A novel, dual pan-PIM/FLT3 inhibitor SEL24 exhibits broad therapeutic potential in acute myeloid leukemia.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in acute myeloid leukemia patients (AML). Although FLT3 tyrosine kinase inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. For such reasons, we have developed SEL24-B489 - a potent, dual PIM and FLT3-ITD inhibitor. SEL24-B489 exhibited significantly broader on-target activity in AML cell lines and primary AML blasts than selective FLT3-ITD or PIM inhibitors. SEL24-B489 also demonstrated marked activity in cells bearing FLT3 tyrosine kinase domain (TKD) mutations that lead to FLT3 inhibitor resistance. Moreover, SEL24-B489 inhibited the growth of a broad panel of AML cell lines in xenograft models with a clear pharmacodynamic-pharmacokinetic relationship. Taken together, our data highlight the unique dual activity of the SEL24-B489 that abrogates the activity of signaling circuits involved in proliferation, inhibition of apoptosis and protein translation/metabolism. These results underscore the therapeutic potential of the dual PIM/FLT3-ITD inhibitor for the treatment of AML.

Anxiogenic effects of developmental bisphenol A exposure are associated with gene expression changes in the juvenile rat amygdala and mitigated by soy.

Early life exposure to Bisphenol A (BPA), a component of polycarbonate plastics and epoxy resins, alters sociosexual behavior in numerous species including humans. The present study focused on the ontogeny of these behavioral effects beginning in adolescence and assessed the underlying molecular changes in the amygdala. We also explored the mitigating potential of a soy-rich diet on these endpoints. Wistar rats were exposed to BPA via drinking water (1 mg/L) from gestation through puberty, and reared on a soy-based or soy-free diet. A group exposed to ethinyl estradiol (50 µg/L) and a soy-free diet was used as a positive estrogenic control. Animals were tested as juveniles or adults for anxiety-like and exploratory behavior. Assessment of serum BPA and genistein (GEN), a soy phytoestrogen, confirmed that internal dose was within a human-relevant range. BPA induced anxiogenic behavior in juveniles and loss of sexual dimorphisms in adult exploratory behavior, but only in the animals reared on the soy-free diet. Expression analysis revealed a suite of genes, including a subset known to mediate sociosexual behavior, associated with BPA-induced juvenile anxiety. Notably, expression of estrogen receptor beta (Esr2) and two melanocortin receptors (Mc3r, Mc4r) were downregulated. Collectively, these results show that behavioral impacts of BPA can manifest during adolescence, but wane in adulthood, and may be mitigated by diet. These data also reveal that, because ERß and melanocortin receptors are crucial to their function, oxytocin/vasopressin signaling pathways, which have previously been linked to human affective disorders, may underlie these behavioral outcomes.

Measurement of bisphenol A, bisphenol A ß-D-glucuronide, genistein, and genistein 4'-ß-D-glucuronide via SPE and HPLC-MS/MS.

Bisphenol A (BPA) is a synthetic industrial reactant used in the production of polycarbonate plastics, and genistein is a natural phytoestrogen abundant in the soybean. Current studies investigating the endocrine-disrupting effects of concomitant exposures to BPA and genistein have warranted the development of an analytical method for the simultaneous measurement of BPA and genistein, as well as their primary metabolites, bisphenol A ß-D-glucuronide (BPA gluc) and genistein 4'-ß-D-glucuronide (genistein gluc), respectively. All four analytes were extracted from rat plasma via solid phase extraction (SPE). Three SPE cartridges and four elution schemes were tested. Plasma extraction using Bond Elut Plexa cartridges with sequential addition of ethyl acetate, methanol, and acetonitrile yielded optimal average recoveries of 98.1 ± 1.8% BPA, 94.9 ± 8.0% genistein, 91.4 ± 6.1% BPA gluc, and 103 ± 6.1% genistein gluc. Identification and quantification of the four analytes were performed by a validated HPLC-MS/MS method using electrospray ionization and selective reaction monitoring. This novel analytical method should be applicable to the measurement of BPA, genistein, BPA gluc, and genistein gluc in urine, cultures, and tissue following in vivo exposures. While reports of the determination of BPA and genistein independently exist, the simultaneous optimized extraction and detection of BPA, genistein, BPA gluc, and genistein gluc have not previously been reported.

ß-Hexachlorocyclohexane levels in serum and risk of Parkinson's disease.

Pesticide exposure has been implicated as an environmental risk factor for the development of Parkinson's disease (PD). However, few studies have identified specific pesticides. Previously, we identified elevated serum levels of the organochlorine pesticide ß-hexachlorocyclohexane (ß-HCH) in PD patients from a small clinical sample. Here, we conducted a case-control study to confirm the association between ß-HCH and PD in a larger sample size (n=283) with serum samples of PD patients and controls obtained from UT Southwestern Medical Center and Emory University. Samples were obtained from two discrete periods at both sites, 2001-2003 and 2006-2008, and were analyzed for ß-HCH levels. Adjusted odds ratios (ORs) for PD were estimated using logistic regression and generalized estimating equations. The mean serum ß-HCH level across all cohorts in this study was 22.3 ng/mg cholesterol (range: 0-376.7), and the levels were significantly higher and samples collected in 2001-2003 vs. 2006-2008. After controlling for age and gender, the OR for increased risk of PD for every 1 ng/mg increase in serum ß-HCH ranged from 1.02 to 1.12 across the four different cohorts, and 1.03 (95% CI: 1.00-1.07, p value=0.031) in the pooled analysis. Furthermore, the OR for increased risk of PD of subjects having serum ß-HCH levels above the inter-quartile range of 39.08 ng/mg cholesterol was 2.85 (95% CI: 1.8, 4.48; p value<0.001). These data are consistent with environmental decreases in ß-HCH levels between 2001 and 2008, but they indicate that elevated levels of serum ß-HCH are still associated with heightened risk for PD.

The antiepileptic drug zonisamide inhibits MAO-B and attenuates MPTP toxicity in mice: clinical relevance.

Zonisamide is an FDA-approved antiepileptic drug that blocks voltage-dependent Na(+) channels and T-type Ca(2+) channels and improves clinical outcome in Parkinson's disease (PD) patients when used as an adjunct to other PD therapies. Zonisamide also modifies dopamine (DA) activity, provides protection in ischemia models and influences antioxidant systems. Thus, we tested it for its ability to protect DA neurons in a mouse model of PD and investigated mechanisms underlying its protection. Concurrent treatment of mice with zonisamide and 1-methyl-4-phenyl-1,2,3,6-tetraydropyridine (MPTP) attenuated the reduction in striatal contents of DA, its metabolite DOPAC and tyrosine hydroxylase (TH). We also discovered that zonisamide inhibited monoamine oxidase B (MAO-B) activity in vitro with an IC(50) of 25 muM, a concentration that is well within the therapeutic range used for treating epilepsy in humans. Moreover, the irreversible binding of systemically administered selegiline to MAO-B in mouse brain was attenuated by zonisamide as measured by ex vivo assays. Zonisamide treatment alone did not produce any lasting effects on ex vivo MAO-B activity, indicating that it is a reversible inhibitor of the enzyme. Consistent with the effects of zonisamide on MAO-B, the striatal content of 1-methyl-4-phenylpyridinium (MPP(+)), which is derived from the administered MPTP via MAO-B actions, was substantially reduced in mice treated with MPTP and zonisamide. The potency and reversibility with which zonisamide blocks MAO-B may contribute to the ability of the drug to improve clinical symptoms in PD patients. The results also suggest that caution in its use may be necessary, especially when administered with other drugs, in the treatment of epilepsy or PD.

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography-tandem mass spectrometry.

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.

Quantification of Paraquat, MPTP, and MPP+ in brain tissue using microwave-assisted solvent extraction (MASE) and high-performance liquid chromatography-mass spectrometry.

Animal models, consistent with the hypothesis of direct interaction of paraquat (PQ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with specific areas of the central nervous system have been developed to study Parkinson's disease (PD) in mice. These models have necessitated the creation of an analytical method for unambiguous identification and quantitation of PQ and structurally similar MPTP and 1-methyl-4-phenylpyridinium ion (MPP+) in brain tissue. A method for determination of these compounds was developed using microwave-assisted solvent extraction (MASE) and liquid chromatography-mass spectrometry. Extraction solvent and microwave conditions such as power and time were optimized to produce recoveries of 90% for PQ 78% for MPTP and 97% for its metabolite MPP+. The chromatographic separation was performed on a C8, column and detection was carried out using an ion trap as an analyzer with electrospray ionization. Mass spectrometer parameters such as heated capillary temperature, spray voltage, capillary voltage and others were also optimized for each analyte. Analysis was done in selective ion-monitoring (SIM) mode using m/z 186 for PQ, m/z 174 for MPTP, and m/z 170 for MPP+. The method detection limit for paraquat in matrix was 100 pg, 40 pg for MPTP, and 20 pg MPP+.

Elevated serum pesticide levels and risk of Parkinson disease.

BACKGROUND: Exposure to pesticides has been reported to increase the risk of Parkinson disease (PD), but identification of the specific pesticides is lacking. Three studies have found elevated levels of organochlorine pesticides in postmortem PD brains. OBJECTIVE: To determine whether elevated levels of organochlorine pesticides are present in the serum of patients with PD. DESIGN: Case-control study. SETTING: An academic medical center. PARTICIPANTS: Fifty patients with PD, 43 controls, and 20 patients with Alzheimer disease. MAIN OUTCOME MEASURES: Levels of 16 organochlorine pesticides in serum samples. RESULTS: beta-Hexachlorocyclohexane (beta-HCH) was more often detectable in patients with PD (76%) compared with controls (40%) and patients with Alzheimer disease (30%). The median level of beta-HCH was higher in patients with PD compared with controls and patients with Alzheimer disease. There were no marked differences in detection between controls and patients with PD concerning any of the other 15 organochlorine pesticides. Finally, we observed a significant odds ratio for the presence of beta-HCH in serum to predict a diagnosis of PD vs control (odds ratio, 4.39; 95% confidence interval, 1.67-11.6) and PD vs Alzheimer disease (odds ratio, 5.20), which provides further evidence for the apparent association between serum beta-HCH and PD. CONCLUSIONS: These data suggest that beta-HCH is associated with a diagnosis of PD. Further research is warranted regarding the potential role of beta-HCH as a etiologic agent for some cases of PD.

Prolonged toxicokinetics and toxicodynamics of paraquat in mouse brain.

BACKGROUND: Paraquat (PQ) has been implicated as a risk factor for the Parkinson disease phenotype (PDP) in humans and mice using epidemiologic or experimental approaches. The toxicokinetics (TK) and toxicodynamics (TD) of PQ in the brain are not well understood. OBJECTIVES: The TK and TD of PQ in brain were measured after single or repeated doses. METHODS: Brain regions were analyzed for PQ levels, amount of lipid peroxidation, and functional activity of the 20S proteasome. RESULTS: Paraquat (10 mg/kg, ip) was found to be persistent in mouse ventral midbrain (VM) with an apparent half-life of approximately 28 days and was cumulative with a linear pattern between one and five doses. PQ was also absorbed orally with a concentration in brain rising linearly after single doses between 10 and 50 mg/kg. The level of tissue lipid peroxides (LPO) was differentially elevated in three regions, being highest in VM, lower in striatum (STR), and least in frontal cortex (FCtx), with the earliest significant elevation detected at 1 day. An elevated level of LPO was still present in VM after 28 days. Despite the cumulative tissue levels of PQ after one, three, and five doses, the level of LPO was not further increased. The activity of the 20S proteasome in the striatum was altered after a single dose and reduced after five doses. CONCLUSIONS: These data have implications for PQ as a risk factor in humans and in rodent models of the PDP.

Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography-tandem mass spectroscopy.

A simple, sensitive and specific LC-MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN-GSH) and N-acetyl cysteine conjugates (SFN-NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extratcion of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid-liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN-GSH and SFN-NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC-MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN-GSH, SFN-NAC and SFN were 8.4, 11.0, and 28.2 min,, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01-0.1 microm depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.

Differential inducibility of rat pulmonary CYP1A1 by cigarette smoke and wood smoke.

Mainstream cigarette smoke (CS) and wood smoke (WS) were compared in terms of their pulmonary CYP1A1 inducibility. The inducibility was assessed in pulmonary microsomes from rats exposed to freshly generated CS or WS and in rat lung explants treated with extracts of CS or WS total particulate matter (TPM). Mutagenicity in Salmonella typhimurium TA98 and TA100, an effect established for CS and WS in previous studies, was also examined as a test of the biological activity of the smoke samples in the present study. Pulmonary microsomal CYP1A1 activity (as measured by ethoxyresorufin O-deethylase), was induced 4.4-fold and 8.3-fold following exposure of rats to smoke from a single cigarette and three cigarettes, respectively, relative to the activity in control rats. The induction was paralleled by elevated CYP1A1 mRNA level (by northern blot analysis). WS, in contrast to CS, induced neither pulmonary CYP1A1 activity nor mRNA in exposed rats. CYP1A1 protein (by western blot analysis) was induced in cultured rat lung explants by extracts of CS TPM or by a high concentration (496 nM) of benzo[a]pyrene (B[a]P) but not by extracts of WS TPM or a low concentration (0.110 nM) of B[a]P. The induction by high B[a]P concentration was inhibited by extracts of CS or WS TPM, with the inhibition by extracts of WS TPM (75%) being greater than that by extracts of CS TPM (31%). Extracts of CS TPM were as mutagenic as extracts of WS TPM to Salmonella typhimurium TA98 but were more mutagenic than extracts of WS TPM to Salmonella typhimurium TA100. The results show that CS and WS are mutagenic but that WS differs from CS in its inability to induce pulmonary CYP1A1.

Solvophobic acceleration of Diels-Alder reactions in supercritical carbon dioxide.

The rate of the Diels-Alder reaction between N-ethylmaleimide and 9-hydroxymethylanthracene in supercritical carbon dioxide (scCO(2)) was determined by following the disappearance of 9-hydroxymethylanthracene with in situ UV/vis absorption spectroscopy. The reaction conditions were 45-75 degrees C and 90-190 bar, which correspond to fluid densities (based on pure carbon dioxide) ranging between approximately 340 and 730 kg m(-3). The measured reaction rate at low scCO(2) fluid densities was nearly 25x faster than that reported in acetonitrile at the same temperature (45 degrees C). An inverse relationship between reaction rate and fluid density/pressure was observed at all temperatures in scCO(2). The apparent activation volumes were large and positive (350 cm(3) mol(-1)) and only a weak function of reduced temperature. A solvophobic mechanism analogous to those observed in conventional solvents is postulated to describe (a) the rate acceleration observed for this reaction in scCO(2) relative to that in acetonitrile, (b) the observed relationship between reaction rate and pressure/temperature/density, and (c) the large, positive activation volumes. Solubility measurements in scCO(2), rate measurements in conventional solvents, and an empirical correlation are used to support this theory. Our results advance the general understanding of reactivity in supercritical fluids and provide a rationale for selecting reactions which can be accelerated when conducted in scCO(2).

In vivo pharmacokinetics and regulation of gene expression profiles by isothiocyanate sulforaphane in the rat.

Sulforaphane (SUL) is one member of the isothiocyanate class of cancer chemopreventive compounds that has been shown to be effective in blocking initiation and progression of carcinogenesis. Previously, many studies have shown that SUL can potently induce phase II detoxifying enzymes, which contributes to its chemopreventive functions. In this study, we used 4967 oligonucleotides microarray to assess the genes that are modulated by SUL in in vivo rat livers, as well as time course of expression of these genes. The pharmacokinetics of SUL was assessed after oral dose of 50 micromol of SUL. The plasma concentration occurred at 1 h and peaked around 20 microM at 4 h after dosing and declined with a half-life of about 2.2 h. Analysis of the gene expression data found various clusters of genes that are important in cellular defense mechanisms and cell cycle regulation. The most robust cluster of genes is the metallothionein-like genes (MT-1/2 and MT-1a), which are increased up to 10-fold by 2 to 4 h after SUL dosing. The second cluster of genes is the glutathione S-transferase-A3-like genes, which include aflatoxin B1 aldehyde reductase and aldehyde oxidase. These genes are increased slightly by 4 h and peaked at 12 h. Real-time polymerase chain reaction was performed to authenticate the mRNA expression of some of these genes. In summary, this in vivo study of SUL provides the first clue as to the plasma concentrations of SUL, in vivo mitogen-activated protein kinase activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes. The results from this study may yield better insights for its chemopreventive functions.

Glucuronides of tea catechins: enzymology of biosynthesis and biological activities.

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are major green tea catechins with antioxidant and anticancer activities. In this study, we characterized the glucuronidation of EGCG and EGC in human, mouse, and rat microsomes and by nine different human UGT 1A and 2B isozymes expressed in insect cells. Six EGCG and EGC glucuronides were biosynthesized, and their structures were identified for the first time. (-)-EGCG-4"-O-glucuronide was the major EGCG glucuronide formed in all incubations. The catalytic efficiency (V(max)/K(m)) for (-)-EGCG-4"-O-glucuronide formation followed the order: mouse intestine > mouse liver > human liver > rat liver >> rat small intestine. The UGT-catalyzed glucuronidation of EGC was much lower than that of EGCG. The V(max)/K(m) for (-)-EGC-3'-O-glucuronide followed the following order: mouse liver > human liver > rat liver > rat and mouse small intestine. Human UGT1A1, 1A8, and 1A9 had high activities with EGCG. UGT1A8, an intestine-specific UGT, had the highest V(max)/K(m) for EGCG but low activity with EGC. Mice appeared to be more similar to humans than rats to humans in the glucuronidation of EGCG and EGC. Some of these catechin glucuronides retained the activities of their parent compounds in radical scavenging and in inhibiting the release of arachidonic acid from HT-29 human colon cancer cells. These results provide foundations for understanding the biotransformation and biological activities of tea catechins.