Search

Background Information on cannabinoids in breast milk and maternal cannabis use is limited. We quantified cannabinoids in plasma and breast milk of breastfeeding mothers and assessed cannabis use patterns. Methods This is a prospective study at a university hospital in a state with legal medical and recreational cannabis. Breast milk and plasma samples along with survey data were collected from volunteers using cannabis in the last 48 h at 2 weeks and 2 months postpartum. Results Twenty subjects were enrolled. Median age (IQR) was 27 (24-34) years. Median (IQR) instances of cannabis use in the last 7 days were visit 1: 17 (6-29) and visit 2: 23 (15-45). Median (IQR) tetrahydrocannabinol (THC) concentrations were: plasma 3.7 ng/ml (0.8-56.8) and breast milk 27.5 ng/ml (0.8-190.5). Median (IQR) cannabidiol (CBD) concentrations were: plasma 0.6 ng/ml (0.5-6.4) and breast milk 1.2 ng/ml (0.5-17.0). Median (IQR) THC M/P: 7.0 (1.8-34.6) and CBD M/P: 2.6. Median breast milk THC concentration increased from visit 1 to visit 2 by 30.2 ng/ml (95% CI 3.05-69.3 ng/ml). Conclusions THC and CBD accumulate in breast milk. Breastfeeding mothers used cannabis frequently and increased use in the early postpartum period. Research on the effects of infant exposure to cannabinoids in breast milk is urgently needed. Impact Cannabis use is increasing in the general population and many nursing mothers use cannabis. THC has been previously detected in breast milk but little is known on how it concentrates relative to plasma. Data on cannabinoids other than THC, reasons for cannabis use, and patterns of use in breastfeeding women are also limited. We detected THC and CBD in breast milk. Both concentrate in breast milk relative to plasma. We showed that breastfeeding mothers increased cannabis use in the weeks after childbirth. Further research is needed to evaluate infant exposure to cannabinoids via breast milk and effects on infant health.

ELQ-300 is a preclinical antimalarial drug candidate that is active against liver, blood, and transmission stages of Plasmodium falciparum. While ELQ-300 is highly effective when administered in a low multi-dose regimen, poor aqueous solubility and high crystallinity have hindered its clinical development. To overcome its challenging physiochemical properties, a number of bioreversible alkoxycarbonate ester prodrugs of ELQ-300 were synthesized. These bioreversible prodrugs are converted to ELQ-300 by host and parasite esterase action in the liver and bloodstream of the host. One such alkoxycarbonate prodrug, ELQ-331, is curative against Plasmodium yoelii with a single low dose of 3 mg/kg in a murine model of patent malaria infection. ELQ-331 is at least as fully protective as ELQ-300 in a murine malaria prophylaxis model when delivered 24 hours before sporozoite inoculation at an oral dose of 1 mg/kg. Here, we show that ELQ-331 is a promising prodrug of ELQ-300 with improved physiochemical and metabolic properties and excellent potential for clinical formulation.

BackgroundThe potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice.MethodsFour trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P.yoelii, assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 hours to 5 ½ months after injection.ResultsA single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4 ½ months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (< 6 hours) and sustained (4-5 months) effective plasma ELQ-300 concentration, maximum ELQ-300 concentrations far below the estimated threshold for toxicity, and a distinctive ELQ-300 concentration vs. time profile. Pharmacokinetic modeling indicates a high-capacity, slow-exchange tissue compartment which serves to accumulate and then slowly redistribute ELQ-300 into blood, and this property facilitates an extremely long period during which ELQ-300 concentration is sustained above a minimum fully-protective threshold (60-80 nM).ConclusionsExtrapolation of these results to humans clearly predicts that ELQ-331 should be capable of meeting and far-exceeding currently published duration-of-effect goals for antimalarial LAI-C. Allometric scaling from mice to humans would predict a several-fold enhancement in the relationship between duration-of-effect and dose, and available drug engineering and formulation technologies would be expected to offer significant improvement over the simple powder in sesame oil used here. Furthermore, the distinctive pharmacokinetic profile of ELQ-300 after treatment with ELQ-331 may facilitate durable protection using a variety of delivery and formulation options, and may enable protection for far longer than 3 months. Particularly in light of the favorable pharmacodynamic profile of ELQ-300, ELQ-331 warrants consideration as a leading prototype for LAI-C.

Objective HLA-B27 associated spondyloarthropathies are associated with an altered intestinal microbiota and bowel inflammation. Therefore, we sought to identify B27-dependent changes in both host and microbial metabolites in the HLA-B27/β2m rat and whether microbiota-derived metabolites could impact disease in this major model of spondyloarthropathy. Methods Cecal contents were collected from 6wk (pre-diseased) and 16wk (diseased) Fischer 344 HLA-B27/β2m transgenic rats and WT controls. Metabolomic profiling was performed by high-throughput gas- and liquid-chromatography-based mass spectrometry. HLA-B27/β2m rats were treated with microbial metabolites propionate or butyrate in drinking water for 10wks and disease activity subsequently assessed. Results Our screen identified 582 metabolites, of which over half were significantly altered by B27 expression at 16wks. Both microbial and host metabolites were altered, with multiple pathways including amino acid, carbohydrate, xenobiotic and medium chain fatty acid metabolism affected. Differences were even observed at 6wks, with upregulation of histidine, tyrosine, spermidine, N-acetylmuramate and glycerate in HLA-B27/β2m rats. Administration of the short chain fatty acid propionate significantly attenuated B27-associated inflammatory disease, albeit was not associated with increased FoxP3+ T cell induction, or altered expression of cytokines IL-10, IL-33 or tight junction protein ZO-1. HLA-B27 expression was also associated with altered host expression of microbial metabolite receptor genes FFAR2, FFAR3 and NIACR1. Conclusion HLA-B27 expression profoundly impacts the intestinal metabolome, with changes evident in rats even at 6wks of age. Critically, we demonstrate a microbial metabolite, propionate attenuates development of B27-associated inflammatory disease. These and other microbiota-derived bioactive mediators may provide novel treatment modalities in B27-associated spondyloathropathies. This article is protected by copyright. All rights reserved.

A relatively new therapy in the treatment of hemorrhoids is transanal hemorrhoidal dearterialization (THD). We report a case of brain abscess caused by Streptococcus milleri following THD. Although a brain abscess after drainage of a perianal abscess has been described in the literature, no report exists of a brain abscess following treatment of hemorrhoids. A healthy 51-year-old man with hemorrhoids underwent THD. Two weeks later he presented with a headache, bradyphrenia, flattened behavior and a left hemiplegia. No perianal complaint and/or perianal abscess was present. A contrast CT scan of the cerebrum showed a right temporoparieto-occipital abscess, with edema and compression of the surrounding tissue and lateral ventricles. MRI showed an abscess with leakage in the right lateral ventricle. Treatment with dexamethasone and intravenous antibiotics was started. Because of progression of symptoms, 3 weeks later ventriculoscopy was performed and the abscess was drained. Culture of the punctuate showed S. milleri. Because of developing hydrocephalus 3 days after ventriculoscopy, first an external ventricle drain and later a ventriculoperitoneal drain was placed. Hereafter the hemiplegia and cognitive disorders improved. This case report describes a severe complication following treatment of hemorrhoids with THD which until now, to our knowledge, has never been described in the literature.

Long term therapeutic drug monitoring and assessment of renal function are required in renal transplant recipients on immunosuppressant therapy such as tacrolimus. Dry blood spots (DBS) have been used successfully in the clinic for many years and offers a convenient, simple and non-invasive method for repeated blood tests. We developed and performed a preliminary validation of a method for the analysis of tacrolimus and creatinine from a single DBS using liquid chromatography-tandem mass spectrometric (LC–MS/MS). Tacrolimus and creatinine were extracted from a 6 mm punch with a mixture of methanol/acetonitrile containing ascomycin and deuterated creatinine as internal standards. A 10 μl aliquot of the extract was analyzed directly after dilution for creatinine with normal phase high performance liquid chromatography and multiple reaction monitoring. The remainder of the extract was processed and analyzed for tacrolimus. The lower limit of quantification for tacrolimus was 1 ng/ml with accuracy of 0.34% bias and precision (CV) of 11.1%. The precision ranged from 1.33% to 7.68% and accuracy from −4.44% to 11.6% bias for the intra- and inter-day analysis. The lower limit of quantification of creatinine was 0.01 mg/dL with precision of 7.94%. Accuracy was based on recovery of additional creatinine spiked into whole blood samples and ranged from −2.45% bias at 5 mg/dL to 3.75% bias at 0.5 mg/dL. Intra- and inter-day precision was from 3.48 to 4.11%. The assay was further validated with DBS prepared from pediatric renal transplant recipients. There was excellent correlation between the levels of tacrolimus and creatinine obtained from the clinical laboratory and the DBS method developed. After additional validation, this assay may have a significant impact on compliance with medication intake as well as potentially lowering the cost associated with intravenous blood draws in clinical laboratories.

New treatments for tuberculosis infection are critical to combat the emergence of multi-drug and extensively-drug resistant Mycobacterium tuberculosis (Mtb). We report the characterization of a diphenylether modified adamantyl 1,2-diamine that we refer to as TBL-140, which has a minimal inhibitory concentration (MIC(99)) of 1.2 μg/mL. TBL-140 is effective against drug-resistant Mtb and non-replicating bacteria. In addition, TBL-140 eliminates expansion of Mtb in cell culture infection assays at its MIC. To define the mechanism of action of this compound, we performed a spontaneous mutant screen and biochemical assays. We determined that TBL-140 treatment impacts the proton motive force (PMF) by perturbing the transmembrane potential (ΔΨ), consistent with a target in the electron transport chain (ETC). As a result, treated bacteria have reduced intracellular ATP levels. We show that TBL-140 exhibits greater metabolic stability than SQ109, a structurally similar compound in clinical trials for treatment of MDR-TB infections. Combined, these results suggest that TBL-140 should be investigated further to assess its potential as an improved therapeutic lead against Mtb.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Obesity is known to suppress CYP3A4/5 activity. • Use of combined oral contraceptives (COC) has been shown to suppress CYP3A4/5 activity in non-obese women. • However, the effect of COC on CYP3A4/5 activity is unknown in obese women who constitute 50% of women of reproductive age. WHAT THIS STUDY ADDS • Although we hypothesized that obesity exposure might amplify the impact on CYP3A4/5 activity in COC users, we found that this was not the case. • This finding is reassuring regarding potential additional drug–drug interactions in obese COC users as CYP3A4/5 is a major player in the metabolism of many marketed drugs. AIM(S) While it is known that CYP3A4/5 activity is decreased with combined oral contraceptive (COC) use and obesity suppresses CYP expression, the combined effects of obesity and COC use on CYP3A4/5 activity are unclear. Therefore, our aim was to examine the effect of COC usage on CYP3A4/5 activity in obese women. METHODS Thirty-four, obese (body mass index, BMI > 30 kg m−2) women of reproductive age (18–35 years old) were placed on a COC pill containing 20 µg ethinylestradiol/100 µg levonorgestrel for 21 days starting at the onset of menses. A midazolam pharmacokinetic study was conducted prior to initiation and after 21 days of COC treatment. Serial blood samples were collected and plasma concentrations of midazolam were measured using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were estimated using a non-compartmental method. RESULTS Midazolam clearance, a surrogate measure of CYP3A4/5 activity, was significantly decreased upon COC use (63.3 l h−1vs. 53.9 l h−1, P < 0.05). A median decrease of 5.6 l h−1 (95% CI −4.1, 13.3 l h−1) was observed. However, the magnitude of change was similar to that reported in women with normal BMI. CONCLUSIONS Although we hypothesized that obesity might amplify the impact on CYP3A4/5 activity in COC users, we found that this was not the case. This finding is reassuring regarding potential additional drug−drug interactions in obese COC users as CYP3A4/5 is a major enzyme in the metabolism of many marketed drugs.

The study explores whether patient satisfaction is increased when a web application is used to inform patients about a surgical intervention. A randomised single centre study was conducted. The study population consisted of 32 patients that underwent an elective colon resection for colon cancer (15 in the experimental and 17 in the control group; patient characteristics were equally divided between both groups). The study group got the information with the web application from the nurse practitioner at least 10 days before surgery; the control group was given the standard oral information by the nurse practitioner. Satisfaction level was measured with a validated patient satisfaction questionnaire directly after giving the information and two weeks after the surgical intervention. The results indicate that patients are more satisfied when information is given by means of a web application. Given that expectations regarding hospital admission are more realistic when information is given in the opti...

Alcohol-induced liver injury (ALI) has been associated with, among other molecular changes, abnormal hepatic methionine metabolism, resulting in decreased levels of S-adenosylmethionine (SAM). Dietary methyl donor supplements such as SAM and betaine mitigate ALI in animal models; however, the mechanisms of protection remain elusive. It has been suggested that methyl donors may act via attenuation of alcohol-induced oxidative stress. We hypothesized that the protective action of methyl donors is mediated by an effect on the oxidative metabolism of alcohol in the liver. Male C57BL/6J mice were administered a control high-fat diet or diet enriched in methyl donors with or without alcohol for 4 weeks using the enteral alcohol feeding model. As expected, attenuation of ALI and an increase in reduced glutathione:oxidized glutathione ratio were achieved with methyl donor supplementation. Interestingly, methyl donors led to a 35% increase in blood alcohol elimination rate, and while there was no effect on alcohol metabolism in the stomach, a profound effect on liver alcohol metabolism was observed. The catalase-dependent pathway of alcohol metabolism was induced, yet the increase in CYP2E1 activity by alcohol was blunted, which may be mitigating production of oxidants. Additional factors contributing to the protective effects of methyl donors in ALI were increased activity of low- and high-K(m) aldehyde dehydrogenases leading to lower hepatic acetaldehyde, maintenance of the efficient mitochondrial energy metabolism, and promotion of peroxisomal beta-oxidation. Profound changes in alcohol metabolism represent additional important mechanism of the protective effect of methyl donors in ALI.

Background and Purpose— Cytochrome P450 epoxygenase metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are produced in the brain and perform important biological functions, including vasodilation and neuroprotection. However, EETs are rapidly metabolized via soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). We tested the hypothesis that sEH gene deletion is protective against focal cerebral ischemia through enhanced collateral blood flow. Methods— sEH knockout (sEHKO) mice with and without EETs antagonist 14, 15 epoxyeicosa-5( Z )-enoic acid (EEZE) were subjected to 2-hour middle cerebral artery occlusion (MCAO), and infarct size was measured at 24 hours of reperfusion and compared to wild-type (WT) mice. Local CBF rates were measured at the end of MCAO using iodoantipyrine (IAP) autoradiography, sEH protein was analyzed by Western blot and immunohistochemistry, and hydrolase activity and levels of EETs/DHETs were measured in brain and plasma using LC-MS/MS and ELISA, respectively. Results— sEH immunoreactivity was detected in WT, but not sEHKO mouse brain, and was localized to vascular and nonvascular cells. 14,15-DHET was abundantly present in WT, but virtually absent in sEHKO mouse plasma. However, hydrolase activity and free 14,15-EET in brain tissue were not different between WT and sEHKO mice. Infarct size was significantly smaller, whereas regional cerebral blood flow rates were significantly higher in sEHKO compared to WT mice. Infarct size reduction was recapitulated by 14,15-EET infusion. However, 14,15-EEZE did not alter infarct size in sEHKO mice. Conclusions— sEH gene deletion is protective against ischemic stroke by a vascular mechanism linked to reduced hydration of circulating EETs.

Nonalcoholic steatohepatitis is prevalent among obese individuals with excessive caloric intake, insulin resistance, and type II diabetes. However, no animal models exist that recapitulate this important association. This study produced and characterized steatohepatitis (SH) caused by intragastric overfeeding in mice. C57BL/6, tumor necrosis factor (TNF) type I receptor-deficient, and genetically matched wild type mice were fed via an implanted gastrostomy tube a high-fat diet for 9 weeks in the increasing amount up to 85% in excess of the standard intake. Animals were examined for weight gain, insulin sensitivity, and histology and biochemistry of liver and white adipose tissue (WAT). Overfed C57BL/6 mice progressively became obese, with 71% larger final body weights. They had increased visceral WAT, hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance, and insulin resistance. Of these mice, 46% developed SH with increased plasma alanine aminotransferase (121 +/- 27 vs. 13 +/- 1 U/L), neutrophilic infiltration, and sinusoidal and pericellular fibrosis. Obese WAT showed increased TNFalpha and leptin expression and reciprocally reduced adiponectin expression. The expression of lipogenic transcription factors (SREBP-1c, PPARgamma, LXRalpha) was increased, whereas that of a lipolytic nuclear factor PPARalpha was reduced in SH. SH was associated with reduced cytochrome P450 (Cyp)2e1 but increased Cyp4a. TNF type I receptor deficiency did not prevent obesity and SH. In conclusion, forced overfeeding with a high-fat diet in mice induces obesity, insulin resistance, and SH in the absence of TNF signaling or Cyp2e1 induction.

A phase I study of fixed-dose 5-fluorouracil (FU) and leucovorin (LCV), with excalating doses of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib, was conducted in 16 patients with advanced colorectal adenocarcinoma. At doses typically used to treat arthritis patients (100-200 mg po BID), celecoxib did not increase toxicities expected from the chemotherapy alone. 5-FU and leucovorin did not affect COX-2 inhibition by celecoxib. Preliminary data suggest it is safe to combine celecoxib with standard chemotherapeutic agents, in treatment of patients with colorectal cancer.

Chronic oxidative stress induced by overexpression of the cytochrome P450 isoform 2E1 (CYP2E1) has been implicated in hepatocyte injury and death. However, the mechanism by which CYP2E1 overexpression may promote cell death is unknown. Acute oxidative stress activates mitogen-activated protein kinases (MAPK), suggesting that chronic oxidant generation by CYP2E1 may regulate cellular responses through these signaling pathways. The effect of CYP2E1 overexpression on MAPK activation and their function in altering death responses of CYP2E1-overexpressing hepatocytes were investigated. Chronic CYP2E1 overexpression led to increased extracellular signal-regulated kinase 1/2 (ERK1/2) activation constitutively and in response to oxidant stress from the superoxide generator menadione. CYP2E1-overexpressing cells were resistant to menadione toxicity through an ERK1/2-dependent mechanism. Similar to menadione, the polyunsaturated fatty acid (PUFA) arachidonic acid (AA) induced an increased activation of ERK1/2 in hepatocytes that overexpressed CYP2E1. However, CYP2E1-overexpressing cells were sensitized to necrotic death from AA and the PUFA gamma-linolenic acid, but not from saturated or monounsaturated fatty acids. Death from PUFA resulted from oxidative stress and was blocked by inhibition of ERK1/2, but not p38 MAPK or activator protein-1 signaling. CYP2E1 expression induced ERK1/2 activation through increased epidermal growth factor receptor (EGFR)/c-Raf signaling. Inhibition of EGFR signaling reversed CYP2E1-induced resistance to menadione and sensitization to AA toxicity. In conclusion, chronic CYP2E1 overexpression leads to sustained ERK1/2 activation mediated by EGFR/c-Raf signaling. This adaptive response in hepatocytes exposed to chronic oxidative stress confers differential effects on cellular survival, protecting against menadione-induced apoptosis, but sensitizing to necrotic death from PUFA.

Cytochrome P450 (CYP) 2E1 is induced by ethanol and is postulated to be a source of reactive oxygen species during alcoholic liver disease. However, there was no difference in liver pathology and radical formation between wild-type and CYP2E1 knockout mice fed ethanol. Other CYP isoforms may contribute these effects if CYP2E1 is inhibited or absent. The purpose of this study was, therefore, to determine if blocking most of the P450 isoforms with 1-aminobenzotriazole (ABT; 100 mg/kg i.g.), has any effect on liver damage and oxidative stress due to alcohol in rats and mice. Male C57BL/6 mice and Wistar rats were fed either high-fat control or ethanol-containing enteral diet for 4 weeks. ABT had a significant inhibitory effect on many P450 isoforms independent of concomitant alcohol administration. However, ABT did not protect against liver damage due to alcohol in either species. Indices of oxidative stress and inflammation were also similar in livers from vehicle-treated and ABT-treated animals fed ethanol. In summary, suppression of P450 activity with ABT had no apparent effect on oxidative stress caused by alcohol in both rats and mice. These data support the hypothesis that oxidative stress and liver damage can occur independently of CYP activities in both rats and mice during early alcohol-induced liver injury.

PC-SPES, a proprietary mixture composed of eight different herbs, is used worldwide as an alternative treatment by prostate cancer patients. It has been suggested that the clinical and in vitro antitumor activity exhibited by PC-SPES may be due to estrogenic activity, which in turn may be mediated by the presence of undeclared prescription drug contaminants. Here, we evaluated the in vivo effects of two different commercial lots of PC-SPES in male and female rats. Our high-pressure liquid chromatography analysis coupled with gas chromatography/mass spectrometry analysis by an independent laboratory suggested that PC-SPES lot 5430125 was contaminated with diethylstilbestrol (DES), whereas lot 5431249 was not. Treatment of male rats with PC-SPES lot 5430125 or DES alone reduced the weight of androgen target organs and decreased circulating levels of sex steroids and luteinizing hormone, whereas lot 5431249 was without effect. In addition, lot 5430125 and DES, but not lot 5431249 increased uterine weight in female rats. These results suggest that the inhibitory effects on androgen targets are mediated through suppression of the hypothalamic-pituitary axis and this suppression is probably due to DES contamination. We assessed the effects of both lots of PC-SPES and DES on hepatic cytochrome P450 expression and activity. Both lots of PC-SPES and DES reduced CYP3A activity and protein levels. Because the response of CYP3A to PC-SPES was not dependent on whether it contained DES, a phytochemical component of PC-SPES is most likely responsible for this effect. Inhibition of CYP3A has important implications for potential herbal-drug interactions.

Increased expression of cytochrome P450 2E1 (CYP2E1) occurs in alcoholic liver disease, and leads to the hepatocellular generation of toxic reactive oxygen intermediates (ROI). Oxidative stress created by CYP2E1 overexpression may promote liver cell injury by sensitizing hepatocytes to oxidant-induced damage from Kupffer cell-produced ROI or cytokines. To determine the effect of CYP2E1 expression on the hepatocellular response to injury, stably transfected hepatocytes expressing increased (S-CYP15) and decreased (AN-CYP10) levels of CYP2E1 were generated from the rat hepatocyte line RALA255-10G. S-CYP15 cells had increased levels of CYP2E1 as demonstrated by Northern blot analysis, immunoblotting, catalytic activity, and increased cell sensitivity to death from acetaminophen. Death in S-CYP15 cells was significantly decreased relative to that in AN-CYP10 cells following treatment with hydrogen peroxide and the superoxide generator menadione. S-CYP15 cells underwent apoptosis in response to these ROI, whereas AN-CYP10 cells died by necrosis. This differential sensitivity to ROI-induced cell death was partly explained by markedly decreased levels of glutathione (GSH) in AN-CYP10 cells. However, chemically induced GSH depletion triggered cell death in S-CYP15 but not AN-CYP10 cells. Increased expression of CYP2E1 conferred hepatocyte resistance to ROI-induced cytotoxicity, which was mediated in part by GSH. However, CYP2E1 overexpression left cells vulnerable to death from GSH depletion.

The use of DBSs for home monitoring has been limited due to unsatisfactory blood sampling and analytical difficulties. The aim of this longitudinal feasibility trial was to assess the utility of DBS to monitor TAC and Cr at home in transplant recipients. A total of 30 participants (2-21 years, mean±SD, 13.6±5.4 year) were enrolled over 12 months. Eighteen were males. Monthly DBS samples were obtained at home and mailed to the central laboratory for analysis of TAC and Cr. Nineteen patients completed the study, and 216 cards were received in the laboratory from a total of 279 cards expected, with 416/519 (80%) blood spots being suitable for analysis. We found a high correlation between blood TAC and Cr levels by DBS and the clinical laboratory, R2=.81 and .95, respectively. Fifteen parents and 15 youth completed measures of satisfaction with and preference for DBS testing. All but one parent/caregiver and youth reported satisfaction and preference for this method of testing over laboratory blood draws. We conclude that home DBS monitoring is a feasible method to monitor TAC and Cr in pediatric transplant recipients.

Diets high in cruciferous vegetables are associated with lower risk of incidence of prostate cancer, including aggressive forms of this disease. Human intervention studies with cruciferous vegetable-rich diets also demonstrate modulation of gene expression in important pathways in prostate cells. Sulforaphane is a constituent of these foods postulated to harbor the anti-neoplastic activity based on multiple tumor models. Our own work demonstrates that sulforaphane inhibits AR signaling in prostate cancer cells. Here, we report results from the first clinical trial of sulforaphane-rich extracts in men with prostate cancer. We treated 20 patients who had recurrent prostate cancer with 200 μmoles/day of sulforaphane-rich extracts for a maximum period of 20 weeks and determined the proportion of patients with ≥50 % PSA declines, the primary endpoint. Only one subject experienced a ≥50 % PSA decline. Thus, the primary endpoint was not achieved. Seven patients experienced smaller PSA declines (

The continuous intragastric enteral feeding protocol in the rat was a major development in alcohol-induced liver injury (ALI) research. Much of what has been learned to date involves inhibitors or nutritional manipulations that may not be specific. Knockout technology avoids these potential problems. Therefore, we used long-term intragastric cannulation in mice to study early ALI. Reactive oxygen species are involved in mechanisms of early ALI; however, their key source remains unclear. Cytochrome P-450 (CYP)2E1 is induced predominantly in hepatocytes by ethanol and could be one source of reactive oxygen species leading to liver injury. We aimed to determine if CYP2E1 was involved in ALI by adapting the enteral alcohol (EA) feeding model to CYP2E1 knockout (−/−) mice. Female CYP2E1 wild-type (+/+) or −/− mice were given a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. All mice gained weight steadily over 4 wk, and there were no significant differences between groups. There were also no differences in ethanol elimination rates between CYP2E1 +/+ and −/− mice after acute ethanol administration to naive mice or mice receiving EA for 4 wk. However, EA stimulated rates 1.4-fold in both groups. EA elevated serum aspartate aminotransferase levels threefold to similar levels over control in both CYP2E1 +/+ and −/− mice. Liver histology was normal in control groups. In contrast, mice given ethanol developed mild steatosis, slight inflammation, and necrosis; however, there were no differences between the CYP2E1 +/+ and −/− groups. Chronic EA induced other CYP families (CYP3A, CYP2A12, CYP1A, and CYP2B) to the same extent in CYP2E1 +/+ and −/− mice. Furthermore, POBN radical adducts were also similar in both groups. Data presented here are consistent with the hypothesis that oxidants from CYP2E1 play only a small role in mechanisms of early ALI in mice. Moreover, this new mouse model illustrates the utility of knockout technology in ALI research.

Bullfrogs (Rana catesbeiana) anesthetized with a large dose of thiopental (42.8 mg/kg) retained movement responses to nociceptor stimuli despite an average plasma drug level of 51 mg/l, of which 63% was bound to plasma proteins. This concentration, when corrected to include only unbound and uncharged drug, was 2-fold greater than those reported to abolish nociceptor response (NR) during surgical anesthesia in man. The median anesthetic dose (AD50) for loss of the righting reflex was 11.2 mg/kg by s.c. injection into the abdominal lymph sac; however, at 54.0 mg/kg, all frogs retained NRs, although otherwise deeply anesthetized. The ratio of NR-blocking dose to light AD was thus >4.8, as compared to

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.

Hepatic CYP2E1 is induced in several models of alcohol administration, but clinically relevant pathology is only observed in rats in a model involving the continuous intragastric administration of an ethanol-containing, corn oil-based, high-fat diet. The level of CYP2E1 correlates with the degree of liver pathology in the intragastric feeding model, which leads to the hypothesis that radical production by CYP2E1 is responsible for the pathology. Destruction of the Kupffer cells with gadolinium chloride (GdCl3) prevented the development of ethanol-dependent pathology and decreased the production of radicals that appeared in the bile of intragastrically alcohol-fed rats. If the induction of CYP2E1 and subsequent formation of oxidant species by the enzyme is causative in the ethanol-dependent hepatic pathology, then protection by GdCl3 could be due an inhibition of CYP2E1 induction. In the current study, ethanol-administration for 4 wk produced marked steatosis, necrosis, and inflammation not seen in control rats. Immunochemically, CYP2E1 was induced 5- to 6-fold in microsomes from the ethanol-treated animals. Rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated approximately 3-fold, consistent with CYP2E1 induction. When GdCl3 was administered with ethanol, there was a decrease of approximately 80% in Kupffer cell receptor expression, and there was a significant decrease in hepatic pathology, which confirms previous studies. However, in the ethanol and GdCl3-treated animals, there was no significant decrease in the induction of CYP2E1. CYP2E1 was elevated approximately 5-fold, as estimated by immunoblot analysis, and rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated 3- to 4-fold in ethanol + GdCl3-treated rats. Thus, these results clearly dissociate the induction of CYP2E1 by intragastric infusion of ethanol from the generation of early alcohol-induced liver disease. It is concluded that Kupffer cells rather than CYP2E1 play the major role in the initiation of hepatocyte damage caused by alcohol.

The role of cytochrome P450 metabolism of fatty acids and lipid peroxidation in the alterations of the fatty acid composition of the liver and liver pathology was investigated. The CYP2E1 inhibitors partially prevented CYP2E1 induction by ethanol and completely blocked lipid peroxidation. However, the liver pathology induced by ethanol was only partially prevented as was the decrease in arachidonic acid in total liver lipid, triglycerides and cholesterol esters. This means that liver peroxidation induced by ethanol can not completely account for the liver pathology or the decrease in arachidonic acid caused by ethanol. Lauric acid omega-1 hydroxidation by the liver microsomes in vitro was increased by ethanol and partially blocked by CYP2E1 inhibitors. However, although ethanol feeding increased the total hydroxidation and epoxidation of arachidonic acid, these were not inhibited by CYP2E1 inhibitors. Thus the ethanol-induced arachidonic acid depletion is not likely due to CYP2E1 metabolism of arachidonic acid, since the severity of liver pathology correlated negatively with the decrease in arachidonic acid in the ethanol-fed rats. The increase in its metabolism by microsomes and decrease in synthesis may be an important mechanism of ethanol-induced liver injury.

Aim Nitric oxide donors, such as isosorbide dinitrate ointment (ISDN), are considered as first-choice agents in the treatment of chronic anal fissure. Injection with botulinum toxin A in the internal anal sphincter is often used as a second-line therapy, although it may give better results and fewer side effects than nitric oxide donors. The aim of this randomized clinical trial was to investigate whether botulinum toxin A (Dysport®) is more effective than ISDN in the primary treatment of chronic anal fissure. Method From April 2005 until October 2009, 60 patients (32 men) with a median age of 42 (25–82) years were randomized to receive either ISDN 10 mg/ml (1%) (n = 33) or injection with 60 units of Dysport® (n = 27). The primary end-point was the percentage of complete fissure healing after 8 weeks. Results After a median of 9 weeks complete fissure healing was noted in 18 of 27 patients in the Dysport® group and in 11 of 33 patients in the ISDN group (P = 0.010). Absolute improvement of pain scores after 9 weeks was similar in both groups (P = 0.733). Patients treated with Dysport® had fewer side effects than patients treated with ISDN (P = 0.028). Of the patients with a healed fissure, 28% of the Dysport® group and 50% of the ISDN group had a recurrence within 1 year (P = 0.286; hazard ratio 2.08; 95% CI = 0.54–7.97). Conclusion Dysport® is more effective than ISDN ointment and has fewer side effects in the primary treatment of chronic anal fissure. The recurrence rate within 1 year in both treatment groups is high.

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies. Cancer Res; 73(11); 3356–70. ©2013 AACR.

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.

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.

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)

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.

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.

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).

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 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.

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.

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.

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.

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.