in subjects with multiple sclerosis (MS) and determine whether Polyphenon E would be futile as a neuroprotective agent. Background Polyphenon E (Mitsui Norin; Shizuoka, Japan) is an extract from the leaves of green tea. Its main active component is epigallocatechin gallate (EGCG), an antioxidant that accumulates within the mitochondria of neurons and increases their resistance to apoptosis under conditions of oxidative stress. Mitochondrial dysfunction leads to axonal injury in mice with experimental autoimmune encephalomyelitis (EAE). EGCG effectively treats EAE in female SJL/J mice. Design/Methods: Ten subjects with relapsing remitting or secondary progressive MS were recruited for this open-label, six-month pilot study of 400 mg of Polyphenon E twice daily. Adverse events (AE) were assessed monthly. N-acetyl aspartate (NAA) levels were measured using 3T magnetic resonance spectroscopy and differences were assessed between baseline and six-months. Plasma levels of epigallocatechin gallate (EGCG) were measured at one month, 3 and 8 hours after the morning dose. Results: No serious AE occurred. All subjects completed the study assessments. One discontinued therapy after mild elevation of liver enzymes. Treatment with EGCG resulted in an increase of 13% [95%,CI(1%-23%) p Conclusions: There were no safety concerns for treating MS patients with Polyphenon E. Our futility endpoint was rejected, as NAA levels increased. This and the correlated changes in NAA and EGCG levels support a potential neuroprotective effect that should be investigated in a larger randomized, placebo-controlled trial measuring NAA levels over a longer follow-up. Supported by: NCCAM K23AT004433; LSUHSC-New Orleans CTRC. Disclosure: Dr. Ramos has nothing to disclose. Dr. Garrison has nothing to disclose. Dr. Koop has nothing to disclose. Dr. Rooney has nothing to disclose. Dr. Bourdette has received personal compensation for activities with Biogen Idec, Serono, and Teva Neurosciences as a speaker. Dr. Lovera has received personal compensation for activities with EMD Serono, Teva Neuroscience, and Biogen Idec as a consultant.

In the present study, the regio- and stereoselective epoxidation of arachidonic acid by cytochromes P450 2C8 and 2C9, two members of the CYP2C gene subfamily expressed in human liver, was determined. Purified P450 isozymes, reconstituted with NADPH:P450 oxidoreductase, cytochrome b5 and lipid, or microsomes isolated from human liver, were incubated with [1-14C]-arachidonic acid. For regioselective analysis, the epoxide metabolites formed, 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (EETs), were resolved by reverse-phase high-performance liquid chromatography. P450 2C8 produces only the 14,15- and 11,12-EETs in a 1.25:1.00 ratio. The two epoxides represent 68% of the total metabolites. P450 2C9 produces 14,15-, 11,12- and 8,9-EETs in a 2.3:1.0:0.5 ratio. The three epoxides represent 69% of the total metabolites. Neither P450 isoform catalyzes the formation of 5,6-EET. For chiral analysis, the two major epoxide metabolites, 14,15- and 11,12-EETs, were derivatized to methyl and pentafluorbenzyl esters, respectively. Enantiomers of 14,15- and 11,12-EET esters were subsequently resolved on Chiralcel OB and OD columns (J.T. Baker, Phillipsburg, PA), respectively. Both P450 2C8 and 2C9 are stereoselective at the 14,15- position, preferentially producing 14(R), 15(S)-EET with 86.2% and 62.5% selectivity, respectively. Both enzymes are also stereoselective at the 11,12-position but have the opposite selectivity. P450 2C8 is 81.1% selective for 11(R), 12(S)-EET; P450 2C9 is 69.4% selective for the 11(S), 12(R)-EET. Immunoinhibition studies performed with anti-2C9 immunoglobulin G (which also reacts with P450 2C8) and hepatic microsomes indicate that these two P450s are important arachidonic acid epoxygenases in human liver.

In the present study, we determined the stereoselective epoxidation of arachidonic acid by cytochrome P-450 (P-450) 2CAA and P-450 2C2, two arachidonic acid epoxygenases found in rabbit renal cortex, by chiral normal-phase high-performance liquid chromatography (HPLC)-analysis. Purified P-450 2CAA reconstituted with P-450 oxidoreductase, lipid and cytochrome b5 or microsomes isolated from COS-1 cells expressing P-450 2C2 were incubated in the presence of [1-14C]arachidonic acid. The epoxide metabolites 14,15- and 11,12-epoxyeicosatrienoic acids (EETs) were purified by reverse-phase HPLC and derivatized to methyl (14,15-EET) and pentafluorobenzyl (11,12-EET) esters. Enantiomers of 14,15-EET-methyl ester and 11,12-EET-pentafluorobenzyl ester were resolved on Chiralcel OB and OD columns, respectively, with a mobile phase of 0.15% 2-propanol in n-hexane. P-450 2CAA and P-450 2C2 produce 11,12- and 14,15-EETs in distinct ratios but are equally stereoselective at the 11,12-position. P-450 2CAA produced 11(S), 12(R)-EET with 63% stereoselectivity, and P-450 2C2 produced the same enantiomer with 61% stereoselectivity. Both enzymes are also stereoselective at the 14,15- position, preferentially producing the 14(R), 15(S)-EET. P-450 2CAA produces this enantiomer with 72% selectivity, and P-450 2C2 produces it with 62% selectivity. The results of this study indicate that P-450 2CAA and P-450 2C2 are not only regioselective but also exhibit a high degree of stereoselectivity.

In the present study we determined the regioselectivity of arachidonic acid epoxidation by several members of the cytochrome P-450 2B subfamily, including rat P-450 2B1, 2B1-WM (an allelic variant of 2B1 expressed in Wistar-Munich rats), 2B2, and rabbit 2B4 and 2B5. The major products formed with all isoforms were the four regioisomeric epoxides, but each isoform produced a distinct distribution of the four epoxides. P-450 2B1 produced predominantly 14,15-epoxyeicosatrienoic acid (EET), while P-450 2B1-WM produced the 11,12-EET as the major product. P-450 2B2, 2B4, and 2B5 catalyzed the formation of all four epoxides in nearly equal amounts. The single Gly-478-->Ala substitution in the variant P-450 2B1-WM was sufficient to cause a dramatic change in the ratio of epoxides when compared with P-450 2B1. The Gly-478-->Ala mutation also changed the regioselective epoxidation of gamma-linolenic acid at the three double bonds. Four site-directed mutants of P-450 2B1 were also evaluated. The mutations included two single mutants where Ile-114 was changed to either Val or Ala and two double mutants where the Ala-478 mutation was coupled with either Val or Ala at position 114. When Ile-114 was mutated to Val, the degree of epoxidation of arachidonic acid at all four double bonds was nearly equal. However, substitution of Ile-114 with Ala, resulted in a significant reduction in the degree of epoxidation at the 14,15- and 11,12-double bonds, and the 8,9- and 5,6-EETs were the major products. When Ala was introduced at position 478 in conjunction with Val at position 114 the regioselective epoxidation of the mutant enzyme more closely resembled P-450 2B1-WM in that 11,12-EET was the major metabolite. The double mutation with Ala at both positions 114 and 478 produced a unique distribution of epoxide products with 5,6-EET as the major metabolite. The results of these studies indicate that residues 114 and 478 in the P-450 2B subfamily are important for the orientation of fatty acids in the active site.

A rapid assay system for measuring the potential of estrogenic drugs is introduced. Luciferase induction could be measured in estrogen-receptor-positive human MCF-7 breast cancer cells, which had been transfected with a novel luciferase reporter plasmid ERE luc. The minimal requirement was 1 h exposure to the inducing drug and 3.5 h of incubation after removal of the drug. The assay system was used to measure the stability of the drug diaqua-[1,2-bis (2,6-dichloro-4-hydroxyphenyl) ethylenediamine] platinum(II) sulfate, containing an estrogenic ligand and reactive platinum. Luciferase activity was observed only when the drug was in the culture medium and cells for short times, whereas the estrogenic ligand alone remained active. It is assumed that binding of the platinum moiety to macromolecular constituents of the culture or cells renders the drug inaccessible for binding to the estrogen receptor.

Abstract 3504 The imatinib paradigm established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKIs). However, once-daily dasatinib (serum half-life: 3–5 h) is clinically effective despite only transient BCR-ABL inhibition, opening an opportunity for in-depth study of the mechanistic requirements for ABL TKI-induced CML cell death. Apoptosis commitment after potent, transient target inhibition is observed with ABL TKIs in vitro (Blood, 114, 2009, 3459–63), although variations in required TKI concentrations relative to their activity against BCR-ABL kinase suggest involvement of previously unrecognized factors. The “oncogenic shock” concept holds that temporary disruption of BCR-ABL-mediated prosurvival and proapoptotic signaling sets up a kinetic imbalance in favor of apoptosis. We have undertaken a comprehensive mechanistic exploration of this issue, wherein CML cells were transiently exposed to the ABL TKIs imatinib (50 and 500 nM), nilotinib (50 and 500 nM), dasatinib (10 and 100 nM), and ponatinib (AP24534; 10 and 100 nM) and then investigated with respect to pathways critical to drug efficacy and intracellular residence time. Cellular studies utilized multi-parameter intracellular FACS and immunoblot analysis, liquid chromatography-mass spectrometry, and high-throughput real-time qPCR expression analysis. Corresponding biochemical studies to determine ABL kinase/inhibitor dissociation parameters were also performed. All four ABL TKIs tested were capable of triggering apoptosis following transient exposure, although nilotinib and imatinib (which feature much narrower kinase target profiles than dasatinib and ponatinib) did so only at high concentrations. In contrast to potent, transient inhibition of BCR-ABL being necessary and sufficient for commitment of CML cells to apoptosis, we found that apoptosis could be reversed under conditions involving extensive additional TKI washout protocols. Consistent with the best indicator of apoptosis induction in our experiments being incomplete restoration of BCR-ABL signaling activity to pre-treatment levels, in all cases for which apoptosis commitment was irreversible, we identified a small, functionally important pool of intracellular TKI after washout of drug from the media. This property correlated with results of ABL kinase/inhibitor dissociation studies. For example, we found that ponatinib is a tight-binding inhibitor with a remarkably slow off-rate (t1/2 >95 h). Transient exposure to ponatinib followed by thorough washout committed CML cells to apoptosis despite very low intracellular concentrations that did not completely inhibit BCR-ABL. Based on these findings, we explored the possibility that effective TKIs are inhibiting additional targets involved in committing cells to an apoptotic fate, and used high-throughput qPCR assays to identify a preliminary profile of 30 apoptosis-related genes differentially expressed in TKI-treatment conditions that do and do not irrevocably commit CML cells to apoptosis. Taken together, our findings reveal that even slightly attenuated restoration of BCR-ABL signaling correlates with apoptosis commitment and that cryptic cellular retention of ABL TKIs is important in mediating this effect, potentially via sustained low-level inhibition of auxiliary targets. By extension, monitoring intracellular drug levels by LC-MS may be informative, especially for short serum half-life kinase inhibitors such as dasatinib. These studies further establish and refine the guiding principles of commitment of CML cells to apoptosis and improve our ability to design kinase inhibitors for CML and other malignancies. Disclosures: Riddle: Life Technologies Corporation: Employment, Equity Ownership. Apgar:BD Biosciences: Employment. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Druker:Bristol-Myers-Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential COI has been reviewed and managed by the OHSU COI in Research Committee & Integrity Program Oversight Council.

The role of arachidonic acid (AA) and its metabolites in vasopressin (AVP)-induced calcium mobilization in A7r5 aortic smooth muscle cells was explored by intracellular calcium monitoring, [14C]AA labeling, and high-performance liquid chromatography (HPLC) techniques. In fura 2-loaded A7r5 cells, AA potentiated AVP-stimulated increase in intracellular free Ca2+ ([Ca2+]i). The cyclooxygenase inhibitor indomethacin reduced both the AA- and AVP-induced influx of extracellular Ca2+. AVP-induced [Ca2+]i transients were not altered by lipoxygenase inhibitors but were reduced in a dose-dependent fashion by ketoconazole, an inhibitor of cytochrome P-450 monooxygenases. Among several epoxygenase metabolites of AA tested, 5,6-epoxyeicosatrienoic acid potentiated AVP-induced [Ca2+]i transients. Reverse-phase HPLC analysis of lipid extracts from A7r5 cells prelabeled with [14C]AA isolated a radioactive peak that did not coelute with established products of cyclooxygenase-, lipoxygenase-, or cytochrome P-450-catalyzed oxidations of AA. This peak was significantly increased after AVP stimulation and was completely blocked by preincubation with ketoconazole. Thus the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several cytoplasmic signaling pathways involving AA metabolite formation through the cyclooxygenase and epoxygenase pathways.